MECHANISMS OF PREIMPLANTATION DEVELOPMENTAL PROGRAMMING BY CHOLINE

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: ACTIVE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1031151
• Grant No.: 2023-67015-40730
• Cumulative Award Amt.: $650,000.00
• Proposal No.: 2022-07897
• Project Start Date: Sep 1, 2023
• Project End Date: Aug 31, 2026
• Grant Year: 2023
• 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
It is the goal of animal scientists to improve the efficiency at which agriculturally-important animals produce products useful for man. Most commonly, enhanced production has been achieved through a combination of two strategies: selection of genes that are optimal for production and provision of a postnatal environment that maximizes the opportunity for those genes to be expressed. There is a third strategy that is only now being explored - regulation of the processes of prenatal development to produce a neonate poised for optimal production. Developmental programming is the phenomenon whereby changes in the environment of the developing organism or of the gametes from which it is derived reprograms aspects of development to modify postnatal phenotype. Exploitation of the phenomenon of developmental programming offers new opportunities for enhancing animal growthwhile also avoiding some negative perceptions by consumers of some other methods to increase muscle growth (e.g., growth promotants that could be considered as hormones by consumers).While the concept of developmental programming is well established, strategies to use this phenomenon to enhance animal production are poorly developed. We still do not understand how specific components of the maternalenvironment act on the developing embryo or fetus to program characteristics of the animal after birth. Here we will do experiemnts to distinguish between two potential mechanisms. One possibility is that choline induces epigenetic changes in DNA or histones that persist into fetal and postnatal life and cause changes in somatic growth. Another possibility is that choline acts to modify processes involved in lineage formation affect organ formation and postnatal growth. To evaluate the first possibility, we will test whether there are aa set of epigenetic marks modified by choline that persist into fetal and postnatal life To test the second possibility, we will test whetther choline causes changes in the distribution of the day 21 embryo into specific cell lineages.Gaining an increased knowledge of how a molecule like choline programs postnatal development could provide insights that inform other strategies for programming.One pertinent question is whether it is the changes in the epigenome of the preimplantation period that drive actions of choline on postnatal phenotype or whether other modifications induced by choline are important. Understanding how the maternal environment of the preimplantation embryo affects its development in the short- and long-term makes the proposed research directly relevant to the goal of the Animal Reproduction Program to study embryonic and fetal development including interaction between the conceptus and its uterine environment.

Animal Health Component

20%

Research Effort Categories

Basic

80%

Applied

20%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
301	3399	1050	90%
301	3410	1050	10%

Knowledge Area
301 - Reproductive Performance of Animals;

Subject Of Investigation
3410 - Dairy cattle, live animal; 3399 - Beef cattle, general/other;

Field Of Science
1050 - Developmental biology;

Keywords

developmental programming

cattle production

choline

Goals / Objectives
The long-term goal of the proposed research is to develop methods to program postnatal growth through manipulation of the environment of the preimplantation embryo. The goal of the current proposal is to understand how one programming agent, choline, acts on the bovine preimplantation embryo to modify postnatal growth. One possibility is that choline induces epigenetic changes in DNA or histones that persist into fetal and postnatal life and cause changes in somatic growth. Another possibility is that choline acts to modify processes involved in lineage formation affect organ formation and postnatal growth. Both of these hypotheses will be tested in the current proposal. There are three objectives:Characterize actions of choline to change the epigenetic landscape of the inner cell mass and trophectoderm of the bovine blastocyst with respect to DNA methylation, histone methylation and gene expression.Using skeletal muscle and liver as model tissues, test the hypothesis that epigenetic changes in DNA and histone methylation caused by preimplantation exposure of the embryo to choline persist through fetal and postnatal development.Employ single-cell RNA-Seq to test whether treatment of the preimplantation embryo with choline alters the relative numbers of various cell lineages in the embryo during gastrulation at Day 21 of development. ?

