Source: MICHIGAN STATE UNIV submitted to NRP
ENDOCANNABINOIDS MODULATE MACROPHAGE INFLAMMATORY PHENOTYPE AND LIPOLYSIS IN PERIPARTURIENT DAIRY COWS’ ADIPOSE TISSUES
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1032595
Grant No.
2024-67011-42947
Cumulative Award Amt.
$180,000.00
Proposal No.
2023-11488
Multistate No.
(N/A)
Project Start Date
Aug 15, 2024
Project End Date
Aug 14, 2027
Grant Year
2024
Program Code
[A7101]- AFRI Predoctoral Fellowships
Recipient Organization
MICHIGAN STATE UNIV
(N/A)
EAST LANSING,MI 48824
Performing Department
VET MED DEAN
Non Technical Summary
Several physiologic conditions drive lipid mobilization (lipolysis) in the adipose tissues (AT) of dairy cows, including periparturient energy deficits and the presence of inflammatory stimuli during infectious disease. Should lipolysis become excessive or dysregulated, AT inflammation results, and cows become susceptible to metabolic and inflammatory diseases, milk yield losses, and reproductive complications. Such challenges come at substantial costs to US producers and compromise the welfare of dairy cows.In addition to energy-dense lipids, cows' fat cells (adipocytes) release endocannabinoids during lipolysis, which are signaling molecules that regulate inflammation and metabolism. We hypothesize that these molecules regulate the inflammatory responses of immune cell populations, and the expression of their receptors vary in AT based on the energy demands faced by the cow. We will test this hypothesis through two aims. Aim 1 will reveal how the stimulation of cannabinoid receptors on immune cells in AT influences their numbers, inflammatory properties, and abilities to regulate lipolysis. For this aim, we will harvest and culture mononuclear cells and adipocytes from dairy cows, expose them to cannabinoid receptor-activating compounds, and evaluate the cells' responses. Aim 2 will investigate the relationships between the expression of cannabinoid receptors and abundance of immune cells in the AT of pre- and post-partum dairy cows. In this aim, we will collect AT from mature dairy cows at -21, +14, and +21 days relative to calving and, using microscopic and molecular biological techniques, assess how the distribution of cannabinoid receptors and abundance of mononuclear cells change relative to one another. Findings from Aims 1 and 2 will be published in peer-reviewed journals, presented at scientific meetings, and shared with industry stakeholders.This project will allow us to elucidate the role of endocannabinoids in the regulation of metabolic and inflammatory responses in the AT of dairy cows. We anticipate that these findings will support the development of targeted nutritional and pharmacological approaches to managing dairy cattle, especially in early lactation. Long-term, these efforts will help improve the health and productivity of dairy cows and, in addition, the sustainability and productivity of the US dairy industry.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
0%
Developmental
0%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3053410104050%
3053410103050%
Knowledge Area
305 - Animal Physiological Processes;

Subject Of Investigation
3410 - Dairy cattle, live animal;

Field Of Science
1040 - Molecular biology; 1030 - Cellular biology;
Goals / Objectives
The primary goal of this project is to determine the molecular mechanisms by which endogenous signaling molecules known as endocannabinoids regulate metabolism and inflammation in the fat of dairy cows. By better understanding these fundamental processes, this project aims to provide insight that may be applied to improve the health, welfare, and productivity of dairy cows and promote dairy industry sustainability and food security. This project will provide comprehensive research training to the PD in advanced molecular biological techniques, data analysis, and scientific communications. The specific objectives of this project consist of: Research objectives1. Investigate the relationships between cannabinoid receptor activation and mononuclear cell proliferation, activation, and regulation of lipolysisDetermine how cannabinoid receptor activation modulates the proliferation and activation of cultured mononuclear cellsAssess the role of cannabinoid receptor signaling on mononuclear cells' capacity to regulate lipolysis in adipocytes2. Characterize the relationships between mononuclear cell abundance, phenotype, and endocannabinoid system component distribution in the adipose tissue of periparturient cowsDetermine how the abundance and phenotype of mononuclear cells change in cows' adipose tissues throughout the periparturient periodEvaluate how the distribution of endocannabinoid system components within adipose tissue relates to mononuclear cell abundance and phenotypeTraining objectives1. Technical skill developmentDevelop expertise in monocyte and adipocyte culture and functional assays to investigate the role of the endocannabinoid system on cell proliferation, inflammatory