Abstract
ABSTRACT: The central goal of the following studies was to understand how FGF2, LIF, and IGF1, a cocktail called 'FLI', influence bovine embryo development by the degree of transcriptomic variation throughout preimplantation development. All embryos were produced in vitro with 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 seven upregulated and six downregulated differentially expressed genes (DEGs). At the morula stage, of the 1,856 DEGs, 580 were upregulated in FLI. Gene ontology analysis showed 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 embryos, 199 upregulated and 545 downregulated DEGs revealed an increase in processes associated with interferon-gamma production and cell differentiation. Overall, FLI modulates many of the regulatory pathways in the developing embryo to drive increased cell survival, cell integrity, and overall embryo development. LAY SUMMARY: This study investigated whether adding three supportive proteins - FGF2, LIF, and IGF1 (together called FLI) - could improve the development of cow embryos grown in vitro. In cattle breeding, embryos are often produced outside the body to enhance fertility and support genetic selection. However, many embryos fail to develop properly under laboratory conditions. To address this, researchers tested whether FLI could create a more favorable environment for early embryo growth. Although embryos grown with and without FLI appeared similar under the microscope, gene expression analysis revealed important differences. Embryos exposed to FLI showed signs of improved cell survival, healthier growth, and reduced stress. These molecular changes suggest that FLI may help embryos become more resilient to key procedures such as freezing and transfer. The findings support the use of FLI as a culture supplement to improve the efficiency and success of in vitro embryo production systems used in livestock reproductive biotechnologies.