IMPROVING PREGNANCY SUCCESS OF IN VITRO DERIVED BOVINE EMBRYOS THROUGH CULTURE MEDIUM MODIFICATIONS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1028048
• Grant No.: 2022-67011-36568
• Cumulative Award Amt.: $120,000.00
• Proposal No.: 2021-09454
• Project Start Date: Dec 15, 2021
• Project End Date: Dec 14, 2023
• Grant Year: 2022
• Program Code: [A7101]- AFRI Predoctoral Fellowships

Recipient Organization
UNIVERSITY OF MISSOURI
(N/A)
COLUMBIA,MO 65211

Performing Department
Animal Sciences

Non Technical Summary
Eighteen percent of the world's beef is produced in the United States by eight percent of the world's cattle. By increasing animal efficiency, less resources are used, and a smaller carbon footprint is left. Better genetics is one component of increasing efficiency. Reproductive technologies, such as in vitro embryo production, are important tools for capitalizing on superior genetics, as well as preserving them. Of the livestock species, cattle use more embryo technologies than any other. Just in 2019, 1,419,336 transferrable embryos were produced worldwide, from which 72.8% were in vitro produced. While this technology is growing in use, its implementation is hindered by low efficiency. The Prather and Roberts laboratories at the University of Missouri identified fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF), and insulin-like growth factor 1 (IGF1) as a molecule cocktail (termed FLI) with the potential to increase the efficiency of in vitro embryo production in livestock. Here, the proposed studies investigate the supplementation of FLI to the in vitro bovine embryo culture system through an analysis of target pathways, such as JAK/STAT and MAPK, while also assessing the ability of these embryos to increase pregnancy establishment. We hypothesize that supplementing FLI during bovine embryo culture increases stimulation of JAK-STAT and MAPK during the preimplantation period, thus promoting embryo development, survival, and pregnancy establishment. Results from these studies will be used to optimize livestock embryo culture systems and increase the use of this reproductive technology.

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	3399	1050	100%

Knowledge Area
301 - Reproductive Performance of Animals;

Subject Of Investigation
3399 - Beef cattle, general/other;

Field Of Science
1050 - Developmental biology;

Keywords

fertility

in vitro derived embryos

embryo culture

pregnancy success

Goals / Objectives
The long term goal of this research is tounderstand mechanisms that regulate embryo development and establishment of pregnancy in cattle. Throughout my training I will be focusing on understanding embryo development at the molecular level and then translating that information into improvement of reproductive technologies and their use in the industry. The central goal of this projectis to understand how specific growth factors modulate preimplantation embryo development and pregnancy outcomes in cattle. The goal of Objective 1 is to elucidate how the combination of Fibroblast Growth Factor 2 (FGF2), Leukemia Inhibitory Factor (LIF), and Insulin-like Growth Factor 1 (IGF1) (termed FLI), improves preimplantation bovine embryo development in vitro. Objective 2 is to determine how the addition of FLI influences the ability of an embryo to establish pregnancy following cryopreservation.

