FGF2 and BMP4 modulate lineage allocation in porcine parthenogenetic embryos cultured in N2B27 medium

Current porcine embryo culture relies on Porcine Zygote Medium 3 (PZM3) supplemented with fetal bovine serum (FBS), which causes batch-to-batch variability due to undefined components. Here, we evaluated N2B27-a chemically defined serum-free medium-as an alternative and compared the developmental outcomes of culture in PZM3 (PZM), PZM3 with 10% (v/v) FBS (PZM + FBS), and N2B27 in parthenogenetic embryos. N2B27 increased the total cell number, blastocyst and hatched blastocyst formation rates, and epiblast, primitive endoderm, and trophectoderm cell counts compared to PZM and PZM + FBS. Epiblast allocation increased relative to PZM but decreased relative to PZM + FBS, whereas primitive endoderm allocation increased relative to PZM and did not differ from PZM + FBS. Ungulates undergo rapid expansion of extraembryonic lineages during pre-implantation; thus we examined the effects of fibroblast growth factor 2 (FGF2) and bone morphogenetic protein 4 (BMP4) on extraembryonic lineage specification. FGF2 increased primitive endoderm cells and decreased epiblast cells, and their allocation. BMP4 increased the number of primitive endodermal cells and their allocation. FGF2 and BMP4 demonstrated combined effects, reducing the epiblast and increasing the primitive endoderm in both cell number and allocation. These results suggest that N2B27 can serve as a serum-free alternative for porcine parthenogenetic in vitro culture and that FGF2 and BMP4 can influence lineage specification toward the primitive endoderm.