Source: UNIV OF PENNSYLVANIA submitted to NRP
DECIPHERING THE ROLE OF INDIVIDUAL METHANOGENS AND THEIR INHIBITION ON HYDROGEN METABOLISM IN THE RUMEN OF DAIRY COWS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1024304
Grant No.
2021-67015-33385
Cumulative Award Amt.
$500,000.00
Proposal No.
2020-02672
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
UNIV OF PENNSYLVANIA
(N/A)
PHILADELPHIA,PA 19104
Performing Department
5805 - Clinical Studies-New Bo
Non Technical Summary
RationaleMethane (CH4), a potent greenhouse gas, is naturally produced in the reticulo-rumen by methanogenic archaea utilizing hydrogen (H2) released during feed fermentation by bacteria. Research indicates that underrepresented methanol-reducing Methanosphaera and methanol- and methylamine-reducing Methanomassiliicoccales may have a greater share in overall CH4 production compared with CO2 reducing Methanobrevibacter; however, knowledge on who the main archaeal players are and their proportional contribution to total CH4 formation in the rumen is lacking. Furthermore, the fate of H2 when CH4 production is inhibited remains unresolved as excessive H2 accumulation can inhibit feed fermentation in the rumen. The goal of this project is to determine the contribution of individual methanogens to total CH4 formation and determine the implications of inhibiting individual methanogens on regulation of H2 metabolism in H2-producing bacteria in the rumen. We propose to use macroalga Asparagopsis taxiformis (AT) as a feed supplement that can serve as potent inhibitor of methanogenesis in the rumenHypothesisThe overarching hypothesis is that Methanosphaera and Methanomassiliicoccales may have a greater share in total CH4 formation than was originally thought and that supplementing dairy cows with the macroalga Asparagopsis taxiformis (AT) may selectively eliminate Methanosphaera in the rumen.Specific ObjectivesObjective 1: Decipher and quantify the relative contributions of Methanobrevibacter, Methanosphaer,a and Methanomassiliicoccales to total CH4 formation in the rumen of dairy cows.The goal of this objective is to determine the functional role of different methanogens, quantify their contribution to total CH4 formation, and determine how AT selectively inhibits Methanosphaera. To achieve this, we will first perform in vitro experiments with pure cultures of individual methanogens and determine their sensitivity to AT. Second, we will perform in vitro rumen fermentation assays with rumen inoculum obtained from dairy cows with high and low CH4-yield phenotypes. Methane emissions and the relative abundance of individual methanogens will be assessed at different times in a 24h incubation period with and without the addition of AT. Finally, an in vivo 4 X 4 Latin square experiment will be conducted using 20 cows with 4 treatments (0, 0.25, 0.5, and 0.75% AT dietary dry matter basis). At the end of each period, rumen samples will be obtained from the cows which will be subjected to amplicon, metagenomic, and metatranscriptomic approaches to determine differences in microbial composition as well as their genes and transcripts. In addition, we will also collect data on CH4 emissions, dissolved and free H2 and fermentation in the rumen, dietary dry matter intake, and milk yield and composition.Objective 2: Determine the fate of H2 under CH4 inhibition in the rumen of dairy cows with different CH4 yield phenotypes with and without AT supplementation.The goal of this objective is to understand the mechanisms involved in H2 production and utilization in the rumen. The hypothesis is that supplementing AT to low CH4-yield cows may be effective in not only reducing CH4 but also in diverting H2 to non-methanogenic bacteria and may improve the energy efficiency of rumen fermentation. Further, by determining the effect of AT on individual methanogens, and tracking hydrogenases and H2 uptake by non-methanogenic sinks, this study will be able to account for H2 that is spared when CH4 is inhibited and will also be able to predict fermentation pathways. We will use both metagenomics and metatranscriptomics to track hydrogenases and H2 uptake by non-methanogenic sinks in the rumen of high and low CH4 emitters supplemented with AT to better understand H2 metabolism in the rumen. Eight to 10 cows each for high and low CH4-yield phenotypes will be identified with half the cows in each group receiving AT (0.5% dietary dry matter) and the remaining cows serving as control. Each experimental period will be 28 d, with 21 d for adaptation to the diet and 7 d for sampling and data collection. Data for CH4, CO2, and H2 emissions, total tract digestibility, urinary nitrogen losses, rumen fermentation (including dissolved H2), rumen microbiota, milk fatty acid profile, and production variables (DMI, milk yield and composition) will be analyzed.Objective 3: Determine the mechanisms by which butyrate production is increased in H2-producing bacteria in the rumen with inhibited methanogenesis using labeled precursors.The goal of this objective is to investigate the mechanisms through which butyrate-producing bacteria sense H2 under inhibited methanogenesis by AT. We will use labeled glucose as a VFA precursor to understand how butyrate production is enhanced under CH4 inhibition with AT and to what extent the labeled glucose is directed to acetate, propionate, and butyrate using in vitro experiments. Total gas production, gas composition, fermentation variables, fiber degradability, and microbial protein synthesis and microbiome composition will be monitored.Expected resultsWe expect to quantify the role of individual methanogens in methanogenesis and total CH4 formation in the rumen. Further, we also anticipate understanding the mechanism of action of AT in inhibition of methanogenesis. By supplementing AT to low CH4-yield dairy cows, we anticipate not only inhibiting methanogenesis but also diverting H2 away from CH4 formation to other sinks that help increase energy efficiency in the rumen. From this project, we anticipate enhancing knowledge on mechanisms by which butyrate-producing bacteria sense changes in H2 concentrations in response to CH4 inhibition. Overall, this project may provide new insights on enhancing energy efficiency while mitigating CH4 emissions from dairy cows.Anticipated ImpactBy understanding the role of individual methanogens and also selectively inhibiting individual methanogens, this project will significantly reduce CH4 emissions from dairy cows without adversely affecting intake and production performance. The project will also shed light on alternate pathways of fermentation in the rumen that have the potential to improve production efficiency in dairy cows.
Animal Health Component
15%
Research Effort Categories
Basic
85%
Applied
15%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
30234101010100%
Knowledge Area
302 - Nutrient Utilization in Animals;

