CONTROL AND DYNAMICS OF FERTILITY TO ESTRUS IN BEEF COWS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: ACTIVE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1028331
• Grant No.: 2022-67015-36839
• Cumulative Award Amt.: $650,000.00
• Proposal No.: 2021-06792
• Project Start Date: Feb 1, 2022
• Project End Date: Jan 31, 2027
• Grant Year: 2022
• Program Code: [A1211]- Animal Health and Production and Animal Products: Animal Reproduction

Recipient Organization
UNIVERSITY OF FLORIDA
G022 MCCARTY HALL
GAINESVILLE,FL 32611

Performing Department
(N/A)

Non Technical Summary
Sustainable beef production requires efficient reproductive performance in the cow herd. Sub-optimal reproductive performance is caused by the inability of females to support the growth of an embryo and maintain a pregnancy. A large proportion of failure occurs during early gestation, because of a non-receptive uterus. There is a critical need to define the nature and the abundance of molecules present in the uterine lumen when a pregnancy succeeds. We propose experiments to compare the endometrial transcriptome and metabolome of animals that succeeded or failed to become pregnant, in sequential pregnancy opportunities (Experiment 1). We also propose to test a strategy to more adequately program uterine function to increase receptivity to the embryo and better support gestation (Experiment 2). The basic and applied aspects of the current proposal have many predicted positive outcomes. For example, we will for the first time characterize the molecular factors associated with embryo receptivity. Such evidence has the potential to lead to the generation of non-genomic markers that can be used for the selection of embryo recipients. Markers may be used in future studies, for example, to study the influence of breeds and crosses, season, stress, lactation, synchronization strategies, nutritional strategies, energy status etc. on the uterine environment and its ability to support pregnancy. Molecular differences between pregnant and non-pregnant animals will be used both as markers for prognostics of pregnancy outcome and as targets to research on uterine function to support pregnancy. New information generated from our pre-estrus programming of uterine receptivity to estrus experiment will serve as the basis for the optimization of strategies for estrus synchronization aiming artificial insemination or embryo transfer. We anticipate having generated new knowledge and tools to be used for basic and applied research, as well as used by beef producers to increase pregnancy success in cow-calf operations. Because reproductive efficiency is a core aspect of sustainable beef cow-calf operations, production of strategies and tools through successful completion of our proposed research is expected to contribute directly to long-range improvement and sustainability of US agriculture and food systems.

Animal Health Component

20%

Research Effort Categories

Basic

80%

Applied

20%

Developmental

0%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
301	3310	1020	60%
301	3310	1040	40%

Knowledge Area
301 - Reproductive Performance of Animals;

Subject Of Investigation
3310 - Beef cattle, live animal;

Field Of Science
1040 - Molecular biology; 1020 - Physiology;

Keywords

beef cattle

embryo

embryo transfer

endometrium

gene expression

metabolomics

pregnancy

Goals / Objectives
Our long-term goal is to contribute towards the development of targeted interventions to increase reproductive success rates of beef females as a way to increase the economic vitality of cow-calf operations. The overall objective in this proposal is to identify molecules in the uterus that are predictive of pregnancy success. The central hypothesis in this application is that the pre-implantation uterine environment contains a molecular signature that defines the outcome of pregnancy.Specific Objective 1: Identify endometrial transcriptome and uterine luminal metabolome signatures that are predictive of a successful pregnancy. The working hypothesis is that endometrial transcriptome and luminal metabolome differ between cows that succeeded or failed tomaintain a pregnancy in sequential cycles of embryo transfer.Specific Objective 2: Determine endometrial transcriptome and uterine luminal metabolome profiles of cows exposed to contrasting concentrations of P4 before estrus. The working hypothesis is that pre-ovulation exposure to increased concentrations of P4 causes molecular changes in the endometrial transcriptome and luminal metabolome that are associated with poor receptivity to the embryo.Upon the successful completion of the proposed project, we expect to have identified emergent patterns of transcripts and metabolites associated with successful pregnancy (Specific Objective 1). A molecular panel of receptivity markers would provide a strong science-based foundation to generate innovative tools for the prognosis of pregnancy outcomes. In addition, we anticipate having tested a strategy to manipulate uterine function (Specific Objective 2). These combined results are expected to have an important positive impact by contributing substantively to improved cow pregnancy success that, in turn, will enhance beef production efficiency and business sustainability.