Project Methods
Objective #1: Elucidating how FLI improves in vitro embryo development 1a) Characterizing FLI modulation early embryonic development: Abattoir derived cumulusoocyte complexes will be matured and fertilized using our lab's standard procedures. Up to 50 putative zygotes will be placed in a 4-well dish containing 500 µl of SOF-BE2 overlaid with 300 µl mineral oil. Half of the zygotes will receive FLI supplementation (FGF2 (40ng/ml), LIF (20ng/ml), and IGF1 (20ng/ml)) and the other half will not. For each treatment, groups of fifteen embryos will be collected at the 4-6 cell, 9-16 cell, morula, and blastocyst stages. At the time of collection, embryos will be placed in pronase (Millipore Sigma, Burlington, MA, USA) for 60 seconds to degrade any cells outside of the zona pellucida. After 2 washes in nuclease free PBSPVP, RNA isolation will be done using PicoPure™ RNA Isolation Kit (Thermo Fisher Scientific, Waltham, MA, USA). Total RNA concentration and integrity will be recorded for all samples. With the assistance of the University of Missouri DNA Core Facility, amplification and reverse transcription will be performed using the Takara ultra-low RNA library preparation method (Clontech). Libraries of cDNA will be sequenced on a single NextSeq High Output Paired End 75 Flow Cell (NGS platform of Illumina). Sequencing will be done at a depth of 30 million reads per sample. Particular attention will be paid to the receptors for each of the cytokines (FGFR2, LIFR, and IGF1R), as well as downstream targets such as NANOG, OCT4, SOX2, CDX2, GATA6, and STAT3. The experiment will be replicated 3 times. 1b) Assessment of MAPK and JAK/STAT activation: To understand if the JAK-STAT and MAPK are activated by FLI, western blot will be used to assess phosphorylation of MAPK and STAT, and their downstream targets. Following fertilization, half of the putative zygotes will be placed in culture medium supplemented with FLI and the other half will not receive supplementation. For each group, 10 blastocysts will be collected and stored at -80°C until analysis. Embryos will be lysed, and proteins separated on a 4-20% SDS-PAGE gel and transferred to polyvinylidene fluoride membranes via an iBlot transfer system. Membranes will be blocked and incubated at 4°C overnight with the appropriate primary antibody: Phospho-Stat3 (Tyr705, 1:2000), Antiphospho-Stat3 (Tyr68, 1:1000), Phospho-p44/42 MAPK (Thr202/Tyr204, 1:2000), p44/42 MAPK (1:2000), or Anti-GAPDH (1:500). Membranes will be washed and incubated in horseradish peroxidase-linked IgG secondary. After washing, peroxide and Luminol/enhancer solutions will be applied, and the blots will be visualized after 5-10 min exposure. Data will be processed using ImageJ. The experiment will be repeated three times. 1c) Generation of receptor knockdown embryos: To understand the signaling of each cytokine (FGF2, LIF, and IGF1), an siRNA will be utilized to transiently silence their respective receptors. The siRNA will be designed and purchased using the RNAi Design Tool of IDT®. For each gene and treatment (+/- FLI), zygotes (one cell stage) will be injected with either a double-stranded siRNA for FGFR2, LIFR, or IGF1R, a scrambled version of the siRNAs (injected-control), or a noninjected control (in vitro system control). Successful knockdown will be confirmed by PCR. Cleavage and blastocyst rate will be assessed on days 3 and 8, respectively. For each group and gene, 100 embryos will be used, and the experiment will be repeated three times. Statistical Analysis: A power analysis was utilized to determine three replicates will be analyzed per experiment. For the RNA Sequencing data (1a), samples will be aligned to the cow genome using Hisat2 and featurecounts will be used to determine the read counts per gene. EdgeR will be utilized to identify differentially expressed genes at an FDR < 0.05. Blastocyst percentage and densitometries data will be analyzed by ANOVA using the PROC GLM procedure of (SAS 9.4). Tukey's honest significant difference test with P < 0.05 as significance will be used to assess differences between means.Objective #2: The effect of FLI on Pregnancy Success following embryo cryopreservation Production and freezing of embryos: Abattoir derived cumulus-oocyte complexes will be matured and fertilized by using our lab's standard procedures. Following fertilization embryos will be cultured to the blastocyst stage either with or without FLI supplementation. Blastocyst stage (stage 6) embryos with a quality grade of 1 (based on the International Embryo Technology Society manual) will be slow-frozen and stored in liquid nitrogen until transfer.Synchronization of females and transfer of embryos: A total of 100 recipient females will be subjected to the synchronization protocol 7-day CoSynch plus CIDR (controlled internal drug release) with heat detection following CIDR removal. Females will have a single frozen embryo (6-1) transferred 7 days following detection of estrus. Females will be randomly assigned the treatment of embryo they receive. Blood Sampling: Blood samples will be taken on days -7 (CIDR insertion), 0 (estrus), and 7 (transfer) to evaluate progesterone levels and ensure that recipient females present normal reproductive cyclicity. Blood Samples taken on days 19 and 20 will evaluate interferon-stimulated genes (ISG), to determine the presence of the conceptus at this stage. In addition, Pregnancy Associated Glycoproteins (PAGs), which are markers of placental functionwill be measured in blood samples at days 24 and 30 blood. Pregnancy will be diagnosed via ultrasonography on days 30 and 60, which are the critical periods of pregnancy loss in cattle. Statistical Analysis: A power analysis indicates that a total of 200 transfers are needed for this study (100 per treatment). To reach 200 transfers, we will do two rounds of 100 transfers, thus utilizing a crossover design. Analysis of data will be done using a logistic regression model with binomial distribution using the PROC GLIMMIX procedure of (SAS 9.4).

