4 Exploring the actions of FLI on in vitro-produced bovine embryo development and viability

The central goal of the following studies was to understand how FGF2, LIF, and IGF1 (40, 20, and 20 ng/mL, respectively), a cocktail called FLI, modulate embryo development and pregnancy outcomes in cattle. In all experiments, abattoir-derived cumulus–oocyte complexes were matured and fertilized using our group’s standard procedures, and putative zygotes were placed in culture either with or without FLI supplementation. In the first experiment, embryos were analysed on Day 3 for cleavage and Day 7 for development, and blastocyst-stage embryos were cryopreserved by slow-rate freezing, thawed, and hatching from the zona pellucida was recorded. A second experiment evaluated the effects of FLI on conceptus elongation and pregnancy outcome following embryo transfer (ET) of slow-rate frozen embryos into synchronized females. In one group, 8 days following ET, uteri were collected and flushed, and conceptus recovery and length were recorded. In a second group, frozen–thawed blastocyst-stage embryos were transferred to recipient females, Day 24 blood samples were collected to quantify pregnancy-associated glycoproteins (PAG), and Day 30 pregnancy was diagnosed via ultrasonography. In a third experiment, embryos in each treatment were collected at the 4–6 cell, 9–16 cell, morula, and blastocyst stages, and transcriptomic analysis was used to capture differences at the RNA level. Last, western blot was used to compare protein levels for STAT3, MAPK, and AKT at the blastocyst stage. Embryo phenotype, PAG, and western blot data were analysed by ANOVA using the PROC GLM procedure of the SAS v9.4 with significance at (P ≤ 0.05). EdgeR was used to identify differentially expressed genes (FDR < 0.05). In experiment 1, FLI embryos had improved zygote cleavage, development to the blastocyst stage, and survival in vitro following freezing. In experiment 2, Day 15 FLI conceptuses had increased PAG2, PAG12, PTGS2, and TKDP4. By Day 24, recipients carrying FLI pregnancies had increased concentrations of circulating PAG. Transcriptomic analysis revealed differences between the two treatments as the embryo becomes more transcriptionally active at the morula and blastocyst stages. Processes increased in FLI embryos included cell differentiation and proliferation, both of which rely on activation of MAPK and ERK pathways. Furthermore, the message for receptors for each cytokine was illustrated in the control embryos such that FGFR2, LIFR, and IGF1R were present throughout pre-implantation development with increased levels at the earliest stages. Last, no difference (P > 0.05) was detected for presence or phosphorylation of proteins involved in the pathways STAT3, MAPK, and AKT at the blastocyst stage. Together, these data help elucidate the implications of FLI supplementation to the bovine embryo culture system and describe effects beyond development to the blastocyst stage. By increasing the number of embryos eligible for freezing/ET, and improving pregnancy maintenance, we can ultimately increase the efficiency of this technology.

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.