Subject Of Investigation
3410 - Dairy cattle, live animal;

Field Of Science
1010 - Nutrition and metabolism;
Goals / Objectives
The overall goal of this project is to determine how methanogenesis can be effectively mitigated such that alternate pathways of fermentation are triggered to increase energy efficiency and ultimately improve rumen function and production responses in dairy cows. Specific objectives are:Objective 1: Decipher and quantify the contributions of individual methanogens to total CH4 formation using a combination of in vitro and in vivo experiments.Objective 2: Determine the fate of H2 under CH4 inhibition in the rumen of dairy cows with different CH4 yield phenotypes with and without AT supplementation.Objective 3: Determine the mechanisms by which butyrate production is increased in H2-producing rumen bacteria in the rumen when methanogenesis is inhibited.
Project Methods
Individual feeds and the total mixed ration will be sampled daily, composited weekly and analyzed for chemical composition using standard laboratory procedures. Milk yield from individual cows will be measured using milk meters twice daily and samples for milk composition analyses will be collected twice (2 a.m. and 2 p.m. milkings) during the last week of each experimental period; composite samples, per cow and period, will be analyzed for milk fatty acids After a 21-d adaptation period, CH4, carbon dioxide, and H2 emissions will be measured using the GreenFeed system (C-Lock Inc., Rapid City, SD). Measurements will take place 8 times in a 3-d period coveringa 24 h feeding cycle as follows: at 0900, 1500, and 2100 h (sampling d 1), 0300, 1200, and 1700 h (sampling d 2), and 0000, and 0500 h (sampling d 3). Sampling of ruminal contents via stomach tubing will take place during 3 sequential days in week 4 of each experimental period; samples will be collected before feeding (t0) and 2-4, 6-8, 10-12, 16, and 22 h after feeding covering a 24 h feeding cycle. These samples will be passed through 3 layers of cheese cloth to separate solid and liquid fractions. The solid and liquid fractions will be used for microbial analysis whereas a subset of liquid fraction samples will be processed for fermentation data including pH, VFA, dissolved H2 and ammonia concentrations., and microbial community using our established procedures. Total tract digestibility of dietary nutrients, urinary nitrogen excretion, and blood variables will be analyzed using routine protocols (i.e., Melgar et al., 2020).Microbial analysis: 16S-based amplicon, metagenomics and metatranscriptomics approachesNucleic acid (DNA and RNA) extraction: Rumen samples will be extracted for DNA using "Repeated Bead Beating and Column" (RBB+C) purification method (Yu and Morrison, 2004). RNA extraction from rumen samples will be performed using the Trizol method. Briefly, samples will be snap-frozen at cow side using liquid nitrogen. Rumen contents will be ground in liquid nitrogen and then bead beaten in Trizol. Subsequent steps involve extractions with isoamylalcohol and chloroform and finally with ethanol precipitations. The extracted RNA will be purified through a column using MEGAClear kit. The RNA quality will be checked on bioanalyzer and samples with RIN >8 will be processed for sequencing.16SrRNA and 16ScDNA amplicon sequencing: The extracted DNA will be amplified for 16S rRNA gene for bacteria and archaea using our established methods (Pitta et al., 2014a, Kumar et al., 2015). The extracted RNA will be used to construct cDNA using Superscript Vilo cDNA synthesis Kit as described in Pitta et al., (2014b) and amplified with same primers used for 16SrRNA. The PCR product will be bead purified using Beckman Coulter Agencourt AMPure XP Beads (Beckman-Coulter, CA) as per our established methods (Pitta et al. 2014a). The 16S rDNA and 16S rRNA libraries will be sequenced using Illumina MiSeq platform.Metagenomics: 1 μg of extracted DNA from each rumen sample will be prepared for whole genome shot gun sequencing using the Nextera DNA Library Prep Kit. The unamplified library (tight insert size of 250 bp for high-throughput sequencing from both ends by 2 × 150 bp) will be sequenced on an Illumina NextSeq500 instrument available at the Center for Host-Microbe interactions, School of Veterinary Medicine, University of Pennsylvania.Metatranscriptomics: Approximately 1 ug of RNA will be considered for further analysis. Ribosomal RNA will be removed from total RNA using Ribogold rRNA removal kit. Double stranded cDNA will be synthesized from the mRNA enriched RNA using the TruSeq® Stranded mRNA kit. Sequencing method is similar to metagenomics. We anticipate achieving approximately 50 million reads per sample in both metagenomics and metatranscriptomics.

