Fetal Bovine Serum Is Essential for Maintaining Viability of Porcine Blastocysts During Extended In Vitro Culture: A Comparative Study With Defined Supplements

Extended in vitro culture (EIVC) of porcine embryos beyond Day 7 is critical for understanding peri-implantation development but remains technically challenging due to the rapid degeneration of blastocysts. While defined serum-free supplements, such as B27 and growth factor cocktails (FGF2, LIF, IGF1; FLI), have successfully supported EIVC in bovine and murine models, their efficacy in porcine embryos has not been systematically evaluated against fetal bovine serum (FBS). This study investigated the effects of these supplements on the long-term viability and molecular integrity of porcine blastocysts. Porcine blastocysts produced in vitro were cultured from Day 7 to Day 11 in Porcine Zygote Medium (PZM) supplemented with either 10% FBS, FLI cocktail, B27 or no supplement (Control). Contrary to findings in other species, supplementation with FLI or B27 failed to support porcine blastocyst survival, resulting in complete degeneration by Day 11, similar to the control group. In contrast, FBS supplementation significantly extended blastocyst viability and maintained structural integrity up to Day 11. Although blastocyst expansion plateaued after Day 9, the FBS group retained a significantly higher total cell number and preserved the expression of pluripotency (OCT4, SOX2) and lineage (CDX2, GATA6) markers compared to other groups. Furthermore, FBS significantly suppressed apoptosis, as evidenced by a lower BAX/BCL2 ratio. These findings demonstrate a distinct species-specific requirement for porcine EIVC; defined supplements effective in other mammals are insufficient for pigs, suggesting that while FBS is essential for preventing rapid degeneration and maintaining cellular viability, further optimisation is required to fully support extended developmental progression comparable to in vivo peri-implantation elongation.