Project Methods
Objective 1:Characterize actions of choline to change the epigenetic landscape of the inner cell mass and trophectoderm of the bovine blastocyst with respect to DNA methylation, histone methylation and gene expression.Female embryos will be produced in vitro using oocytes harvested from Brahman cows by transvaginal ultrasound guided aspiration (oocyte pickup; OPU) that are fertilized with X-sorted semen using a single Brahman bull. Embryos will be cultured from the zygote to blastocyst stages in culture medium containing either vehicle (1.8 mM NaCl) or choline (1.8 mM extra choline chloride). Blastocysts will be graded for morphological quality using standards of the International Embryo Technology Society (Robertson and Nelson, 1998). Grade 1 blastocysts will be bisected into halves consisting of TE or ICM. Pools of ICM and TE will be separately prepared from 10 female blastocysts and used for transcriptome analysis, whole genome bisulfite sequencing for DNA methylation and CUT&RUN (TheCleavageUnderTargets andReleaseUsingNuclease method that builds upon ChIP-seq) approach to identify DNA associated with histone H3K4me3 (histone methylation associated with gene activation) and H3K27me3 (repressive histone mark). The resultant data will be analyzed to identify modifications caused by choline. Additional analyses will be conducted to determine the relationship between gene expression and each epigenetic feature and to evaluate how differentiation of the blastocyst into ICM and TE is associated with alterations in the transcriptome and epigenome.Objective 2:Using skeletal muscle and liver as model tissues, test the hypothesis that epigenetic changes in DNA and histone methylation caused by preimplantation exposure of the embryo to choline persist through fetal and postnatal development.Analysis of gene expression, DNA methylation and genomic regions associated with H3K4me3 and H3K27me3 will be assessed at four points of development - day 90 and 180 of gestation and day 90 and 180 of postnatal life. Brahman embryos will be produced in vitro in culture medium + 1.8 mM choline and grade 1 blastocysts transferred at the blastocyst stage into synchronized recipients. Fetal tissues will be collected after slaughter of the dam. Postnatal samples of skeletal muscle and liver will be collected by biopsy. All tissues will then be analyzed by RNA-Seq, whole genome bisulfite sequencing, and Chip-Seq using antibodies against H3K4me3 and H3K27me3. Changes caused by choline treatment will be identified and data will be assessed to determine whether there are a set of epigenetic marks that are conserved between stages of development including as early as the blastocyst period.Objective 3: Employ single-cell RNA-Seq to test whether treatment of the preimplantation embryo with choline alters the relative numbers of various cell lineages in the embryonic disc and trophoblast of the elongating embryo during gastrulation at Day 16 of development.Embryos produced in vitro in culture medium + 1.8 mM choline will be transferred to recipient females. After slaughter of recipients at day 21 of gestation, the embryo will be recovered manually from the uterus. The embryo proper and trophoblast will be separately subjected to single cell RNA-Seq to identify specific cell types and quantify their relative abundance in vehicle-treated and choline-treated embryos.

Progress 09/01/24 to 08/31/25

Outputs
Target Audience:Scientists, ag consultants and veterinarians, farmers Changes/Problems:We plan on asking for a one-year no-cost extension to complete experiments. What opportunities for training and professional development has the project provided?Two PhD students (Lane Haimon and Masroor Sagheer) have obtained PhD degrees while working on the project. In addition, one postdoctoral student (Mariangela Maldonado) worked on the project. How have the results been disseminated to communities of interest?SCIENTIFIC PRESENTATIONS From zygote to blastocyst: setting the stage for subsequent development. Billie A. Field Memorial Lectureship Series at the University of Illinois, College of Veterinary Medicine, April 16, 2024. The future of assisted reproductive technologies in cattle. Chappel Memorial Lecture, Ontario Veterinary College, University of Guelph, May 21, 2024. Pressing needs and recent advances to enhance in vitro embryo production. 37th Annual Meeting of the Sociedade Brasileira de Technologia de Embriões. Atibaia, SP, Brazil, August 22, 2024. Importance of the preimplantation period for the future life of the embryo: evidence from the cow. Dept. of Reproduction, Anatomy and Genomics of Animals, University of Agriculture, in Krakow, April 24, 2024. From zygote to blastocyst: Setting the stage for subsequent embryo development. Robert Godke Translational Reproductive and Developmental Biology Seminar, Louisiana State University, Baton Rouge, LA, February 12, 2025. From zygote to blastocyst: Setting the stage for subsequent embryo development. University College Dublin, School of Agriculture and Food Science, Lyons Estate Research Farm, Co. Kildare, Ireland, June 6, 2025. EXTENSION PRESENTATION Developmental programming in cattle - how experiences of the embryo and fetus can affect postnatal phenotype and performance. Dr. Harvey Rubin Memorial Food Animal Veterinary Medical Conference, Florida Veterinary Medical Assn., Gainesville, Florida, Feb 15, 2025. PODCAST RP Choline During Peri-Conceptional Period Programs Postnatal Phenotype: Drs. Sagheer & Hansen, UF. Balchem Real Science Exchange. https://youtu.be/yOaPWoAFM7Y?si=fG67B7YcAYUnmKrD What do you plan to do during the next reporting period to accomplish the goals?Continue the experiments described above and initate studies to evaluate effects of choline on protein synthesis by the embryo.