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

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?4 PhD students and 2 postdocs were engaged in this project. More than 10 presentations were presented at ADSA conference. How have the results been disseminated to communities of interest?Publications and Conferences. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? All experiments have been completed, and the resulting data were either published or currently in review.

Publications

  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2025 Citation: 4. Stepanchenko, N., D. E. Wasson, S.F. Cueva, L. F. Martins, D. W. Pitta, and A. N. Hristov. 2024. Effects of enteric methane inhibitors on ruminal fermentation patterns in vitro using inoculum from cows with high and low methane yield phenotype. J. Dairy Sci. 107 (Suppl. 1):117.
  • Type: Peer Reviewed Journal Articles Status: Accepted Year Published: 2025 Citation: 1. N Indugu, KN Narayan, HA Stefenoni, ML Hennessy, B Vecchiarelli, JB Bender, R Shah, G Dai, S Garapati, C Yarish, SC Welchez, SE R�is�nen, D Wasson, C Lage, A Melgar, AN Hristov, DW Pitta* (2024). Elucidating the mechanistic basis of Asparagopsis taxiformis, a seaweed when effective in curbing enteric methane emissions from dairy cattle. Mbio 15(8):e0078224.
  • Type: Other Status: Awaiting Publication Year Published: 2025 Citation: 3. Stepanchenko, N., , N. Indugu, S. F. Cueva, L. Martins, K. Narayan, D.W. Pitta, A. N. Hristov. 2025. Effects of methane inhibitors on ruminal fermentation and microbial composition in vitro using inoculum from phenotypically high- and low-enteric methane emitting cows. J. Dairy Sci.
  • Type: Peer Reviewed Journal Articles Status: Accepted Year Published: 2024 Citation: 2. DE Wasson, H Stefenoni, S Cueva, C Lage, SE R�is�nen, A Melgar, M Fetter, M Hennessy, K Narayan, N Indugu, DW Pitta, C Yarish, AN Hristov* (2023). Screening of macroalgae species for enteric methane mitigation effect in vitro. Scientific Reports. Jun 17;13(1):9835.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: 1. Wasson, D. E., C. V. Almeida, S. F. Cueva, C. Eifert, S. Issabekova, L. F. Martins, A. Richards, N. Stepanchencko, and A. N. Hristov. 2024. Effects of brown macroalgae on enteric methane emissions and lactational performance of dairy cattle. J. Dairy Sci. 107 (Suppl. 1):336.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: 2. Wasson, D. E., C. V. Almeida, S. F. Cueva, L. F. Martins, A. Richards, N. Stepanchencko, and A. N. Hristov. 2024. Effects of a red macroalga supplementation on enteric methane emission and lactational performance of dairy cattle. J. Dairy Sci. 107 (Suppl. 1):83.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: 3. Wasson, D. E., S. F. Cueva, L. F. Martins, N. Stepanchencko, K. Welter, N. Indugu, D. Pitta and A. N. Hristov. 2024. Effects of Asparagopsis taxiformis supplementation on enteric gas emission and expression of methyl coenzyme reductase in dairy cows. J. Dairy Sci. 107 (Suppl. 1):144.


