Progress 07/01/24 to 06/30/25

Outputs
Target Audience:During this reporting period, our efforts were primarily focused on the foundational research outlined in Objectives 1, 2, and 3 of the project, as well as the initial development of educational materials for Objective 4. Consequently, the target audience most directly impacted and engaged by our work were: 1) Researchers: This includes our internal project team (Principal Investigators, graduate students, and postdoctoral researchers) and our immediate scientific collaborators within the University of Wisconsin-Madison and the U.S. Dairy Forage Research Center. We also had interactions with collaborators from other academic institutions, such as the University of Florida, Michigan State University, and The Ohio State University. Through scientific meetings (e.g., Dairy Symposium, Annual Meeting of the American Dairy Science Association), the activities of the project also extend to the broader community of researchers interested in ruminant nutrition, microbiology, genetics, and environmental sustainability. Our project is generating novel, science-based knowledge concerning the complex interplay of rumen microbiome function, host genetics, and dietary interventions on methane emissions from dairy cattle and manure. This foundational research is crucial for advancing the scientific understanding required to develop effective mitigation strategies. Engaging with fellow researchers facilitates critical peer review, fosters knowledge exchange, and identifies opportunities for future cooperative efforts, ultimately strengthening the scientific basis for more efficient dairy farming systems. Our work also contributes to the global body of knowledge for scientists tackling similar challenges. The efforts conducted during the reporting period included internal project meetings, collaborative data collection and analysis, and initial presentations of methodologies and experimental approaches within academic and research groups. 2) Educational institutions (students and faculty directly involved): Specifically, this audience comprises graduate and undergraduate students, as well as faculty members, within the Animal and Dairy Sciences and Bacteriology departments at the University of Wisconsin-Madison, in addition to the U.S. Dairy Forage Research Center. Leveraging the existing resources at the University of Wisconsin-Madison, our project is serving as a platform for hands-on training and experiential learning. Students and faculty directly involved are gaining opportunities to learn and apply advanced techniques in methane emission monitoring, metatranscriptomics, and microbiome analysis. This direct engagement ensures the development of a highly skilled workforce equipped to address future challenges in sustainable agriculture and contributes to the long-term goal of improving the efficiency of U.S. dairy farming. Activities include mentorship in research methodologies, involvement in thesis and dissertation research directly related to the project objectives, and discussion of the research objectives and experimental approaches in graduate and undergraduate courses. 3) Agricultural extension professionals: This includes extension agents and agricultural educators affiliated with the UW-Extension program. These professionals are key intermediaries for disseminating science-based knowledge to dairy producers and the broader agricultural community. Initial engagement with this group is vital for ensuring that the future extension program's materials and strategies are relevant, accessible, and effectively tailored to the needs of the farming community, maximizing the project's impact on real-world practices. We are maintaining ongoing conversations and collaborations regarding the development of educational materials related to the project themes for integration into the UW-Extension program. Co-PIs Ferraretto and Larson work directly with the extension team at UW-Madison and are already planning the expansion of such activities in the next few months. 4) Dairy producers: One of the core missions of this project is to evaluate effective management strategies that directly benefit dairy farmers. The findings from our research, particularly on improving the productivity of dairy cows and reducing emissions from enteric fermentation and manure, are directly relevant to their economic viability and environmental stewardship. For example, our results will provide insights into how diet influences milk yield and emission traits. Preliminary engagement has been conducted using both in-person and virtual approaches. We are striving to incorporate the practical needs of dairy farmers and existing management practices into our research design, and Co-PI Ferraretto has presented relevant insights related to the animal trial described in Objective 2 through written materials or in-person presentations. Additionally, co-PI Larson has engaged with participants of the 2025 Midwest Manure Summit held to discuss manure emissions reduction strategies. 