Source: CORNELL UNIVERSITY submitted to NRP
UNDERSTANDING UTERINE OXYGEN-GLUCOSE HOMEOSTASIS TOWARDS PREIMPLANTATION EMBRYO SURVIVAL IN CATTLE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1032151
Grant No.
2024-67015-42377
Cumulative Award Amt.
$650,000.00
Proposal No.
2023-08329
Multistate No.
(N/A)
Project Start Date
Jul 1, 2024
Project End Date
Jun 30, 2028
Grant Year
2024
Program Code
[A1211]- Animal Health and Production and Animal Products: Animal Reproduction
Recipient Organization
CORNELL UNIVERSITY
(N/A)
ITHACA,NY 14853
Performing Department
(N/A)
Non Technical Summary
This research project focuses on improving how often dairy cows successfully have calves, an important area for U.S. agriculture. Specifically, the project looks at why high-producing dairy cows (those that give a lot of milk) often have problems with pregnancies that do not go to term, particularly due to early death of embryos. This issue is costly for farmers and stems from complex biological problems not yet fully understood. Lactating cows experience changes in their body energy due to intense milk production right after giving birth. These changes can negatively affect the cows' reproductive organs and embryo health.The overall hypothesis of this research is that the balance of oxygen and sugar levels, controlled by the environment inside the uterus, plays a crucial role in the early development of embryos and their stem cells, ultimately influencing whether they can successfully attach to the uterus and continue developing.The study has two main goals:1. Understanding Embryo Needs: By measuring sugar and oxygen levels in the uterus, the research aims to find out what embryos need to develop healthily and if these needs are different in cows that are lactating compared to younger heifers. This could lead to better understanding of how to support embryo growth during early stages.2. Studying Gene Activity: The project also plans to examine how sugar and oxygen levels influence embryo development at the genetic level. This involves looking at how genes in the embryos are turned on or off by these conditions and could tell us more about why some embryos fail to develop properly.The findings from this research could lead to better ways to help cows have successful pregnancies and improve methods for creating embryos in the lab (in vitro). This would not only help reduce financial losses for farmers but also enhance the productivity and sustainability of dairy farming.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
30134101020100%
Knowledge Area
301 - Reproductive Performance of Animals;

Subject Of Investigation
3410 - Dairy cattle, live animal;

Field Of Science
1020 - Physiology;
Goals / Objectives
This research addresses reproduction efficiency in dairy cattle by investigating embryonic development, a research focus in the USDA animal reproduction priority area. In contrast to heifers, high-producing dairy cows suffer increased pregnancy failures due to early embryo death. This is strongly associated with compromised embryo development and/or failed uterine attachment (implantation). Although the economic loss is quite significant for farms, there are no known fixes to this problem as underlying mechanisms remain largely unknown. Metabolic challenges of lactation such as negative energy balance are known to occur in the postpartum background in cows, but not in heifers during the breeding period. Yet, concomitant influence of such systemic energy partitioning on uterine function remains uncharacterized. In this project, we investigate the uterine environment to understand the favorable vs challenging metabolic settings and assess impact on developing embryos.Aim 1: By measuring in vivo uterine glucose and oxygen levels, we will uncover basic embryonic needs, and possible differences in lactating cows vs heifers. Concurrently, we will identify the optimal oxygen-calibrated glucose demands for rapid proliferation of embryonic trophoblasts (placental precursor cells) representative of growth during preimplantation embryo development.Aim 2: By mapping the transcriptional regulatory landscape of embryo growth and metabolism, we will define the influence of glucose and oxygen on the developmental regulation of trophoblasts (capturing both immediate embryonic and future possible placental problems).This research will advance the efficiency of US agriculture by uncovering mechanisms towards recovering high fertility in modern dairy cows, and providing information for biomimicry promoting superior in vitro embryo production systems.
Project Methods
The research project aims to enhance understanding of the conditions that support the early stages of embryo development in dairy cows. It is structured around two main aims, each with distinct methodologies:Aim 1: Determining Uterine Luminal Oxygen and Glucose LevelsMethods: This aim involves directly measuring in vivo levels of oxygen and aspects of glucose homeostasis within the uterus of both cycling and early pregnant dairy cows and heifers. We will use a fiber-optic oxygen sensing probe with built in temperature compensation to directly read out dissolved oxygen availability for the embryo within the uterine fluid at different stages of the preimplantation period. This approach of in vivo measurements will provide a clear understanding of the metabolic impact in this microenvironment and optimal conditions needed for preimplantation embryo development.Aim 2: Defining the Influence of Oxygen and Glucose on Trophoblast Transcriptional ProgramsMethods: The team will employ Precision Run-On sequencing (PRO-seq) to analyze gene loci that are transcriptionally active and are poised for stimulus-dependent activation in trophoblast stem cells. This technique will specifically assess how these loci are regulated under conditions of low oxygen or physiological hypoxia and varying glucose levels. The approach combines advanced sequencing techniques with novel bioinformatics tools developed at Cornell University.Together, these methods will provide critical insights into the environmental and genetic factors influencing embryo development and pave the way for technological advances in embryo production practices.