Project Methods
Specific Objective 1: Identify endometrial transcriptome and uterine luminal metabolome signatures that are predictive of a successful pregnancy. Research Design:Reproductive managementFifty Bos indicus-influenced cross-bred, mainly Brangus, multiparous, non-lactating, cyclic cows will be used for this objective (please see power analysis below). Experiment will consist of eight sequential replications of a modular design. Each module will last 91 days and the whole experiment will last two years. The same animals will be used in all modules, unless they have to be removed for health issues or death. Modules will be designed to coincide with the calendar seasons. The first module of each year will start in January, which coincides with the breeding season adopted in many beef operations in the Southeastern USA. This modular design will allow us to determine the temporal dynamics of fertility to estrus in regards to year and season. Winter season will contain modules from October to March and Summer season will contain modules from April to September. Moreover, we will be able generate original information on the dynamics of fertility to estrus of individual cows. Each module will consist of a 12-day synchronization period (module days ?12 to 0), a 45-day gestation period (module days 0 to 45) and a 34-day recovery period (module days 46 to 79). In the synchronization period, cows at random stages of the estrous cycle will receive PGF2α (e.g., 25 mg dinoprost, Lutalyse, Zoetis) on day (D) -12 of the module (D?12); on D?9, a P4-releasing device (e.g., 1.38 g progesterone, Eazi-breed CIDR, Zoetis) will be inserted and GnRH (e.g., 50 mg gonadorelin, Factrel, Zoetis) injected. On D?3, the device will be removed, PGF2? will be injected, and an estrus detection patch (e.g., Estrotect) will be applied. Estrus behavior will be observed twice daily between D?2 and D1. We expect that 80% of the animals will be detected in heat between D?0.5 and D1. The day of heat will be considered the first day of the gestation period. Seven days after observed heat (designated experimental D7) animals with a CL detectable by ultrasonography will be submitted to embryo transfer. Two in vitro-produced, grade-1 blastocysts, loaded in a single straw and freeze-thawed will be transferred to recipients previously observed in heat using non-surgical direct transfer by an experienced technician. The reason we will transfer two embryos is to minimize the effect of the embryo on the pregnancy outcome. In this proposal, we are specifically interested in evaluating the ability of the uterus to support a pregnancy. Thus, we intend to reduce the chance of a single, developmentally compromised embryo to result in a failed pregnancy despite a supportive uterine environment. Pregnancy diagnosis will be performed on D20 (day 20 of pregnancy) by Doppler ultrasonography of the CL (absence of abundant vascularization of the CL is indicative of a failed pregnancy) and by retrospective evaluation of the expression of interferon-stimulated genes (ISGs) in peripheral blood mononuclear cells (PBMCs), and on D25, D35 and D45 by transrectal ultrasonography and detection of a heart-beat of the fetus. Presence of multiple embryonic vesicles will be noted. After pregnancy diagnosis on D45, all cows will receive two injections of PGF2α, 12 hours apart, to induce abortion. Cows will not be submitted to any manipulation during the recovery period, from D46 to D79. The 34-day recovery period is sufficient to allow a proper involution of the previously pregnant reproductive tract so that cows will be ready to start the subsequent module.Specific Objective 2: Determine endometrial transcriptome and uterine luminal metabolome profiles of cows exposed to contrasting concentrations of P4 before estrus.Research Design:Reproductive management: Forty Bos indicus-influenced cross-bred, mainly Brangus, multiparous, non-lactating, cyclic cows will be used in this experiment (please see power analysis presented below). Feeding and facilities will be similar to those described under Objective 1. Experiment will consist of a 24-day pre-synchronization period (days ?33 to ?9), a 9-day synchronization period (days ?9 to 0) and a 21-d sample collection period (days 0 to 21). There will be no gestations (i.e., no embryo transfer) in this experiment. Estrus synchronization will be similar to Objective 1, to produce groups with contrasting concentrations of P4 prior to ovulation. Animals will receive two injections of PGF2α 14 days apart starting on D?33. On D?9, 10 days after the second PGF injection, animals will be expected to be between days 5 and 7 of the estrous cycle. Thus, they are expected to have a PGF-responsive CL and a GnRH-responsive first-wave dominant follicle. On this day, all animals will receive a GnRH injection and be assigned to the experimental groups. The Elevated Progesterone group (Elevated P4) will receive no additional injection and will receive a P4-releasing device not used previously. The Reduced Progesterone group (Reduced P4) will receive a luteolytic injection of PGF2α and a P4-releasing device used previously for 12 days. The utilization of previously used P4-releasing devices will result in a smaller increase in plasma P4 concentrations in blood compared to the use of a new device. This strategy has been used widely as a means to modulate P4 release in specific situations, such as the synchronization of ovulation in heifers or Bos indicus-influenced cows. Based on our previous experiments, there should be a 3 to 4 ng/ml difference in P4 concentrations between groups at CIDR removal. In all animals, the P4-releasing devices will be removed six days later, on D?3, an injection of PGF2α will be given and an estrus detection patch (Estrotect) will be applied. Estrus behavior will be observed twice daily from 36 to 72 h after device removal, and we expect that 80% of the animals will be detected in estrus in this time frame. The day of estrus will be considered D0 of the sample collection period. Of critical relevance to this proposal is the fact animals from both groups will be allowed to show estrus, which is the hallmark for fertility in field conditions following both artificial insemination and embryo transfer, and associated positively with conception. Due to the inhibitory effects of P4 on the Elevated P4 group, the interval between removal of the P4-releasing device and estrus is expected to be longer than the Reduced P4 group.