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

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One postdoc, Two doctoral students have been part of this project. Publications and abstracts are the consequence of their efforts. How have the results been disseminated to communities of interest?Publications and Conference presentations What do you plan to do during the next reporting period to accomplish the goals?Complete data analysis and publications.

Impacts
What was accomplished under these goals? All animal experiments are complete. Data analysis and manuscript preparation in progress.

Publications

  • Type: Journal Articles Status: Under Review Year Published: 2024 Citation: 1. KS Narayan, ACB Johnson, N Indugu, JS Bender, HA Stefenoni, AN Hristov, A Melgar, DW Pitta.Real-time PCR quantification of representative methanogens under normal and inhibited methanogenesis using two inhibitors in the rumen of dairy cattle. Submitted to Applied and Environmental Microbiology (In review)
  • Type: Journal Articles Status: Under Review Year Published: 2024 Citation: 2. N Indugu, KN Narayan, HA Stefenoni, ML Hennessy, B Vecchiarelli, JB Bender, R Shah, G Dai, S Garapati, C Yarish, SC Welchez, SE R�is�nen, D Wasson, C Lage, A Melgar, AN Hristov, DW Pitta. Elucidating the mechanistic basis of Asparagopsis taxiformis, a seaweed when effective in curbing enteric methane emissions from dairy cattle. Submitted to Mbio (In review)
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: 1. KS Narayan ?, A Yelampalli, N Indugu, A Castaneda, A Johnson, A Post, T Webb, Pitta D. Understanding the effect of halogenated compounds on methanogen isolates and methane production. American Dairy Science Association (ADSA) scheduled June 1619, 2024, in West Palm Beach, Florida, United States of America.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: 2. KS Narayan ?, N Indugu, A Castaneda, A Johnson, A Post, T Webb, JB Bender, LD Baker, S Rassler, Pitta D. Investigating the anti-methanogenic potential of seaweed varieties in manipulating the rumen fermentation and methane production. American Dairy Science Association (ADSA) scheduled June 1619, 2024, in West Palm Beach, Florida, United States of America
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: 3. N Stepanchenko ?, DE Wasson, SF Welchez, LF Martin1, DW Pitta, and AN Hristov. Effects of enteric methane inhibitors on ruminal fermentation patterns in vitro using inoculum from cows with high and low methane yield phenotype. American Dairy Science Association (ADSA) scheduled June 16-19, 2024, West Palm Beach, Florida, United States of America.


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

Outputs
Target Audience:Animal nutritionists, Dairy producers, livestock industries, Graduate andPostgraduatestudents and faculty associated with Animal Science Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Engagement of graduates and postdocs in research activities Summer internships for high school students and undergraduates How have the results been disseminated to communities of interest?Annual Dairy Science meetings, Penn Nutritional Symposium, Penn State Nutritional Symposium, Publications, Invited talks What do you plan to do during the next reporting period to accomplish the goals?Continue sample analysis for Objective 2 and 3.

