ELUCIDATING THE ROLE OF INTERLEUKIN-6 ON EARLY EMBRYONIC DEVELOPMENT IN CATTLE

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1026439
• Grant No.: 2021-67034-35149
• Cumulative Award Amt.: $120,000.00
• Proposal No.: 2020-10039
• Project Start Date: Jul 1, 2021
• Project End Date: Jun 30, 2023
• Grant Year: 2021
• Program Code: [A7101]- AFRI Predoctoral Fellowships

Recipient Organization
VIRGINIA POLYTECHNIC INSTITUTE
(N/A)
BLACKSBURG,VA 24061

Performing Department
Animal and Poultry Sciences

Non Technical Summary
Early pregnancy loss is a major hurdle that the cattle industry faces.Indeed, nearly halfof beef cows will be nonpregnant at day 30 following a single insemination. Similarly, early pregnancy loss in lactating dairy cows can be as great as 46%. This leads to negative economic effects that stem from an increased number of nonpregnant cows accruing maintenance costs, fewer total pounds of calf at weaning, reduced lifetime milk production and increased culling in dairy cows. Substantial embryonic losses (13-24%) occur during blastocyst hatching and embryo elongation, but little progress has been made to elucidate factor(s) responsible for these losses. This proposal works to investigate embryonic losses occurring at this time point in development.Based on preliminary data from our lab investigating an embryokine called Interleukin-6 (IL6), the project director developed acentral hypothesis of this proposal whichis thatIL6 functions as a key facilitator of early embryonic development in cattle and thus, it is essential for normal development in vivo. Shewill objectively test this central hypothesis using CRISPR-Cas9 gene editing and RNA sequencing technology in addition to other biochemical and molecular techniques. Collectively, the experiments contained within this proposal will provide novel insights into inner cell mass development and blastocyst elongation in cattle.

Animal Health Component

0%

Research Effort Categories

Basic

100%

Applied

0%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
301	3310	1050	50%
301	3410	1050	50%

Knowledge Area
301 - Reproductive Performance of Animals;

Subject Of Investigation
3310 - Beef cattle, live animal; 3410 - Dairy cattle, live animal;

Field Of Science
1050 - Developmental biology;

Keywords

cattle

elongation

embryo

embryonic loss

Goals / Objectives
This predoctoral fellowship proposal addresses the animal health, production, and animal products priority area of the AFRI Farm Bill by investigating the ability of Interleukin-6 (IL6) to improve bovine in vitro embryo production systems. The ultimate goals of this research are to 1) better understand the cause of embryonic losses occurring at the elongation stage, and specifically those associated with inner cell mass (ICM) development, and 2) provide data that may lead to the development of strategies on how to decrease these losses. Recently, our lab was the first to investigate IL6 culture supplementation in bovine in vitro produced (IVP) embryos. From our studies, we found IL6 improves bovine ICM development and has restorative properties in regard to increasing IVP blastocyst formation. Based on these findings, the central hypothesis of this proposal is IL6 functions as a key facilitator of early embryonic development in cattle and thus, it is essential for normal development in vivo. I will objectively test my central hypothesis using CRISPR-Cas9 gene editing and RNA sequencing technology in addition to other biochemical and molecular techniques. Objective 1: Determine the role of IL6 in ICM formation. Rationale: All indications thus far implicate IL6 as a major player in ICM formation in cattle; however, studies that focus on functional removal of IL6 or IL6s signaling capabilities are necessary to demonstrate that IL6 truly impacts ICM formation. Objective 2: Determine if IL6 supplementation improves embryo elongation in vivo. Rationale: The ICM of IVP embryos experience increased cell death as culture duration increases, which likely influences the inability of blastocysts to elongate in vitro. Impairments in embryo elongation likely results in failed pregnancies. I will be able to determine if IL6 is favorable for embryo elongation in vivo by evaluating conceptuses (at day 15 of gestation) that originated from IL6 treated embryos. Collectively, the experiments contained within this proposal will provide novel insights into ICM development and blastocyst elongation in the bovine.

Project Methods
General Scientific Methods:General methods will include in vitro embryo maturation, fertilization and culture, electroporation, CRISPR-Cas9 gene editing, immunofluorescent detection of proteins, DNA cloning, genomic PCR, sanger sequencing, RNA sequencing, artificial insemination, and embryo transfer.Efforts:Efforts will be made to share research data and outcomes with peers through both oral and writtencommunication. Examples of oral communication includes having the program director present at seminars andat conferences via poster or oral presentations. Written communication will be accomplished by publishing resulting work as manuscript(s) in peer-reviewed journals. Foundational knowledge that results from this research can also be shared with students in a classroom setting.Evaluation:Indicators of success will be if the program director was able to answer the hypotheses, which specificially includes1) if the role of IL6 in ICM formation was able to be determined, and 2) if the project director was able to determine if IL6 supplementation improves embryo elongation in vivo. The primary mentor will meet individually at least weekly with the project director to evaluate progress and to give guidance and suggestions.