5) General public: This audience includes K-12 students, parents, and the wider community, as exemplified by participants in public outreach events. Engaging the general public is crucial for raising broader awareness about improving the efficiency of livestock production and for showcasing the science-based solutions being developed. As part of this project, we aim to build public support for innovative research and encourage informed decision-making regarding food systems and improving the productivity of dairy cattle, while minimizing their impact on the environment. As part of this effort, we participated in the UW-Madison Science Expeditions event on April 6th, 2024, in Madison, WI, which attracted over 700 attendees. As the project progresses and the extension program develops and is deployed, the target audience will expand to include a broader community of dairy producers, extensionists, and industry stakeholders. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has fostered interdepartmental collaboration, providing unique and valuable training opportunities for both graduate and undergraduate students across various disciplines. The Soil and Environmental Sciences graduate student responsible for the manure storage and soil application phases of this project (Josh Mirabella) gained new experience with animal and manure handling, improving practical skills and the capacity to connect the areas of animal and field management. Negin Sheibany, Daniel Vieira, and Alice Assumpcao, are PhD students in Animal and Dairy Sciences at UW-Madison, directly involved in the animal experiments under Objectives 1 and 2. These students are gaining skills in sample collection, sample processing, data analysis, and data interpretation. Training and professional development opportunities are being provided for conducting animal nutrition experiments, performing laboratory work, and developing technical proficiency in data management, statistical analysis, data visualization, and the application of bioinformatic tools for meta-taxonomic analysis and metabolomics. Pranay Tiwary, a junior in the Molecular and Cell Biology major, received hands-on training in DNA extraction protocols to process ruminal and fecal samples collected in this project. The project also involves Fiorella Viquez-Umana, a PhD student in Animal and Dairy Sciences, is actively contributing to the development of instructional materials to translate complex scientific findings into accessible information for diverse stakeholders, including dairy farmers and agricultural consultants (Objective 4). Two additional undergraduate students are receiving training under the direct mentorship of Co-PI Li. Their training covers biological sample collection, storage, RNA extraction, and subsequent quality check. Additionally, they received training on RNA sequencing experiment design, library preparation, and subsequent quality check of the finished libraries. These collaborative and training initiatives are key for advancing the specific aims of this USDA-NIFA-funded project and provide significant contributions to the development of the next generation of scientists and extension professionals equipped to foster more efficient and profitable ruminant livestock systems. How have the results been disseminated to communities of interest?Dissemination of the results related to the project has been done through different formats, including written materials, in-person presentations, outreach activities, and virtual presentations (webinars). A summary of the activities developed to disseminate science-based knowledge and results of the project are listed below: 1) Written content: a) Ferraretto, L., M. Pupo, and K. Juckem. 2024. Reducing methane emissions from cows is a major challenge for ag industry and researchers. Wisconsin State Farmer August 16th, 2024. https://www.wisfarmer.com/story/opinion/columnists/2024/08/16/how-is-the-ag-industry-working-to-shrink-cows-methane-emissions/74784191007/ Wisconsin State Farmer is the state's most comprehensive and longest running farm newspaper that serves the statewide agricultural community across the state and beyond. The target audience was dairy farmers and consultants. b) Ferraretto, L. 2024. Starch level may be a tool to mitigate methane. Hoard's Dairyman Intel, August 26, 2024. https://hoards.com/article-35516-starch-level-may-be-a-tool-to-mitigate-methane.html The Hoard's Dairyman is an American agricultural trade publication that focuses on dairy farming. The target audience was dairy farmers and consultants. 2) In-person presentations: a) Ferraretto, L. F. 2024. Modulating cow performance and feeding behavior. RP Nutrients. Fort Atkinson, WI. (08/14; 8 attendees). The target clientele was dairy nutritionists, and the presentation included a discussion of the effects of starch concentration in the diet on methane emissions. b) Emissions mitigation strategies were also included in the 2025 Midwest Manure Summit held in Lambeau Field, WI. Presentations on manure emissions reduction strategies and ongoing discussion on future research were presented to 100 participants by co-PI Larson. 