Progress 02/01/24 to 01/31/25

Outputs
Target Audience:Reproductive physiology scientists, beef cattle producers, veterinarians, graduate students, undergraduate students. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Experiment regarding Specific Objective 2 was integral for the doctoral dissertation of one student (Dr. Felipe Silva), that has obtained his PhD degree and is now a faculty member at the North Carolina State University. Two doctoral students (Dr. Cecilia Rocha and Mr. Abdul Waheed), two master students (Mrs. Meghan Campbell and Mrs. Alexandra Bennet) and three post-doctoral fellows (Dr. Leonardo Resende, Dr. Fernando Mesquita and Dr. Mariângela Cordeiro) provided technical assistance to the project. Nine undergraduate students helped occasionally. Experiment regarding Specific Objective 1 was integral for the doctoral dissertation of three students. Dr. Cecilia Rocha obtained her PhD degree and is now a faculty member at the University of Missouri, Mr. Abdul Waheed is the third year of his PhD program, Mrs. Alexandra Bennett obtained her Masters degree is now a Livestock Agent with UF/IFAS. Three post-doctoral fellows (Dr. Leonardo Resende, Dr. Fernando Mesquita and Dr. Mariângela Cordeiro) provided technical assistance to the project. Nine undergraduate students and one high school student helped occasionally. How have the results been disseminated to communities of interest?Seven abstracts were presented at scientific meetings. Two manuscripts were published. What do you plan to do during the next reporting period to accomplish the goals?Start transcriptomics, metabolomics and bioinformatics portions of Specific Objective 1. Write and submit four manuscripts.

Impacts
What was accomplished under these goals? The experimental portion, collection of samples, processing and storage of samples regarding Specific Objective 1 was completed as planned. Ovarian and uterine data, hormonal data, cell culture data and molecular data were collected and analyzed. Omics and bioinformatics analyses are underway. Partial results were presented on scientific meetings, papers were published. A PhD dissertation was defended. All experimental portion, collection of samples, processing and storage of samples regarding Specific Objective 2 was completed successfully. Ovarian data, hormonal data, transcriptomics data and metabolomics data were analyzed. Bioinformatics analyses were completed. Results were presented on scientific meetings, papers were published. A PhD dissertation and a Master's thesis were defended.

Publications

• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: 6. Rocha, C. C., Cordeiro, A. L. L., Campbell, M., Maldonado, M. B. C., Silva, F. A. C. C., Bennett, A., Waheed, A., Hansen, T., & Binelli, M. (2024). In vitro reminiscence: uterine programming in vivo affects respective luminal epithelial cells function in vitro . Biology of reproduction, 111(3), 600612. https://doi.org/10.1093/biolre/ioae093
• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: 4. Binelli, M., Rocha, C. C., Bennett, A., Waheed, A., Sultana, H., Maldonado, M. B. C., & Mesquita, F. S. (2024). Solutions to the fertility equation in beef embryo recipients. Animal reproduction, 21(3), e20240041. https://doi.org/10.1590/1984-3143-AR2024-00414. Binelli, M., Rocha, C. C., Bennett, A., Waheed, A., Sultana, H., Maldonado, M. B. C., & Mesquita, F. S. (2024). Solutions to the fertility equation in beef embryo recipients. Animal reproduction, 21(3), e20240041. https://doi.org/10.1590/1984-3143-AR2024-0041
• Type: Theses/Dissertations Status: Published Year Published: 2024 Citation: Rocha, C.C.(2024). ENDOMETRIAL RESPONSES THAT AFFECT FERTILITY IN BEEF COWS. Dissertation.
• Type: Theses/Dissertations Status: Published Year Published: 2024 Citation: Bennett, A. (2024). ASSESSING THE ABUNDANCE OF PREGNNACY MARKERS AMONG FERTILITY CLASSIFIED BEEF CATTLE. Dissertation
• Type: Theses/Dissertations Status: Published Year Published: 2023 Citation: Silva, F.A.C.C. (2023). MODULATION OF UTERINE FUNCTION IN BEEF CATTLE. Dissertation.

