Progress 06/15/24 to 06/14/25

Outputs
Target Audience:Undergraduate Students: Undergraduate students were an important target audience during this reporting period, as early exposure to research concepts and hands-on skills fosters interest in careers in animal science, microbiome research, or biology, and helps strengthen the future workforce. Eleven undergraduates participated in laboratory instruction sessions related to this project, where they gained experience in animal handling, sample collection, microbiome analysis, and animal behavior observation. Of these, seven students received training through enrollment in the experiential learning course ADS 4440/GA4800 Research Experience Practicum under the PD's supervision, and four participated as undergraduate interns. In addition, three ADS undergraduate students enrolled in the PD-taught ADS 4723 Animal Microbiome course received classroom instruction on the gut-microbiome-brain axis, further aligning their learning with the project's objectives. Graduate Students: Our primary target audience also consisted of graduate students in Animal and Dairy Sciences. This group was targeted because they represent the next generation of researchers and professionals who will apply microbiome-related knowledge to animal welfare. Two graduate students, who are the main contributors to this project, were engaged through laboratory-based instruction, gaining hands-on experience in animal handling, sample collection, blood parameter measurement, microbiome analysis, and behavioral video analysis. Under the PD's supervision, both enrolled in ADS 9000 Research in Animal and Dairy Sciences to further develop their research skills. An additional thirteen graduate students enrolled in the Spring 2025 ADS 6723 Animal Microbiome course taught by the PD were reached through formal classroom instruction. These students were introduced to the concept of the gut-microbiome-brain axis and the latest research on the rumen-microbiome-brain axis, aligning with the project's focus on microbiome-mediated animal health improvements. Academic Communities: The findings of these studies were disseminated to academic communities through presentations at internal and national conferences, including the 2024 MSU Undergraduate Research Summer Showcase, 2024 MSU Fall Graduate Research Symposium, 2025 Spring MSU Undergraduate Research Symposium, 2025 Conference of Southern Graduate Schools, and the 2025 Southern/Western Joint Meeting of the American Society of Animal Science (ASAS). Local and Regional Producers: The findings of these studies were shared with local and regional producers through extension events, including the 2024 LegenDairy Farm Friday and the 2025 Spring Symposium - Food Science and Industry in Mississippi: Adding Value & Feeding the World. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Training activities: This project involves diverse collaborations with professionals in veterinary science, animal behavior, and computer science. For animal physiological measurements and sample collection, two graduate students and four undergraduate interns participating in this project were trained by a veterinarian from the Mississippi State University (MSU) College of Veterinary Medicine (CVM) in heat stress scoring and rumen contraction measurements for the 2024 summer trial. An additional six undergraduate students were trained for these procedures by the Project Director (PD) and graduate students in preparation for the 2025 spring trial. A junior clinical veterinarian involved in the project received training from a senior clinical veterinarian in rumen fluid collection using rumenocentesis. Two graduate students and three undergraduate interns were trained by the clinical veterinarian in blood sample collection. For animal behavior analysis, two graduate students were trained by collaborators from the MSU Department of Animal and Dairy Sciences in camera setup, ethogram development for cattle behavior, and machine learning techniques for behavioral analysis. For microbiome analysis, two graduate students and three undergraduate interns were trained by the PD and a collaborator from the MSU Institute for Genomics, Biocomputing & Biotechnology (IGBB) in microbial DNA extraction and library preparation. In addition, two graduate students and one undergraduate intern received further training from the PD and collaborators from MSU HPC in using the MSU High Performance Computing (HPC) system for bioinformatics analysis. For blood parameter analysis, two graduate students were trained in Enzyme-Linked Immunosorbent Assay (ELISA) techniques by the co-Principal Investigator (co-PI) and a postdoctoral researcher in the co-PI's laboratory. Two graduate students and ten undergraduate students received training in statistical analysis from the PD or from graduate students. The PD also mentored the two graduate students in abstract and manuscript writing, as well as in creating posters and presentation slides for research symposiums and conferences. Additionally, the PD and graduate students mentored six undergraduate students in writing abstracts and preparing posters for similar presentations. Training activities included one-on-one mentorship with the PD, co-PI, collaborators, and graduate students, as well as participation in the ADS 4440: Research Experience and Practice course led by the PD. Professional development Professional development for undergraduate students involved in this project: One undergraduate student from MSU Department of Animal and Dairy Sciences attended the 2025 Southern/Western Joint Meeting of the American Society of Animal Science (ASAS) and participated the undergraduate 3MT competition with the findings from this project. Two undergraduate students from MSU Department of Biochemistry, Nutrition, and Health Promotion attended the 2024 MSU Undergraduate Summer showcase and 2025 MSU Spring Undergraduate Research Symposium to present the findings from this study (animal