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

Outputs
Target Audience:This project is designed to directly benefit multiple stakeholder groups central to the sustainability and productivity of the United States (U.S.) dairy industry. For this reporting period, the primary audience includes: 1. Students and Trainees in Animal and Veterinary Sciences As part of an academic research program, this project also servedas a training platform for graduate and undergraduate students. Through exposure to interdisciplinary methods ranging from in vivo physiology to transcriptomics and genome editing. This played a crucial role in buildingcapacity in next-generation reproductive research. 2. Animal Reproductive Biologists and Veterinary Scientists The workaddresseda critical gapin the understanding of bovine preimplantation development and trophoblast biology. With novel data on uterine oxygen and its role onregulatory influence on embryo metabolism, this research offeredfoundational insights into species-specific reproductive physiology. The findings are highly relevant to all academic researchers, veterinarians, and specialists in animal reproduction. 3. Embryo technology and IVF industry The commercial bovine IVF sector will benefit from the project's focus on biomimicry. The identification of in vivo oxygen conditions essential for early embryo development provides a blueprint for designing more physiologically relevant culture systems and haspotential to improve embryo viability and reduce gestational complications associated with IVF-derived calves. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has supported the training of two undergraduate students, a doctoral student, and a postdoctoral associate in advanced reproductive physiology, techniques for transcervical device placement, and in vivo measurement methodologies. The students wereinvolved in probe design, optimization, and execution of experimental procedures under close supervision, gaining experience in experimental animal handling, transrectal ultrasonography, and troubleshooting. Additionally, undergraduate trainees were introduced to physiological data collection methods and analysis. The multidisciplinary nature of the project also facilitated cross-training in bioengineering (device design), systems biology (uterus), and embryophysiology (microenvironment and metabolic state) for all involved personnel. How have the results been disseminated to communities of interest?Initial results from this study have been shared with the broader scientific and agricultural communities through invited presentations and internal symposia. Specifically, the findings on uterine oxygen levels and their implications for in vitro embryo production were presented in departmental seminars and work-in-progress reportsaimed at improving IVF practices in cattle. A manuscript detailing these results, titled"Sustenance of a quasi-anoxic uterine lumen in cattle: implications for in vitro embryo production and transfer", is currently in preparation for submission to a peer-reviewed journal. Additionally, informal discussions with industry partners and reproductive biotechnology companies are being initiated to explore how these physiological insights could enhance embryo culture systems and IVF media formulation for commercial use. What do you plan to do during the next reporting period to accomplish the goals?Establish an in vitro trophoblast stem cell (TSC) system mimicking in vivo conditions: Using physiologic oxygen levels determined from in vivo measurements and adjusted metabolic conditions, we will culture bovine trophoblast stem cells under quasi-anoxicenvironments. We will assess the impact of these variables on TSC proliferation, metabolic phenotype, and differentiation capacity using transcriptomic profiling. These experiments will set the stage for adjustments to embryo culture media essential for in vivo biomimicy.

Impacts
What was accomplished under these goals? During the first year of this project, we successfully completed a major objective under Aim 1: the direct in vivo measurement of uterine luminal oxygen concentrations in cycling and early pregnant dairy heifers. Using a custom-fabricated transcervical probe holder and a fiber-optic oxygen sensing system, we demonstrated for the first time that the uterine fluid in cattle maintains an extremely low oxygen environment--below 1 mmHg, equivalent to <0.13% O?--across multiple time points of the estrous cycle and early pregnancy. These findings establish that the bovine uterus exists in a "quasi-anoxic" state, a condition that likely exerts significant influence on preimplantation embryo metabolism and development. We further validated this physiological state using radiocontrast perfusion and micro-CT imaging of uterine vasculature, revealing spiral artery organization and extensive microvasculature that supports this unique low-oxygen niche. These data set the stage for testing how this oxygen tensionregulates trophoblast stem cell function and preimplantation embryo metabolismrelevant for pregnancy success.

Publications