Impacts
What was accomplished under these goals? Experiments are underway to evaluate DNA methylation of the preimplantation embryo as affected by choline. One experiment to evaluate effect of choline on gene expression and DNA methylation in the blastocyst has been performed. Choline caused differential expression of 263 genes, with 208 downregulated and 55 upregulated. A large percent of downregulated genes were involved in translation and ribosome biogenesis. A total of 586,950 regions of DNA were evaluated for changes in DNA methylation.A total of 7,983 differentially methylated regions were identified, with 6,174 hypermethylated (choline>vehicle) and 1,809 hypomethylated. The correlation between gene expression and DNA methylation was low. It was tentatively concluded that actions of choline to program prostnatal development are likely to involve changes in embryogenesis distal to blastocyst formation and possibly driven by a decrease in protein synthesis caused by changes in expression of genes involved in translation. A second experiment is underway to compare the changes in DNA methylation in the blastocyst with changes in DNA methylation in the resultant calves. The calves will be born in August 2025. Two experiments have been completed to test whether feeding choline causessimilar programming of the embryo as does provision of choline to the cultured embryo. One experiment was conducted withbeef cattle. Feeding choline from 1 day before anticipated ovulation through 7 days after ovulation caused a decrease in weaning weight of the resultant calves. Thus, feeding choline programs development in the opposite direction than provisionof choline to culture medium. A second experiment conducted in sheep in which choline was delivered via oral bolus from 1day before until 7 days after estrus was conducted. In this case, there was no effect of choline on weaning weight but choline altered the sex ratio of the resultant lambs to favor females. The mechanism for this effect would be worthy of further investigation. We have completed an experiment to sample sample uterine fluid from living cattle without theneed to dilute uterine fluid. This technique has allowed us to measure choline concentrations in uterine fluid. Resultsobtained to date indicate that concentrations of choline in uterine fluid are over a hundred times higher than in blood plasma and that the concentrations of choline causing developmental programming are within the physiological range. One of the observations from the two choline feeding experiments was that feeding choline does not cause an increase in concentrations in the blood. Accordingly, we have started to evaluate effects of feeding another methyl donor, the amino acid methionine, on the developmental program of the bovine female. In a preliminary experiment, we characterized consequences of varying methionine concentrations on development of the embryo. We are now in the middle of an experiment to test whetther postnatal phenotype of calves produced from embryos cultured with 114 micromolar methionine is different than that for calves derived from embryos cultured with 50 micromolar methionine. .

Publications

• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2025 Citation: Sagheer, M., Haimon, M.L.J., Montoya, S.H., Heredia, D., Tarnonsky, F., Venturini, M.E., Gonella-Diaza, A., DiLorenzo, N., McFadden, J.W., Dalmaso de Melo, G., Pohler, K.G., and Hansen, P.J. Feeding rumen-protected choline during the periconceptional period programs postnatal phenotype of suckled beef calves. J. Anim. Sci. Biotechnol. 16, 48.
• Type: Peer Reviewed Journal Articles Status: Awaiting Publication Year Published: 2025 Citation: Sagheer, M., Carballo, D., Maia, T.S., and Hansen, P.J. (2025) Consequences of varying methionine concentrations on development of the bovine embryo in vitro. Biol Reprod., in press doi: 10.1093/biolre/ioaf111.

Progress 09/01/23 to 08/31/24

Outputs
Target Audience:Experiments have been initated to evaluate DNA methylation of the preimplantation embryo as affected by choline. DNA methylation analysis is underway. In addition, two experiments have been completed to test whether feeding choline causes similar programming of the embryo as does provision of choline to the cultured embryo. One experiment was conducted with beef cattle. Feeding choline from 1 day before anticipated ovulation through 7 days after ovulation caused a decrease in weaning weight of the resultant calves. Thus, feeding choline programs development in the opposite direction than provision of choline to culture medium. A second experiment conducted in sheep in which choline was delivered via oral bolus from 1 day before until 7 days after estrus was conducted. In this case, there was no effect of choline on weaning weight. An additional replicate of this experiment is underway. Recently, we have developed a technique that, for the first time, allows sampling of uterine fluid from living cattle without the need to dilute uterine fluid. This technique has allowed us to measure choline concentrations in uterine fluid. Results obtained to date indicate that concentrations of choline in uterine fluid are over a hundred times higher than in blood plasma. Additional experimentation to assess concentrations is continuing but one implication is that the change in choline concentrations in the blood caused by feeding choline are unlikely to cause a large change in uterine concentrations of choline. An experiment to test this idea is underway. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?one graduate student, Lane Haimon, recently finished her PhD. A second student, Masroor Sagheer, is currently working towards the PhD while performing research for the grant. A third student, Ashley Sproull, started a MS degree in fall 2024 and will be working on the project. How have the results been disseminated to communities of interest?research has been reported at the International Embryo Technology Society, the Society for the Study of Reproduction and at producer meetings in Florida (Beef Cattle Short Course; Small Ruminant Short Course). What do you plan to do during the next reporting period to accomplish the goals?We will continue to make progress on all three objectives.

Impacts
What was accomplished under these goals? Progress has been made in completely objective 1. Experiments for objectives 2 and 3 will be initated in year 2 of the project.

Publications