Embryo lipid profile is affected by in vitro culture conditions, that lead to an increase in lipids. Efforts have been made to optimize embryo lipid composition as it is associated with their quality. The objective of this study was to evaluate whether the diet supplementation of donor cows (n-3 or n-6 PUFA), or the slow freezing protocols (ethylene glycol sucrose EG-S vs. glycerol trehalose GLY-TRE), or the physiological stage of the donor (nulliparous heifers vs. primiparous lactating cows) may impact the bovine embryo lipid profile. Lipid extracts of 97 embryos were individually analysed by liquid chromatography-high resolution mass spectrometry, highlighting 246 lipids including 85% being overabundant in cow embryos compared to heifer embryos. Among 105 differential lipids, 72 were overabundant after EG-S protocol, including a single glycerophosphate PA(32:1) representing 27.3% of the significantly modulated lipids, suggesting that it is degraded when GLY-TRE is used. No lipids were different according to the n-3 or n-6 supplementation of the donor cows. In conclusion, the embryonic lipid profile was mainly affected by the physiological stage of the donors and the slow freezing protocols. The overabundance of lipids in lactating cow embryos and the resulting lower quality of these embryos is consistent with the lower pregnancy rate observed in cows compared to heifers. Unlike GLY-TRE protocol, EG-S freezing allowed to preserve glycerophospholipids potentially improving the slow freezing of in vitro-produced embryos. Further studies are required to modulate embryo quality and freezability by modulating the lipidome and integrating all stages of embryonic production.