Progress 07/01/21 to 05/31/23

Outputs
Target Audience:Target audiences include students and professionals in an academic setting as well as industry scientists and cattle producers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This pre-doctoral project allowed the program director, Savannah, many opportunities for training and professional development. First, much of this research required expertise and skills from within and outside our lab. Due to this, Savannah had a lot of experience collaborating with other scientists and labs. In addition, travel funds were used to send Savannah to various conferences where she presented oral posters, and co-chaired various sessions with the Society for the Study of Reproduction. How have the results been disseminated to communities of interest?The two research manuscripts covering each aimof this research are currently being finalized with plans to submit them soon to the journal called Biology of Reproduction. Arguably the top reproduction-specific journal in our field. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? We were able to accomplish our goals during the award period for this pre-doctoral project proposal. Through our research, we were able to answer the two main goals including 1) better understandingthe causes of embryonic losses occruing at the elongation stage and 2) provide data that could lead to developmental strategies to decrease these losses. We discovered supplementing recombinant interleukin-6 during in vitro embryo culture was able to increase the length of conceptuses. More importantly, IL6 supplementation also numerically increased survival by 20%. Therefore, it appears something as simple as supplementing in vitro embryo culture media with IL6 could decrease embryonic losses. However, more research is needed to follow these pregnancies longer through gestation to ensure the health of mother and offspring before asking producers to implement this. In addition, we identified that part of the IL6 receptor, interleukin-6 signal transducer (IL6ST) is required for early embryonic development in the cow. When IL6ST was disrupted, the entire blastocyst was impaired. Not only did it fail to develop on average, but when they did develop, they had decrease inner cell mass and trophectoderm cell numbers. In summary, IL6 through IL6ST signaling was discovered to be a requirement for sustained development in the cow.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2023 Citation: Speckhart, Savannah L., Mary A. Oliver, and Alan D. Ealy. 2023. "Developmental Hurdles That Can Compromise Pregnancy during the First Month of Gestation in Cattle" Animals 13, no. 11: 1760. https://doi.org/10.3390/ani13111760
• Type: Journal Articles Status: Accepted Year Published: 2023 Citation: Savannah L. Speckhart, Lydia K. Wooldridge, Alan D. Ealy, An updated protocol for in�vitro bovine embryo production, STAR Protocols, Volume 4, Issue 1, 2023, 101924, ISSN 2666-1667, https://doi.org/10.1016/j.xpro.2022.101924.

Progress 07/01/21 to 06/30/22

Outputs
Target Audience:Target audience includes animal scientists, beef and dairy cattle producers, embryo transfer companies, and regional and state agricultural extension agents. Efforts to reach the target audiences included formal and informal classrooom instruction of graduate level classes, experimental instruction to both undergraduate and graduate students, and seminar presentations at invited speaking engagements. Data from this project will be shared by poster or oral presentation at the 2022 Annual Meeting for the Society for the Study of Reproduction. Changes/Problems:COVID-19 has delayed this project; however, we feel confident to have the project completed by the end of the award period. What opportunities for training and professional development has the project provided?By performing this project, I have received many new trainings related to techniques needed to accomplish the endpoints of this research. Additionally, I will present data from this research at the 2022 SSR annual meeting this summer. Whatsmore, my work on this project has been an interest to many outside of our field, which has allowed me to present it at the University of Kansas Medical Center and Washington University. Both of those experiences provided me networking opportunities. How have the results been disseminated to communities of interest?Results are being disseminated by attending and presenting findings at national conferences, undertaking invited speaking opportunities at other institutions, and eventual publication in peer-reviewed journals. What do you plan to do during the next reporting period to accomplish the goals?My plan is to finalize data from Objective 1 and put that data into a manuscript for submission to a peer-reviewed journal. In addition, my plan is to complete Objective 2 and put that data into a separate manuscript for publication.

Impacts
What was accomplished under these goals? By completing Objective 1, we have made much more progress on how IL6 serves as an embryokine in bovine embryos. Our data is the first to show that IL6 signaling through its beta-subunit, IL6ST,is needed for blastocyst development in cattle. This provides additional evidence to IL6 acting as an embryokine in cattle. We are currently working on Objective 2 now.

Publications

• Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Speckhart S. L., K. Farrell, L. K. Wooldridge, F. H. Biase, K. Lee, and A. D. Ealy. 2022. Interleukin-6 signal transducer is involved in blastocyst development in cattle. Abstract. Society for the Study of Reproduction, Spokane, Washington, July 26-29.