Progress 12/15/21 to 12/14/23

Outputs
Target Audience: Our efforts have reached target audiences including embryo transfer and production companies, bovine embryo transfer practitioners, researchers, and the dairy and beef cattle industries. These audiences are groups that will be directly using the technology/research described in this project, as well as groups that will use findings from this to further understand embryo development and the in vitro embryo production system. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has providing training in laboratory techniques such as radioimmunoassay, western blotting, microinjection, and RNA isolation. Training in data computation, siRNA and primer design, as well as field skills such as ovum pickup, embryo transfer, and ultrasonography have also been provided. Attendance to multiple IETS Preconference Symposia provided hands on training for embryo production, collection, freezing, and transfer. This project has provided professional development opportunities including attending and presenting at the annual International Embryo Technology Society Conference, the annual Brazilian Embryo Technology Society meeting, and American Embryo Transfer Association annual meeting. These events provided opportunity for data dissemination as well as invaluable networking opportunities. This project provided opportunity to meet and discuss with the MU Livestock Engineering Team. How have the results been disseminated to communities of interest? The target audiences were reached through conference attendance and presentation, local seminars, and published abstracts and manuscripts. Direct community interaction with practitioners and producers helped these results reach the beef and dairy industries. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? This project aims to improve cattle production through improving embryo technologies, specifically in vitro embryo production (IVEP). Information from this project will directly help cattle producers, as well as embryo technology companies and practitioners. To do this, the project was approached from both a basic and applied aspect. For the basic or molecular approach, the first goal accomplished was to understand transcriptomic differences between groups of embryos treated with or without the cytokine combination, FLI (FGF2, LIF, IGF1). All embryos were producedin vitrowith or without FLI supplementation at the beginning of culture. For each treatment, embryos were collected at the 4-6 cell, 9-16 cell, morula, or blastocyst stages and RNA was isolated and sequenced at a depth of 50 million reads per sample. In the FLI group, at the 9-16 cell stage, there were 7 upregulated and 6 downregulated differentially expressed genes (DEGs). Of those messages,ISG15andUSP18, mediators of interferon signaling were downregulated while message forMGLL,a gene involved in lipid regulation and the PI3K-ATK pathway was upregulated. At the morula stage, 1856 DEGs (580 upregulated in FLI) increased MAPK signaling, TGF-beta signaling, and Hippo signaling which all help regulate cell adhesion, lineage commitment, and growth regulation in the developing embryo. In FLI blastocyst stage embryo, 199 upregulated and 545 downregulated DEGs revealed an increase in processes associated with interferon gamma production and cell differentiation. Many of the downregulated DEGs are involved in MAPK signaling and WNT signaling pathways warranting further research of the fine regulation and interplay of these mechanisms. The addition of FLI at the beginning of culture initiates changes in the early embryo that are reflected during the entire preimplantation period. FLI modulates many of the regulatory pathways in the developing embryo to drive increased cell survival, cell integrity, and overall embryo development. Our second goal was to understand if FLI mediates bovine embryo development through the binding of FGF2 to FGFR2, LIF to LIFR, and IGF1 to IGF1R. To test this, message expression for FGFR2, LIFR, and IGF1R was reduced by siRNA-mediated gene knockdown. Embryos were microinjected with siRNA to knockdownFGFR2,LIFR, andIGF1R(KD) or a scrambled sequence (control). Half of the zygotes injected with siRNA were supplemented with FLI at the moment they were placed in the culture plate (KD +FLI). Transcript expression forFGFR2,LIFR, andIGF1Rwas reduced (P < 0.05) in KD and KD +FLI compared to control. Moreover, compared to the control group development to the blastocyst stage was decreased (P = 0.01) in both KD and KD +FLI. Together this reveals the necessity for these receptors during embryo development and demonstrates that FGF2, LIF, and IGF1 act through these receptors to induce the changes observed in the bovine embryo. The inability of FLI, as well as, other endogenous and exogenous embryokines to bind to the receptors and activate regulatory pathways, is detrimental to embryo development and viability. For an applied or field trial approach embryos were producedin vitroand cultured to the blastocyst stage with or without FLI. Embryos with a quality grade of 1 (6-1 or 5-1) were cryopreserved by slow-rate freezing. A single frozen-thawed embryo was transferred to synchronized recipient females (n = 192). Blood samples were collected on Days 7, 19, 20, and 24 and pregnancy diagnosis was performed using transrectal ultrasonography after Day 28. This experiment was carried out across three locations and pregnancies per ET were similar between both groups at all locations (P = 0.3). Progesterone on days 7, 19, and 24, and day 19 and 20 interferon-stimulated genes (ISG15,MX2,OAS1) expression were similar among both treatments (P > 0.05). There were increased (P = 0.0061) circulating concentrations of PAGs in recipients receiving FLI embryos suggesting advanced placental development and possible improvements in viability beyond day 30. No adverse effects of FLI supplementation on early embryo development were detected in these studies, thus, this is a promising addition to bovine in vitro embryo production systems. Overall, from this research, we can conclude that FLI supplementation is a positive modification to the bovine culture medium. By increasing the number of zygotes that reach the blastocyst stage, we can increase the proportion of embryos eligible for cryopreservation and/or embryo transfer. Even though pregnancy rate at day 30 was not higher in the FLI group, there were more FLI embryos that reached the blastocyst stage and thus could be transferred. Additionally, with no adverse effects of FLI observed, it is a promising improvement for the IVEP system. It will be of particular interest as systems move away from undefined components in the media, such as serum and BSA. The use of IVEP in bovine will only continue to grow, and while the pregnancy rate is good, it is not good enough: research like this will provide a more rounded understanding of embryo development and lead to the overall improvement of this technology.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: McDonald K, Prather R, Ortega MS. (2023) Exploring the actions of FLI on in vitro produced bovine embryo development and viability. Reproduction, Fertility and Development 36(2) 151. https://doi.org/10.1071/RDv36n2Ab4.
• Type: Conference Papers and Presentations Status: Other Year Published: 2023 Citation: Katy McDonald, A.M. Gonella-Diaza, J.G.N. Moraes, R.S. Prather, M.S. Ortega. (2023) Effect of FLI supplementation on pregnancy success following embryo transfer of IVP embryos. AETA
• Type: Theses/Dissertations Status: Other Year Published: 2023 Citation: McDonald K. (2023) Modulation Of Bovine Preimplantation Embryonic Development And Pregnancy By FGF2, LIF, And IGF1. Doctoral Dissertation. University of Missouri-Columbia.
• Type: Journal Articles Status: Awaiting Publication Year Published: 2024 Citation: McDonald K., Moraes J.G.N, Gonella-Diaza, A.M, Prather R.S, Ortega M.S. Effect Of FGF2, LIF, IGF1 Supplementation on Pregnancy Success Following Embryo Transfer Of In Vitro Derived Embryos.
• Type: Journal Articles Status: Awaiting Publication Year Published: 2024 Citation: McDonald K., Prather R.S., Ortega M.S. Cytokine Supplementation at The Beginning Of Culture Influences Bovine Embryo Transcriptome Throughout The Preimplantation Period.
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Stoecklein K, Prather R, Ortega MS. (2022) Cytokine supplementation influences transcriptome differences at various stages of bovine embryo development. Reproduction, Fertility, and Development. https://doi.org/10.1071/RDv35n2Ab116.