3) Outreach activities: UW-Madison Science Expeditions - Cows, burps, and microbes: A journey into sustainable livestock. April 6th, 2025, Madison, WI. (>700 participants, attendees: K-12 and general public). Our exploration station offered a range of interactive exhibits and hands-on activities designed to demonstrate the impact of the rumen microbiome on methane emissions and strategies for mitigating the environmental footprint of dairy cows. We developed the following activities: 1) Feed fermentation model: Participants witnessed a live demonstration showcasing the fermentation of feedstuffs by rumen microorganisms. Using variables such as feed composition, participants explored the factors influencing methane production from enteric fermentation and potential sustainable solutions; 2) Meet the microbes: Using microscopes, and interactive displays, participants examined anatomical features of a cow's stomach and used samples of rumen fluid to observe live rumen microbes and learn about their unique characteristics and functions; 3) Methane mitigation challenge: Participants answered trivia questions and explored multifaceted approaches to sustainable livestock management, including innovative strategies for mitigating methane production such as feed additives, genomic selection, and precision feeding technologies. 4) Badger Dairy Insights Webinar https://dairy.extension.wisc.edu/articles/nutritional-strategies-to-reduce-methane-emissions-in-lactating-cows/. We also organized a webinar to discuss the importance of methane emissions and the nutritional strategies available. The target audience were dairy farmers, nutritionists, and agricultural educators. What do you plan to do during the next reporting period to accomplish the goals?During the upcoming reporting period, our team will focus on several key activities to advance the project toward its overarching goals of enhancing ruminant livestock productivity and sustainability, particularly through the reduction of inefficiencies during ruminal fermentation. These activities encompass continued data collection, the execution of critical experimental phases, in-depth sample analysis, and the expansion of our outreach efforts. We will continue the monitoring of methane emissions from individual dairy cows utilizing the GreenFeed system. This ongoing data collection is essential for identifying and characterizing animals with high- and low-methane emission phenotypes, which is foundational to understanding the underlying mechanisms of methane production and the inefficiencies associated with ruminal fermentation. Concurrently, we will collect biological samples (e.g., rumen fluid, fecal samples) essential for evaluating the abundance of methanogens and characterizing the composition of both rumen and fecal microbial communities. This continuous effort directly supports Objective 1 by providing the necessary biological samples to unravel microbial contributions to overall rumen fermentation and their association with methanogenic activity. Another focus will be the execution of the second phase of the animal trial under Objective 2. This phase is designed to compare the effects of different levels of water-soluble carbohydrates in dairy cow diets. The experiment will evaluate the impact of these dietary interventions on critical production parameters, including milk yield, milk composition, and feed efficiency. Simultaneously, we will measure enteric and manure methane emissions associated with these dietary treatments. This will provide insights into how specific dietary modifications can influence both productivity and environmental footprint, directly addressing the project's goal of developing effective management strategies. Building upon our sample collection, a key activity will involve the processing of samples from our concluded and ongoing animal trials for RNA extraction. This will enable us to perform metatranscriptomic analysis of the active rumen microbial community. This will allow us to move beyond just microbial composition to understand the functional activity of the microbes, particularly those involved in fermentation losses, under different dietary treatments. Analyses of microbial gene expressions will provide unparalleled insights into the ecological processes driving methane production, directly addressing the core aims of Objective 1. In line with Objective 4, we plan a significant expansion of our extension activities to promote enhanced environmental health awareness among target audiences. This will include updating existing fact sheets and developing new, comprehensive fact sheets that disseminate findings on nutritional interventions aimed at reducing carbon losses and improving the productivity of dairy cattle. Furthermore, we will continue to actively develop both face-to-face and online extension activities. These presentations will be specifically tailored to reach and engage dairy farmers and agricultural consultants, providing them with practical, science-based knowledge and management strategies to implement on their farms. This direct engagement is vital for translating our research findings into actionable practices that contribute to more sustainable livestock systems. Additionally, the conclusion of the manure incubation and the start and finish of the soil application incubation will take place, allowing the creation of a holistic assessment of the impact of starch level and individual cow variation on downstream emissions of CH4, CO2, N2O, and NH3.

