The combination of FLI, ITS, pFF, and citrate improves the developmental potential of gilt oocytes during in vitro maturation.

The intrinsic quality of porcine oocytes is a critical determinant of early embryonic development during in vitro production (IVP). This study investigated the effects of chemically defined in vitro maturation (IVM) systems supplemented with glutamine, GlutaMAX, insulin-transferrin-selenium (ITS), porcine follicular fluid (pFF), citrate, and growth factors (FGF2, LIF, IGF1, termed FLI) on the cumulus diffusion and maturation of oocytes derived from different diameters follicles. Moreover, four IVM media were systematically assessed for their effects on oocyte nuclear and cytoplasmic maturation and subsequent embryonic development. Results showed that the addition of pFF or FLI alone significantly enhanced cumulus expansion (P < 0.05). FLI medium combined with ITS, citrate, and pFF, exhibited higher average cumulus diffusion diameters and rates of first polar body extrusion. Notably, the synergistic combination of ITS, citrate, and pFF with FLI medium upregulated expression of cumulus expansion makers (HAS2, PTX3, CX43, TNFAIP6) and oocyte maturation makers (GDF9 and BMP15), improved cortical granule distribution, spindle morphology, chromosome alignment, and elevated glutathione (GSH) levels. This combination also enhanced cleavage and blastocyst rates, increased inner cell mass cell counts in blastocysts, and improved enucleation and fusion efficiency of oocytes following somatic cell nuclear transfer (SCNT). Collectively, the combination of FLI medium with ITS, pFF, and citrate synergistically promoted the maturation and developmental potential of porcine oocytes derived from prepubertal gilts, including those from small follicles. This optimized IVM system provides a robust foundation for advancing porcine IVP in reproductive biotechnology.