Progress 12/15/21 to 12/14/22

Outputs
Target Audience:During this reporting period, our efforts have reached target audiences including embryo transfer and production companies, bovine embryo transfer practitioners, researchers, and the dairy and beef cattle industries. These audiences are groups that will be directly using the technology/research described in this project, as well as groups that will use findings from this to further understand embryo development and the in vitro embryo production system. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has providing training in laboratory techniques such as radioimmunoassay, western blotting, and RNA isolation.Training in data computation as well as field skills such as ovum pickup, embryo transfer, and ultrasonography have also been provided. Attendance at the IETS Preconference Symposium (The Life and Travels of the IVF Embryo: From Donor to Recipient Focus on the Practitioner) provided hands on training by Dr. M.B. Wheeler and Dr. B.R. Lindsey on embryo production, collection, freezing, and transfer. This project has provided professional development opportunities including attending and presenting at the annual International Embryo Technology Society Conference and the annual Brazilian Embryo Technology Society meeting. Data from this project was also presented at the MU Livestock Engineering Team meeting. How have the results been disseminated to communities of interest?The target audiences were reached through conference attendance and presentation, local seminars, and published abstracts and manuscripts. Direct community interaction with practitioners and producers helped these results reach the beef and dairy industries. What do you plan to do during the next reporting period to accomplish the goals?To continue this project from a basic or molecular aspect, an SiRNA approach will be used to silence genes within the developing embryo that are hypothesized to have a role in the action of the three cytokines, FLI. Using this genetic engineering technique, we will be able to understand the function of these genes for embryo viability. To continue with aim two or the applied approach, increased embryo transfers will take place with both groups of embryos and pregnancy rate at day 30 will be recorded. Blood samples taken from recipient females at days 7, 19, 20, and 24 will be analyzed for interform stimulated genes, progesterone, and pregnancy associated glycoproteins to give a better look at embryo loss during the first 30 days of pregnancy. Results from both aims will be prepared and submitted for publication, as well as presented at the 49th Annual Conference of the IETS in Lima, Peru. Work from this project has been selected for poster presentation and the Domestic Animal Biomedical Embryology SLAM competition.