Impacts
What was accomplished under these goals? During the reporting period, we collected data on methane emission measurements as well as rumen and fecal samples from 22 animal trials across 2 research stations (Emmons Blaine Arlington Dairy Research Center, Arlington, WI, and Marshfield Agricultural Research Station, Stratford, WI) and one commercial farm (Rosy Lane Holsteins, Watertown, WI). These activities are related to Objective 1 of the project. The methane and milk production data were collected for a minimum of 5 weeks using the GreenFeed system (Objective 1), involving a total of 815 dairy cows (as of June 2025). Ranking of animals is based on residual methane intensity (RMI) calculated as CH4 regressed on milk energy and metabolic body weights, and residual methane yield as CH4 regressed on dry matter intake. The models also include days in milk (DIM) and treatments. Animals ranked as high or low methane emitters will be used for subsequent nucleic acid extraction and characterization of the active rumen microbial community, microbiome composition, and abundance of methanogens as they relate to host phenotypes. The first animal experiment related to Objective 2, investigating how dietary changes influence milk production and methane emissions, was concluded in May 2025, under an approved protocol by the Animal Care and Use Committee of the College of Agriculture and Life Sciences at the University of Wisconsin-Madison. Sixty-four multiparous Holstein cows (88 ± 24 days in milk and 722 ± 81 kg of body weight at the start of the experiment; mean ± SD) were housed in a pen equipped with 32 electronic gate feeders (RIC system, Isentec, Marknesse, the Netherlands) in the University of Wisconsin-Madison sand-bedded freestall barn (Emmons Blaine Dairy Research Center, Arlington, WI). Cows (experimental unit) and gate feeders were randomly assigned to 1 of 2 treatments in a crossover design experiment with 35-d periods. Cows were allowed 1 week to acclimate to gate feeders before the start of the trial, during which all the cows received a common diet. At the start of the 10-week treatment period, cows were switched to their assigned treatment diets, and at the end of week 5, treatments were crossed over between the two groups of 32 cows. Cows had free access to water at all times. Cows were allowed access to all gate feeders assigned to their respective treatment group per period (32 cows allowed access to the 16 gate feeders assigned per treatment) during the 2 periods. Treatment diets were formulated to contain high starch concentration (30% of dry matter; HS) and reduced starch concentration (20% of dry matter; RS). Dietary starch concentrations of 20% and 30% were achieved by adjusting the proportions of dried ground corn and soyhulls in the diet. Throughout each experimental period, individual milk yield, milk components, dry matter intake, feeding behavior, body weights, body condition score, and methane, hydrogen, and carbon dioxide emissions were measured. Production of methane was measured on an individual cow basis using the GreenFeed systems (C-Lock Inc., Rapid City, South Dakota) for the entire experiment period. Data collected from the GreenFeed system throughout this period was also used to rank cows based on their methane emissions. Rumen and fecal samples were collected in the last week of each experimental period via oro-esophageal tubing or rectal palpation, respectively. We are currently analyzing the data for performance parameters, and DNA extractions of rumen and fecal samples will start soon. Preliminary results from this animal trial show that cows fed a high-starch diet have lower dry matter intake (DMI, 30.9 kg/d) and higher milk yield compared to the low-starch group (32.4 kg/d) (P < 0.01). In addition, lower milk urea nitrogen and higher feed efficiency (expressed as kg of milk per kg of DMI) were observed in cows fed high-starch (HS) diets than in reduced-starch (RS) cows (P<0.0001). Additionally, cows fed the HS diet produced 13.4% less methane (g/d) than cows fed the RS diet (P=0.001). Cows in the HS treatment also had lower methane yield (g CH4/kg dry matter) and lower methane intensity (g CH4/kg milk produced). We are still evaluating the data to determine the impact of the dietary changes on methane emission from cows ranked as high and low emitters, but these preliminary results support the premise that diet formulation can be a critical factor in developing better nutritional management strategies to improve efficiency and reduce the environmental impact of dairy farming. Spearman's rank correlations were also conducted based on three traits: methane production (CH4), residual methane intensity (RMI, methane production adjusted for milk energy and mBW), and residual methane yield (RMY, methane production adjusted for intake). The correlations were calculated by treatment sequence, from HS to LS or from LS to HS, each treatment sequence with n = 32 cows (per period in the crossover design). Cows from HS to LS had a strong correlation (>0.81) with the parameters listed above, which was higher than observed for cows moving from LS to the HS diet, in which the correlations were between 0.47-0.68. Cows from HS to LS changed their CH4 production from 435 g/d in HS to 471 g/d in LS, while cows from LS to HS did not change their CH4 production, around 448 g/d regardless of the treatment. Analysis of the microbial communities associated with high and low methane-emitting cows across experimental periods will document the diversity and abundance of ruminal microorganisms in animals with disparate emission traits. For objective 3, manure samples were also collected from cows in both treatment groups described in Objective 2 (high and reduced starch levels) to evaluate emissions from manure storage and land application. Two manure storage incubations were established to measure emissions of CH4, CO2, N2O, and NH3. Each incubation consists of manure samples from 32 dairy cows, half of which received a high starch diet in the first feeding period and the other half of which received a high starch diet in the second feeding period. Cumulative gas emissions over several months were measured, and the relationships between cumulative emissions during storage, starch level in feed, and enteric CH4 emissions during the feeding period are currently being determined. The incubations are ongoing, and soil incubation is planned to begin in July 2025, which will investigate the downstream impacts of animal and manure management practices on soil emissions. Manure emissions will also be linked to distinct cow phenotypes to evaluate the link between enteric and manure emissions under different dietary conditions.

Publications