phenotype, and lipolysisProvide hands-on training in advanced molecular biological techniques including gene expression analysis (RT-qPCR, RNA sequencing, sm-FISH), cell morphology and surface marker expression quantitation (flow cytometry), lipidomic and proteomic analyses, capillary electrophoresis, and immunohistochemistry2. Data analysisGain proficiency in bioinformatics tools and statistical analysis software for analyzing gene expression data, protein expression, image analysis, and cell phenotyping resultsIntegrate and interpret complex datasets to draw accurate and meaningful biological conclusions3. Scientific communicationAdvance the ability to communicate scientific findings through the preparation of research manuscripts in peer-reviewed journals and conference proceedingsProvide opportunities to generate figures and graphics and present research findings at scientific conferences, departmental seminars, and stakeholder outreach eventsPromote mentorship and leadership skills through training of undergraduate and graduate research students and attendance of workshops and seminars
Project Methods
To determine how endocannabinoid (eCB) receptor activation influences immune cell activity and fat breakdown in cows, we will collect blood and fat samples.Aim 1: We will examine how eCB receptor activation alters the behavior of immune cells and mobilization of lipids from fat cells. For the first experiment, we will culture immune cells derived from the blood and expose them to different cannabinoid receptor-targeting agents and observe how they differ in number, type, and function. In the second experiment, we will grow fat cells harvested from cows' fat along with the treated immune cells to observe the effects on fat breakdown and inflammatory responses.Aim 2: We will analyze fat samples from pre- and post-partum cows to understand how the relationships between eCB system components and the characteristics of immune cells change in the tissue throughout this time. Fat samples will be collected from cows prior to and following calving, and we will study the expression and distribution of eCB-associated genes and proteins.The data generated from this project will be shared in peer-reviewed scientific publication and poster and oral presentation format at scientific conferences and stakeholder meetings. The results of this project will help clarify how the eCB system may regulate inflammation and metabolism in the fat of cows, which may improve cow health and productivity.

Progress 08/15/24 to 08/14/25

Outputs
Target Audience:During this reporting period, my research and professional development activities reached diverse audiences across academic, industry, and scientific communities, as well as students and early-career researchers. Scientific and Academic Audiences: I presented my research on endocannabinoid system regulation of adipose tissue metabolism and inflammation in dairy cows at multiple national and international conferences, including the American Dairy Science Association (ADSA) Annual Meeting (2025), the International Cannabinoid Research Society Symposium (2025), the International Veterinary Immunology Symposium (2025), and the Conference of Research Workers in Animal Disease (CRWAD, 2025). These meetings included academic researchers, industry scientists, veterinarians, graduate students, and trainees from around the world. My presentations focused on the metabolic and inflammatory roles of endocannabinoids and bioactive lipid mediators in dairy cattle, contributing to knowledge exchange in comparative medicine, veterinary science, and livestock production. I also participated in the Big-10 Academic Alliance Lipids Conference (2025), where my research reached lipid biologists and translational metabolism researchers, and the National Association of Veterinary Scholars Combined Degree Colloquium (2025), engaging veterinary clinician-scientists and students in combined DVM/PhD training programs. Industry Stakeholders and Dairy Professionals: Through research abstracts presented at ADSA and the Michigan Dairy Health Symposium (2024), I disseminated findings to dairy industry professionals, extension specialists, nutritionists, and veterinarians working to improve dairy cow health, welfare, and productivity. My collaborative research with domestic and international partners (Israel, Lebanon, Colombia, USA) also connected with stakeholders interested in sustainable livestock management and innovative feed additive strategies. Undergraduate and Graduate Student Audiences: As a BRUSH Program Fellow and program co-coordinator, I mentored undergraduate students conducting summer research in veterinary and biomedical sciences. I provided feedback on study design, data analysis, and scientific communication, helping students prepare abstracts, posters, and oral presentations. I also contributed to candidate selection, faculty mentor pairing, and program administration to enhance the student research experience. Within the