Impacts
What was accomplished under these goals? This project aims to improve cattle production through improving embryo technologies, specifically in vitro embryo production. Information from this project will directly help cattle producers, as well as embryo technology companies and practitioners. To do this, the project was approached from both a basic and applied aspect. For the basic or molecular approach, the first goal accomplished was to understand transcriptomic differences between groups of embryos treated with or without the cytokine combination, FLI. Results from this showed that there are numerous differences in gene expression at the two later stages of embryo development, morula and blastocyst, indicating that early supplementation of FLI influences embryo development up to the time of transfer. The next step is to understand what those differences in gene expression mean. Western blot was used to analyze the activation of regulatory pathways within the developing embryo. Blastocyst stage embryos from groups of embryos treated with or without FLI were evaluated. These data have been collected, but not yet statistically analyzed. To summarize results from aim one, transcriptomic differences between the two groups are apparent in morphologically similar embryos from the 9-16 cell stage until the blastocyst stage with increased variation from days six to eight. FLI supplementation at the beginning of embryo culture could initiate changes in the early embryo that increase development to the blastocyst stage and overall viability. For an applied or field trial approach, a single frozen-thawed embryo was transferred to a recipient female 7 days following detection of estrus and pregnancy diagnosis was performed on day 30 using transrectal ultrasonography. So far, a total of 130 transfers have been completed with 68 of those being the transfer of control (embryos produced under normal conditions) embryos and 62 being supplemented with FLI (normal conditions plus cytokine addition). The pregnancy rate per embryo transfer was 30.88 ± 5.6 % in the control group and 30.65 ± 5.9 % in the FLI supplemented group (P = 0.98). We concluded that embryos cultured with or without FLI and cryopreserved by slow-rate freezing have similar developmental competence up to day 30 of pregnancy. It was previously known that FLI supplementation improves development to the blastocyst stage, thus making more embryos eligible for transfer. Information from this part of the project helps us further understand that FLI supplementation does not affect pregnancy establishment following transfer of in vitro produced cryopreserved cattle embryos. Information from these accomplishments is helping increase the knowledge base of embryo development as well as improve protocols for culturing embryos in vitro and freezing in vitro produced embryos. This information helps livestock producers and industry professionals better understand and use embryo technologies.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Stoecklein K, Drum J, Garc�a-Guerra A, Duran B, Moraes J, Spate L, Prather R, Ortega MS. (2021) Cytokine supplementation to improve developmental competence of bovine embryos following slow-rate freezing. Reproduction, Fertility, and Development. https://doi.org/10.1071/RDv34n2Ab79.
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Stoecklein K, Drum J, Garc�a-Guerra A, Duran B, Moraes J, Spate L, Prather R, Ortega MS. (2022) Effect of FGF2, LIF, and IGF1 supplementation on pregnancy success following embryo transfer of IVP embryos. Anim Reprod, e22141, 2022
• Type: Journal Articles Status: Awaiting Publication Year Published: 2022 Citation: Stoecklein K, Garc�a-Guerra A, Duran B, Prather R, Ortega MS. (2022) Actions of FGF2, LIF, and IGF1 on Bovine Embryo Survival and Conceptus Elongation following Slow-rate Freezing. Front. Anim. Sci. doi: 10.3389/fanim.2022.1040064.