116 Cytokine supplementation influences transcriptome differences at various stages of bovine embryo development

The supplementation of growth factors in the in vitro embryo culture environment has been widely explored, but little is known beyond the phenotypic effects of these additions. The addition of FGF2, LIF, and IGF1 (termed FLI) was previously shown to increase embryo development to the blastocyst stage and improve survival following slow-rate freezing, but the mechanisms that regulate these changes represent a gap in our knowledge. The objective of this study was to investigate the influence of FLI at various stages of pre-implantation embryo development and determine the degree of transcriptomic variation by using RNA sequencing. Abattoir-derived cumulus-oocyte complexes were matured and fertilised by using our laboratory’s standard procedures. Putative zygotes were placed in culture either with or without FLI (FGF2 [40 ng/mL], LIF [20 ng/mL], and IGF1 [20 ng/mL]). For each treatment, groups of 15 embryos were collected at the 4–6 cell, 9–16 cell, morula, and blastocyst stages. At the time of collection, embryos were placed in pronase for 60 s, washed in nuclease-free PBS-PVP, and RNA isolation was done by using PicoPure™ RNA Isolation Kit. Amplification and reverse transcription were performed by using the Takara ultra-low RNA library preparation method and libraries of cDNA were sequenced on a single NovaSeq S4 - PE100 Flow Cell. Sequencing was done at a depth of 50 million reads per sample. After quality control, transcriptome of samples was aligned to the bovine genome by using Hisat2 and FeatureCounts was used to determine the read counts per gene. EdgeR was used to identify differentially expressed genes (FDR < 0.05). Gene ontology (GO) enrichment analysis (ShinyGO 0.76) was used to identify processes associated with the differentially expressed genes (DEG). As anticipated, at the 4–6 cell stage, there were no DEGs detected. At the 9–16 cell stage, 13 DEGs (7 upregulated and 6 downregulated in FLI) represented terms associated with processes such as peptidase activity, cell adhesion, and ion channel activity. At the morula stage, 1,856 DEGs (580 upregulated and 1,276 downregulated in FLI) were in enriched in functions including protein-containing complex assembly, mitochondria function, autophagy, and oxidative phosphorylation. Lastly, 744 (199 upregulated and 545 downregulated in FLI) DEGs at the blastocyst stage represented processes such as transcription regulation, embryo development, cell differentiation, and protein metabolism. To summarise, 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 as lineage commitment occurs and the embryo becomes more transcriptionally active. 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.

This research was supported by the Clifton Murphy Scholarship Fund, USDA NIFA National Needs Fellowship Grant 2019-38420-28972, and USDA NIFA Predoctoral Fellowship Grant 2022-67011-36568.