Contreras Laboratory, I trained undergraduate researchers and visiting scholars in adipose tissue experimental techniques, including adipocyte isolation, cell culture, RNA extraction, and image analysis. I also guided students in understanding lipid mediator biology and endocannabinoid system research, supporting their professional growth and confidence in research settings. Professional Peers and Collaborators: I engaged with veterinarians, nutritionists, and scientists through membership and active participation in professional societies, including the American Dairy Science Association (ADSA), American Association of Bovine Practitioners (AABP), International Cannabinoid Research Society (ICRS), and the American Association of Veterinary Immunologists (AAVI). My oral and poster presentations, combined with published peer-reviewed manuscripts and abstracts, advanced scientific communication and collaborative opportunities. Publications and Knowledge Dissemination: I contributed as first author of one accepted and one in-review publication, and co-authored on peer-reviewed manuscripts in high-impact journals, including the Journal of Dairy Science, Journal of Animal Science & Biotechnology, and Frontiers in Immunology. These publications reached academic researchers, clinicians, and industry professionals interested in dairy science, immunology, and comparative medicine. Competitions and Outreach: I shared my research with broad audiences by participating in scientific communication competitions, including the 3-Minute Thesis Competition at CRWAD 2025 (second place) and the ADSA Midwest Branch Young Scholar Award (2025). These events provided opportunities to translate complex scientific findings into accessible formats for mixed academic and industry audiences. Through these combined efforts, I reached audiences spanning veterinary students, undergraduate researchers, graduate trainees, academic scientists, dairy professionals, and international collaborators. My presentations, publications, mentoring, and program leadership directly supported knowledge transfer, workforce development, and collaborative research in the fields of veterinary science, dairy production, and comparative metabolism. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Technical Skill Development Gained advanced expertise in adipocyte culture, functional lipolysis assays, and lipidomic analyses (LC-MS/MS), leading to multiple first-author publications. Completed advanced workshops, including the Scientific Illustration Masterclass (Dec. 2024), which improved figure design and communication of complex datasets. Data Analysis Performed bioinformatic and statistical analyses for RNA-seq, lipidomics, and pathway analyses (IPA, KEGG, Reactome), integrating results to predict metabolic and inflammatory signaling changes. Created publication-ready figures and analyses presented at ADSA, CRWAD, and ICRS conferences. Scientific Communication Published peer-reviewed manuscripts and presented multiple abstracts at major scientific meetings. Received recognition through awards such as the 3-Minute Thesis competition at CRWAD 2025, the ADSA Midwest Branch Young Scholar Award, and the AAVI Travel Award. Mentorship & Leadership Continued serving as a BRUSH Program Fellow, providing mentorship and guidance to undergraduate researchers and supporting program management and student training. Delivered feedback on scientific writing and presentations, further strengthening communication skills. Served as an evaluator at MidSURE 2025, a research conference for undergraduate summer research students to share their results in poster format. How have the results been disseminated to communities of interest? Disseminated findings through two first-author peer-reviewed publications and multiple co-authored abstracts and manuscripts. Presented results at national and international meetings, including ADSA 2024 and 2025 Annual Meetings, CRWAD 2025, NAVS Combined Degree Colloquium, ICRS 2025, the Big-10 Academic Alliance Lipids Conference, and the PHAT/TRINS joint symposia. Shared research outcomes with collaborators and stakeholders in dairy science, comparative medicine, and immunology through presentations, abstracts, and manuscripts. What do you plan to do during the next reporting period to accomplish the goals? Complete analysis of adipose tissue mononuclear cell abundance and phenotype using flow cytometry, RT-qPCR, and immunohistochemistry. Finalize integration of RNA-seq and lipidomic data to identify molecular pathways linking ECS signaling to adipose tissue immune function and metabolism. Prepare and submit additional manuscripts focused on immune and metabolic adaptations of bovine adipose tissue under endotoxemia. Present new findings at national and international conferences to engage with academic, clinical, and industry audiences. Continue mentoring undergraduate researchers through the BRUSH program and further develop professional skills through workshops and presentations.

