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INVESTIGATOR: Newton, G.

PERFORMING INSTITUTION:
PRAIRIE VIEW A&M UNIVERSITY
P.O. Box 519, MS 2001
PRAIRIE VIEW, TEXAS 77446

MICROBIOME RESEARCH PROGRAM DEVELOPMENT

NON-TECHNICAL SUMMARY: A long-term goal of the International Goat Research Center (IGRC) is to use biotechnology and genomics to improve animal breeding strategies that enhance and sustain the livelihoods of our cliental in Texas, the Gulf Coast region and developing countries abroad. This proposal establishes a new area of research collaboration between Prairie View A&M University, Texas A&M University (TAMU) and Baylor College of Medicine (BCM) that will define microbial community structures and functions important to reproductive biology. Our working hypothesis is the upper female reproductive tract (UFRT) contains a unique population of commensal bacteria that influences uterine epithelial (UE) cell function and protects against invasion by pathogenic bacteria that may infiltrate the uterus during breeding. Specific objectives of this proposal are to: a) Identify temporal changes in microbial communities in the UFRT during the estrous cycle and compare the microbial communities found in the UFRT during the estrous cycle with those found on equivalent days of early pregnancy, b) Evaluate changes in the uterine luminal environment by measuring specific substances that can be attributed to known microbial communities metabolic reactions, c) Assess the uterine environment and related changes in endometrial lymphocyte and macrophage populations during the estrous cycle and early pregnancy and d) Characterize the content of exosomes (potential mediators of the inflammation reaction) found in seminal plasma. All of these objectives will provide undergraduate students in the College of Agriculture and Human Sciences (CAHS) with experiential learning experiences in reproductive physiology, biotechnology and metagenomics.

OBJECTIVES: The microbiome is a term for the 100 trillion microorganisms that live in the environment, including those that inhabit the bodies of mammals. Sometimes referred to as our 'second genome', commensal bacteria and their metabolites may impact animal health and disease in a variety of ways. Studies in the literature sugggest that, in the gut, commensal bacteria, epithelial cells, and innate immune cells interact to create a protective mechanism against infection caused by pathogenic bacteria Additional studies also suggest that the healthy upper female reproductive tract (UFRT), which was previously thought to be a sterile, harbors unique populations of bacteria in the absence of infection that are associated with diminished implantation rates and reduced pregnancy outcomes after in vitro fertilization. Furthermore, a unique low-abundance placental microbiome has been identified that may influence the subsequent health and wellbeing of the offspring. Our preliminary data, generated using 16s rRNA gene profiling, suggests that distinct bacterial community composition and structural differences exist between vaginal and uterine samples obtained on day three of the goat estrous cycle. Therefore, the role commensal bacteria play in reproductive tract function, especially during the earliest stages of pregnancy, is the focus of this investigation.Our goal is to determine if the UFRT contains a unique population of commensal bacteria that could influence uterine epithelial (UE) cell function and protects against invasion by pathogenic bacteria that may infiltrate the uterus during breeding. Specific objectives of this proposal are to:1) Identify temporal changes in microbial communities in the UFRT during the estrous cycle and compare the microbial communities found in the UFRT during the estrous cycle with those found on equivalent days of early pregnancy,2) Evaluate changes in the uterine luminal environment by measuring specific substances that can be attributed to known microbial communities metabolic reactions,3) Assess the uterine environment and related changes in endometrial lymphocyte and macrophage populations during the estrous cycle and early pregnancy, and4) Characterize the content of exosomes (potential mediators of the inflammation reaction) found in seminal plasma.

APPROACH: Vaginal swabs and endometrial explants (500 mg) will be collected on Days 1, 5, 10 and 16 of the estrous cycle or on Days 1, 5, 10 and 16 after breeding (n=5/day). All samples will be collected under aseptic conditions and processed for 16s rRNA gene profiling at the CORE facility in The Alkek Center for Metagenomics and Microbiome Research in the Molecular Virology and Microbiology Department, Baylor College of Medicine. Metagenomic analysis will determine bacterial community relationships in the uterus and test whether community composition and structure differs in bred and non-bred animals and between vaginal and uterine samples.We will correlate changes in microbial community structures during the estrous cycle and early pregnancy with changes in the bacterial metabolome by assessing uterine flushings. We will partner with the Integrated Metabolomics Analysis Core (IMAC) at Texas A&M University to perform specific metabolomics workflows for the identification, characterization and quantification of small molecules that are produced by microbiota. We will utilize the IMAC to determine if potential markers of microbial community metabolism can be detected uterine flushings.We will use immunohistochemistry to evaluate endometrial changes in lymphocyte populations during the estrous cycle and after breeding to establish whether there are important changes in lymphocyte trafficking after exposure to seminal plasma at breeding. And finally we will evaluate the seminal plasma for factors that may stimulate lymphocyte recruitment into the endometrium, including the content of exosomes, using deep miRNA sequencing.We will employ formative, process, summative and outcome based evaluations. Evaluation will measure achievement of annual measurable outcomes, per the program objectives and incorporate feedback to facilitate project modifications. Formative evaluation will be employed to track effectiveness of improving research capabilities and productivity and student success in hypothesis-driven undergraduate research at PVAMU. The formative evaluations will include: a) assessing progress achieving the research goals, b) student academic progress, which will monitored during the weekly lab meetings/journal club, and c) number of presentations and publications. Summative evaluation will include annual discussions with collaborators and students to assess data on measurable outcomes. Summative evaluations will include: a) total number and quality of the research presentations and publications, b) the number of students entering professional programs, and c) the number and quality of new research proposals submitted. The annual summative reports will also describe unanticipated results and how these will impact the project in the subsequent year.

PROGRESS: 2019/07 TO 2020/06
Target Audience:Undergraduate research assistants are receiving training in animal reproduction and management. Laboratory instruction and data management activities arealso provided. The scientific community is engaged through presentations and posters. Changes/Problems:There is a changein personnel resulting from aco-investigator's retirement and separation fromthe university. What opportunities for training and professional development has the project provided?Two underrepresented undergraduate research assistants have received training in animal reproduction and managent. Laboratory training opportunities occurred during the fall semester but were halted during the spring semester. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?We will finish proteomic analyses and a metabolomic assessmentof the uterine flushings. We will finish analysis of the metagenomic data generated from endometrial and vaginal swabs. We will also finish assessing endometriallymphocyte and macrophage populations in endometrial tissues collected during various days of the estrous cycle and early pregnancy.We will begin assessing seminal plasma for factors that may influence endometrial function.

IMPACT: 2019/07 TO 2020/06
What was accomplished under these goals? Biological sample collection was completed in the fall of 2019. Endometrial and vagoinal swabs werewere submitted for metagenomic analysis. Data assessment and analysis is currently underway. Analysis of lymphocyte and macrophage populations in endometrial tissues using immunohystochemistry was initiated upon completion of biological sample collection.Analysis of other biological samples has been affected by the COVID-19 shutdown of university laboratories.

PUBLICATIONS (not previously reported): 2019/07 TO 2020/06
Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: o Foxworth, W.B., Horner, S., Nuti, L., Ho-Watson, A., Gilmore, I., Gutierrez, K., Lewis S.K., and Newton, G.R. Comparison of Transcervical and Intracervical Artificial Insemination Techniques for Fixed-Time Artificial Insemination (FTAI) in the Goat. Proceedings of the American Society of Animal Scientists Annual Conference. Austin, Texas. 2019.