physiological differences under heat stress and heat stress-relieved condition, capabilities of rumen microbes in synthesizing neurotransmitters) as posters. One student also participated in the section of three-minute thesis (3MT). One student won the Honorable Mention at 2025 George Hopper Excellence in Undergraduate Research Competition. Two undergraduate students from MSU Department of Biological Sciences and Department of Biochemistry, Nutrition, and Health Promotion attended the 2025 Spring Symposium - Food Science and Industry in Mississippi: Adding Value & Feeding the World to present the findings from this project (behavioral differences between Holstein and Jersey cows) as posters. One student won second place in the poster competition. Professional development for graduate students involved in this project: One graduate student attended the 2025 Southern/Western Joint Meeting of the American Society of Animal Science (ASAS) and participated the undergraduate 3MT competition with the findings from this project. One graduate student attended the 2024 Fall MSU Graduate Research Symposium and gave poster and oral presentation and participated in the 3MT competition with the findings from this study. This student won second place for the oral presentation and grand champion for the 3MT competition, and further represent MSU in the 3MT competition at the 2025 Conference of Southern Graduate Schools. One graduate student attended the 2025 Southern/Western Joint Meeting of the American Society of Animal Science (ASAS) and participated the graduate 3MT competition with the findings from this project. Professional development for PD: PD attended the 2024 International Symposium on Ruminant Physiology and 2025 Southern/Western Joint Meeting of the ASAS, where she shared research ideas and findings with other attendees and built collaborations. PD also served as judge for Academic Quadrathlon Oral Presentations Section with the topic about the animal microbiome on 2025 Southern/Western Joint Meeting of the ASAS. How have the results been disseminated to communities of interest?1. The findings from this project were disseminated to national academic communities through oral presentations at the 2025 Southern/Western Joint Meeting of the American Society of Animal Science (ASAS) (Title: In vitro Assessment of the Neuroactive Potential of the Rumen Microbiome; Assessing the Effects of Menthol-based Heat Stress Mitigation Strategy via Vagus Nerve Stimulation on the Behavior of Dairy Cattle). The abstracts were published in the respective conference proceedings. These findings contribute to advancing the understanding of the rumen microbiota, particularly their roles in producing neuroactive compounds. 2. The findings from this project were shared with regional and local stakeholders through outreach events such as the 2024 LegenDairy Farm Friday as well as through poster presentations at the 2025 Spring Symposium - Food Science and Industry in Mississippi: Adding Value & Feeding the World (Titles: Examining Behavioral Differences of Holstein and Jersey Cows to Novel Stimuli; Examining the Association of Parlor Behavior and Milk Production Between Holstein and Jersey Lactating Cows), hosted by Mississippi State University. These events helped raise awareness of key issues in the dairy industry, including heat stress, breed-specific behavioral differences (particularly between Holstein and Jersey cows), the relationships among cattle behavior, rumen microbiota, and milk production, and the emerging understanding of how gastrointestinal microbiota influence animal behavior. 3. The findings from this project were shared with early-career scientists through poster and oral presentations, as well as Three-Minute Thesis (3MT) competitions at the MSU Graduate and Undergraduate Research Symposiums and the 39th Annual MANRRS Training Conference & Career Expo. One graduate student earned first place in the 2024 MSU Graduate 3MT Competition with results from the heat stress project and went on to represent MSU in the 3MT competition at the 2025 Conference of Southern Graduate Schools. 4. The findings of this study were also integrated into the teaching materials for the PD's undergraduate and graduate-level course, ADS 4723/6723 Animal Microbiome. Specifically, they were included in Module 3.3.2 (Effects of Gut Microbiota on the Nervous and Endocrine Systems) and Module 9.3 (Animal Microbiome and Animal Welfare). This integration enhances students' understanding of the roles of the gastrointestinal microbiome in regulating animal behavior, welfare, and associated physiological mechanisms. What do you plan to do during the next reporting period to accomplish the goals?1. We will complete the animal behavior analysis using the current trained machine-learning models to identify breed-associated behaviors under both thermoneutral and heat stress conditions, and compare behavioral patterns across thermoneutral, heat stress, and heat stress-relieved conditions. 2. We will complete rumen microbiome and neurotransmitter analyses on samples collected under thermoneutral conditions. 3. We will collect nervous system tissue samples from dairy cattle under thermoneutral and heat stress conditions, and conduct histological and RNA-Seq analyses to investigate breed- and heat stress-associated differences, as well as the connections among the enteric nervous system, microbiome, and central nervous system. 4. We will carry out statistical analyses of the collected data, particularly for the associations between microbiome profiles and animal behavioral and physiological parameters. 5. The microbiome sequencing data and tissue RNA-Seq data generated from this project will be submitted to NCBI database. 6. We will summarize the results and prepare manuscripts for submission to peer-reviewed journals. 7. We will disseminate project findings to the academic community and producers through conference presentations and extension events on an ongoing basis.