Impacts
What was accomplished under these goals? Research Objective 1: Published a first-author manuscript in the Journal of Animal Science and Biotechnology showing that lipolysis pathways modulate lipid mediator release and endocannabinoid system signaling in dairy cows' adipocytes. Published a first-author manuscript in Frontiers in Immunology demonstrating that endotoxemia alters circulating levels of fatty acid-derived lipid mediators known as oxylipins. Presented related findings at national and international meetings, including ADSA 2024 and 2025 Annual Meetings, CRWAD 2025, NAVS Combined Degree Colloquium, and ICRS 2025. Research Objective 2: Generated new data on endotoxemia-induced alterations in plasma endocannabinoid and ethanolamide profiles, presented at ADSA 2025, the Big-10 Academic Alliance Lipids Conference, the PHAT/TRINS joint symposia, and ICRS 2025. Shared findings on systemic and adipocyte-derived oxylipin profile changes during endotoxemia in multiple conference proceedings. Prepared manuscripts integrating RNA-seq data and adipocyte size distribution to evaluate immune and metabolic adaptations of adipose tissue under stress.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: Endotoxemia Alters Systemic and Adipocyte-Derived Oxylipin Profiles in Dairy Cows. MN Myers, M Chirivi, J Parales-Gir�n, J dos Santos Neto, LC Worden, AL Lock, GA Contreras. Big-10 Academic Alliance Lipids Conference 2025, Preventative Health of Adipose tissue (PHAT) & Translational Research in NeuroMetabolism Symposium (TRINS) Joint Symposia Proceedings at The Ohio State University.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: Endotoxemia modulates endocannabinoid and ethanolamide profiles in dairy cows plasma. MN Myers, M Chirivi, JC Gandy, J Parales-Gir�n, J dos Santos Neto, AL Lock, GA Contreras. CRWAD 2025 abstract booklet, Chicago, IL.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Lipolysis pathways modulate the production of bioactive lipid mediators in dairy cows adipocytes. MN Myers, M Chirivi, JC Gandy, J Tam, M Zachut, GA Contreras. 2024 Phi Zeta Research Day at Michigan State University abstract booklet.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Endocannabinoid production is modulated by lipolysis pathways in dairy cows adipocytes. MN Myers, M Chirivi, JC Gandy, J Tam, M Zachut, GA Contreras. National Association of Veterinary Scholars 2024 Combined Degree Colloquium, St. Paul, MN; program booklet p.27.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Abomasal infusion of docosahexaenoic acid alters plasma oxylipin profiles in lactating dairy cows. MN Myers, M Chirivi, U Abou-Rjeileh, HL Reisinger, ML Miller, AL Lock, BJ Bradford, GA Contreras. Proceedings of the 2024 ADSA Annual Meeting, J. Dairy Sci. Vol. 107 Suppl. 1 p.158.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Lipolysis pathways modulate endocannabinoid production and release by dairy cows adipocytes. MN Myers, M Chirivi, JC Gandy, J Tam, M Zachut, GA Contreras. Proceedings of the 2024 ADSA Annual Meeting, J. Dairy Sci. Vol. 107 (Suppl. 1): p. 221.
  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2025 Citation: Dynamics of oxylipin biosynthesis in systemic inflammation: Insights from a large animal model of endotoxemia. MN Myers, M Chirivi, JM dos Santos Neto, J Parales-Gir�n, L Worden, AL Lock, GA Contreras. Front. Immunol. Vol 16:1595888(2025).
  • Type: Peer Reviewed Journal Articles Status: Submitted Year Published: 2024 Citation: Lipolysis pathways modulate lipid mediator release and endocannabinoid system signaling in dairy cows adipocytes. MN Myers, M Chirivi, JC Gandy, J Tam, M Zachut, GA Contreras. J. Animal Sci. & Biotechnol. Vol 15:103(2024).
  • Type: Peer Reviewed Journal Articles Status: Submitted Year Published: 2025 Citation: TLR4 and prostaglandin pathways at the crossroads of endotoxemia-induced lipolysis. M Chirivi, U Abou-Rjeileh, MN Myers, J Parales-Gir�n, L Worden, AL Lock, GA Contreras. Front. Immunol. Vol 16:1591210(2025).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: Endotoxemia alters endocannabinoid and ethanolamide profiles in the plasma of dairy cows. MN Myers, M Chirivi, J Parales-Gir�n, J dos Santos Neto, LC Worden, AL Lock, GA Contreras. International Cannabinoid Research Societys Annual Symposium 2025. Bloomington, IN. Abstract Proceedings booklet, p. 80.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: Endotoxemia Alters Plasma Endocannabinoid and Ethanolamide Profiles in Dairy Cows. MN Myers, M Chirivi, J Parales-Gir�n, J dos Santos Neto, LC Worden, AL Lock, GA Contreras. American Dairy Science Association 2025 Annual Meeting, Louisville, KY. J. Dairy Sci. Vol. 108(Suppl. 1):96.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: Endotoxemia induces systemic and adipocyte-specific alterations in oxylipin profiles in dairy cows. MN Myers, M Chirivi, J Parales-Gir�n, J dos Santos Neto, LC Worden, AL Lock, GA Contreras. American Dairy Science Association 2025 Annual Meeting, Louisville, KY. J. Dairy Sci. Vol. 108(Suppl. 1):236.