Progress 02/01/23 to 01/31/24

Outputs
Target Audience:Reproductive physiology scientists. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Experiment regarding Specific Objective 2 is integral for the doctoral dissertation of one student (Dr. Felipe Silva), that has defended and obtained his PhD degree. Three doctoral students (Mrs. Cecilia Rocha, Mr. Abdul Waheed and Mr. Andrey Lopes) and three post-doctoral fellows (Dr. Thiago Martins, Dr. Fernando Mesquita and Dr. Mariângela Cordeiro) provided technical assistance to the project. Nine undergraduate students helped occasionally. How have the results been disseminated to communities of interest?Two abstract was presented at a scientific meeting. Two manuscripts were published. What do you plan to do during the next reporting period to accomplish the goals?Finalize experimental portion, start transcriptomics, metabolomics and bioinformatics portions of Specific Objective 1. Write and submit two manuscripts.

Impacts
What was accomplished under these goals? The experimental portion, collection of samples, processing and storage of samples regarding Specific Objective 1 is underway (75% completed). Ovarian and uterine data, hormonal data, cell culture data and molecular data are being collected and analyzed. Omics and bioinformatics analyses have not started. Partial results were presented on scientific meetings. All experimental portion, collection of samples, processing and storage of samples regarding Specific Objective 2 was completed successfully. Ovarian data, hormonal data, transcriptomics data and metabolomics data were analyzed. Bioinformatics analyses were completed. Results were presented on scientific meetings, papers were written and are now published.

Publications

• Type: Journal Articles Status: Published Year Published: 2023 Citation: Silva FACC, Martins T, Sponchiado M, Rocha CC, Pohler KG, Pe�agaricano F, Binelli M. Hormonal profile prior to luteolysis modulates the uterine luminal transcriptome in the subsequent cycle in beef cross-bred cows . Biol Reprod. 2023 Jun 9;108(6):922-935. doi: 10.1093/biolre/ioad035. PMID: 36930057.
• Type: Journal Articles Status: Published Year Published: 2023 Citation: Silva FACC, Martins T, Sponchiado M, Rocha CC, Ashrafi N, Graham SF, Pohler KG, Pe�agaricano F, Gonella-Diaza AM, Binelli M. Pre-estrus progesterone does not affect post-estrus luminal metabolome in cross-bred beef cows. Reproduction. 2023 Jun 21;166(2):99-116. doi: 10.1530/REP-22-0372. PMID: 37224090.

Progress 02/01/22 to 01/31/23

Outputs
Target Audience:Reproductive physiology scientists. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Objective 1: Doctoral student Cecilia Rocha is responsible for the overall reproductive management of the experiment. Doctoral student Abdul Waheed is responsible for transcriptomics, metabolomics and bioinformatics. Master student Alexandra Bennett is responsible for analyses of pregnancy outcomes. Personnel from different levels have been involved on the implementation and execution of this experiment, contributing with different levels of expertise. Objective 2: Doctoral Student Felipe Silva conducted and analyzed data from the experiment related to this objective. He defended his dissertation based on this experiment. He submitted two papers for publication, which are current under evaluation. Post-doctoral associate Dr. Thiago Martins and doctoral student Cecilia Rocha, assisted with the execution of the experiment and data analyses. How have the results been disseminated to communities of interest?The PI has presented partial results in scientific seminars directed to reproductive biologists in the US and internationally. Dr. Felipe Silva presented is results in Departmental Seminars at the University of Florida. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: field experiment is ongoing. During 2023, four of the six remaining modules are expected to be completed. Partial analysis of data will be conducted. Objective 2: complete publication process of two papers that have been submitted.

Impacts
What was accomplished under these goals? Objective 1: This is a 2-year long experiment consisting of 8 modules. The 2 modules scheduled for 2022 were completed successfully: facilities were made operational, supporting materials were acquired, team was trained, animals were enrolled, samples were collected, and partial analyses conducted. Co-PI Moriel is heavily involved on nutritional management. Co-PI Hansen is coordinating embryo handling prior to transfer. Objective 2: the experiment was completed, samples were analyzed and 2 scientific papers were submitted. Co-PI Peñagaricano has provided expertise with data analyses and bioinformatics.

Publications

• Type: Journal Articles Status: Published Year Published: 2022 Citation: Binelli, M., Silva, F. A. C. C., Rocha, C. C., Martins, T., Sponchiado, M., Van Hoeck, V., Cordeiro, A., Campbell, M., Leroy, J. L. M. R., Pe�agaricano, F., & Pugliesi, G. (2022). Endometrial receptivity in cattle: the mutual reprogramming paradigm. Animal Reproduction, 19(4), e20220097. https://doi.org/10.1590/1984-3143-AR2022-0097
• Type: Theses/Dissertations Status: Published Year Published: 2022 Citation: Silva, F. A. C. C. (2022). Modulation of Uterine Function in Beef Cattle. Doctoral Dissertation, University of Florida.