Impacts
What was accomplished under these goals? This project aims to mitigate heat stress in dairy cattle by regulating the nervous system, particularly through the rumen-microbiome-brain axis. In the United States, heat stress costs the dairy industry over $1.5 billion annually due to losses in production and reproductive performance. Certain intestinal microbes can produce neurotransmitters and influence animal behavior and stress responses. This study focuses on evaluating the potential of rumen microbes to produce neuroactive compounds and on examining their associations with heat stress responses in Holstein and Jersey cattle. This project targets the training of the next generation of animal scientists and related STEM students in research involving the rumen-microbiome-brain axis and animal welfare. It also aims to raise awareness within the academic community about the neuroactive potential of rumen microbes and to provide dairy producers with knowledge and strategies that help reduce the impact of heat stress in cattle. Objective 1: Major activities completed / experiments conducted: In summer 2024, we conducted a 14-day animal trial following a 7-day adaptation period, which included three groups: heat stressed Holstein lactating cows (n =12); heat stressed Jersey lactating cows (n = 12), both with limited access to fan and sprinkler system; and heat-relieved Holstein lactating cows (n = 12) that had free access to fan and sprinkler system. In spring 2025, we conducted a 2-month animal trial to better understand breed differences under thermoneutral conditions and heat stress conditions, which included two groups: Holstein lactating cows (n = 12) and Jersey lactating cows (n = 12). In addition, we applied a machine learning approach to identify heat stress-associated rumen bacterial genera in Holstein lactating cows by analyzing publicly available raw sequencing data from previous studies. Data collected: Animal behavior was continuously recorded using video cameras. Pen environmental conditions (temperature-humidity index, THI) and animal physiological parameters (e.g., respiratory rate, rectal temperature, rumen contraction frequency, milk yield, feed intake) were routinely measured. Blood, milk, and rumen samples were collected on day 0, day 7, and day 14 of the 2024 summer trial and on day 0, day 30, and day 60 of the 2025 spring trial. Blood parameters, including cortisol and heat shock protein (HSP) 70, as well as milk composition, were measured for 2024 summer trial. The rumen pH was measured for both trials. The long-read rumen microbiome data for 2024 summer trials were generated. Summary statistics and discussion of results: For the 2024 summer trial, the fan and sprinkler system did not significantly affect the pen THI (P > 0.05). However, respiratory rate and rectal temperature were significantly higher in the heat-stressed groups compared with the heat stress-relieved group, while feed intake was significantly lower in the heat-stressed groups. These findings are consistent with previous studies showing that fan and sprinkler systems do not alter ambient THI but enhance convective heat loss and evaporative cooling from the skin surface, thereby alleviating heat stress. Notably, the respiratory rate and morning rectal temperature of heat-stressed Jersey cows were significantly higher than those of heat-stressed Holstein cows, while rumen contraction rates were also higher in Jersey cows, although this difference did not reach statistical significance. There were no significant differences in rumen pH among the three groups. We are now in the middle of analyzing the differences of these parameters in thermoneutral conditions during the 2025 spring trial. Additionally, with the publicly available short-read based raw 16S amplicon sequencing data, the random forest machine learning model using the relative abundance of rumen microbial taxa showed a much higher performance for heat stress prediction, compared to the model without rumen microbiota profile (Area Under the Curve: 0.851 vs 0.440), demonstrating the impact of heat stress on rumen microbiota. We are now working on statistical analysis of our long-read based microbiota data. Objective 2: Major activities completed / experiments conducted: The animal trials were conducted as mentioned in Objective 1. The neurotransmitter profile in rumen fluid samples and a focal group of blood samples from 2024 Summer trial were measured. Additionally, as the detected neurotransmitters in rumen fluid could be derived from both animal