Impacts
What was accomplished under these goals? For objective 1A: We have identified and validated primers for 15 different methanogens and investigated the effect of different inhibitors on individual methanogens in the rumen of dairy cows supplemented with 3-NOP and Asparagopsis taxiformis. We standardized the growth conditions for Methanobrevibacter ruminantium and Methanosphaera stadtmaniae. For objective 1B: In vitro rumen fermentation assays with inoculum from high and low methane emitting cows with supplementation of AT is completed. An abstract on fermentation data and microbial analysis has been submitted to ADSA 2023 (attached below). For objective 2: The in vivo study has been completed. Sample analysis is in progress. Objective 3: The in vitro protocol for labeling study has been completed. Sample analysis is in progress. Publications: KS Narayan?, N Indugu, M Hennessy, B Vecchiarelli, JB Bender, H stefnoni, A Hristov, M Audino, DW Pitta. Effects of methanogenic inhibitors on individual methanogens in the rumen of dairy cattle. American Dairy Science Association (ADSA) scheduled June 25-28,2023, Ottawa, Ontario, Canada. N Indugu?, HA Stefenoni , M Hennessy, B Vecchiarelli , J Bender, R Shah, S Garapati, C Yarish, SC Welchez, SE Räisänen, DE Wasson, C Lage, A Melgar, AN Hristov, DW Pitta. The effects of supplementing a seaweed Asparagopsis taxiformis on the rumen microbiome in dairy cows. American Dairy Science Association (ADSA) scheduled June 25-28,2023, Ottawa, Ontario, Canada. D. Pitta, H. Stefenoni, N. Indugu, M. Hennessy, B. Vecchiarelli, V. Shabtai, C. Welchez, S. Räisänen, D. Wasson, A. Melgar, M. Fetter, and A. Hristov. 2020. Differences in methanogenesis pathways and microbial diversity in the rumen of low- and high-methane-yield phenotype dairy cows. J. Dairy. Sci. Vol. 103, Suppl. 1. p. 159. N. Stepanchenko, D.E Wasson, S. Welchez, T. Silvestre, L. Martins, D. W. Pitta, A. N. Hristov. 2022. Effect of methane inhibitors on in vitro fermentation and CH4 production using rumen inoculum from cows with high and low methane yield phenotype. J. Dairy. Sci. Vol. 105, Suppl. 1. p. 316. N. Stepanchenko, D.E Wasson, S. Welchez, L. Martins, D. W. Pitta, A. N. Hristov. 2023. Lactational performance and enteric methane emissions of phenotypically high and low methane emitting dairy cows fed bromoform. J. Dairy. Sci. Vol. 106, Suppl. 1. p. TBD. N. Stepanchenko, H. Stefenoni, M. Hennessy, N. Indugu, D.E Wasson, S. Welchez, S. Räisänen, C. D. Dechow, D. W. Pitta, A. N. Hristov. 2023. Microbial composition, rumen fermentation parameters, enteric methane emissions, and lactational performance of phenotypically high- and low-methane emitting dairy cows. J. Dairy. Sci. Vol. 106, p. TBD. N Stepanchenko, H Stefenoni, M Hennessy, N Indugu, DE Wasson, S Cueva, S Räisänen, C Dechow, DW Pitta, AN Hristov (2023). Microbial composition, rumen fermentation parameters, enteric methane emissions, and lactational performance of phenotypically high- and low-methane emitting dairy cows. Journal of Dairy Science (accepted).

Publications


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

    Outputs
    Target Audience:Animal nutritionists, Rumen microbiologists, Environmental scientists, Dairy producers, livestock industries, Feed companies, Graduate and Postgraduate students and faculty associated with Animal and Dairy Sciences. Changes/Problems:For Objective 1: It took longer time to get the facilities and protocols up and running to culture methanogens. We are beginning to culture Methanobrevibacter ruminantium and Methanosphaera stadtmanae. We anticipate generating transcriptome data of individual methanogenic isolates in the next six months. For Objective 2: Screening the cows for high and low methane emissions to coincide with availability of Asparagopsis has been a challenge. Because of this, the in vivo animal experiment is moved to the second year of this project. What opportunities for training and professional development has the project provided?2 PhD students and 1 post-doc are working on this project. How have the results been disseminated to communities of interest?conference papers and seminars What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

    Impacts
    What was accomplished under these goals? For objective 1A: In vitro methanogen culture: The setup to work with methanogens is complete. We have begun culturing Methanobrevibacter ruminantium and Methanosphaera stadtamane. For objective 1B: In vitro rumen fermentation assays with inoculum from high and low methane emitting cows with supplementation of AT is completed. An abstract on fermentation data has been submitted to ADSA 2022 (attached below). Microbial analysis is in progress. For objective 1C: The in vivo study is in progress. Objective 3: The in vitro protocol for labeling study has been finalized. The experiment will occur in March/April of 2022.

    Publications

    • Type: Conference Papers and Presentations Status: Submitted Year Published: 2022 Citation: Stepanchenko, N., D.E Wasson, S. Welchez, T. Silvestre, L. Martins, D. W. Pitta, and A. N. Hristov. 2022. Effect of methane inhibitors on in vitro fermentation and methane production using rumen inoculum from cows with high and low methane yield phenotype. J. Dairy Sci. 105 (Suppl. 1) (submitted).