host and microbes, to determine the contribution of the rumen microbes in synthesizing the neurotransmitters and identify the responsible bacterial genes and bacterial host, we collected rumen fluid from cannulated cattle for 48-hour in vitro culture and collected sub-samples at different timepoints to detect the neurotransmitter profiles and analyzed the rumen microbiome. Data collected: The neurotransmitters in rumen fluid samples and a focal group of blood plasma samples for 2024 Summer trial, including glutamate, gamma-aminobutyric acid (GABA), serotonin, dopamine, and norepinephrine were analyzed. For the in vitro trial, the concentration of neurotransmitters in rumen fluid samples collected at different timepoints were analyzed. The microbial genes encoding the key enzymes for neurotransmitter production and catabolism and the microbial hosts of these genes were identified. Summary statistics and discussion of results: We are now in the middle of conducting statistical analysis for the neurotransmitter data collected from the 2024 Summer trial. For the in vitro trial, we found the concentration of glutamate was the highest in rumen fluid samples and fluctuated during the in vitro culture (34,344 - 55,106 ng/mL), followed by GABA (425 - 1097.1 ng/mL) that gradually increased from 0 h to 24 h but decreased at 48 h, while the concentration of other neurotransmitters was much lower or not detectable. Consistently, the relative abundance of microbial genes encoding key enzymes for glutamate synthesis and catabolism (glutamine synthetase, glutamate synthase, and glutamate dehydrogenase) were the most abundant and remained stable during in vitro culture. The relative abundance of genes encoding enzymes for GABA synthesis, such as glutamate decarboxylase and aldehyde dehydrogenase, increased from 0 h to 24 h and maintained stable at 48 h, while relative abundance of genes involved in GABA catabolism increased slightly from 0 to 24 h but dramatically increased at 48 h. A significantly less abundance of microbial genes involved in the synthesis of acetylcholine, serotonin, or dopamine were detected. The in vitro trial demonstrates the capability of rumen microbes in producing certain neurotransmitters and their dynamic genetic potential in neurotransmitter production. Objective 3: The animal trails and data collections were conducted as mentioned in Objective 1-2. We will integrate all the data collected from this study to understand the uniqueness of the rumen-microbiota-brain axis in heat-tolerant Jersey cows compared to heat-susceptible Holstein cows. Key outcomes: We confirmed the capability of rumen microbes in synthesizing certain neurotransmitters, especially glutamate and GABA, and identified critical microbial genes involved in these pathways, and their microbial hosts. The differences in animal physiology were observed between heat stress groups and heat-stress-relived group, and between Holstein and Jersey breeds. The heat-stress associated rumen microbial taxa (phylum to genus level) were identified by reanalyzing the publicly available 16S amplicon sequencing data with machine learning approach. We trained 11 undergraduate students and 2 graduate students. We produced one peer-reviewed journal article, along with 10 abstracts, 7 posters, and 7 oral presentations, which were shared at internal research symposiums and external academic conferences to present the findings from this project.

Publications

• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2025 Citation: Bernard B, Joshi H, Fan P. Menthol in Livestock: Unveiling Its Multifaceted Properties and Future Potential for Sustainable Agriculture. International Journal of Molecular Sciences. 2025 Mar 17;26(6):2679.
• Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: Joshi H, Bernard B, Lemley CO, Rude BJ, Fan P. 36 In vitro assessment of the neuroactive potential of the rumen microbiome. Journal of Animal Science. 2025 Jun;103(Supplement_2):142.
• Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: Reon LJ, Joshi H, Hawkins JG, Bernard B, McBride A, Bethini A, Lemley CO, Woolums A, Brett J, Jumper WI, McGee M. 53 Assessing the effects of menthol-based heat stress mitigation strategy via vagus nerve stimulation on the behavior of dairy cattle. Journal of Animal Science. 2025 Jun;103(Supplement_2):145.
• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: H. Joshi, L. Reon, M. Caprio, and P. Fan. 2024. Rumen microbiome signature-based machine learning model for heat stress prediction.J. Dairy Sci. 107(Suppl. 1): 188. (Abstr. 2027)