Despite the progressive increase in the use of Assisted Reproductive Technologies (ART) (2.9 % of births in France in 2012), the safety of ART associated procedures remains a matter of concern. Especially, an increase in the prevalence of imprinting disorders has been reported among ART issued children, pointing to potential epigenetic defaults induced by such procedures. To eliminate confounding factors and precisely isolate the impact of embryo culture conditions on embryonic epigenome, we chose the rabbit embryo as a model since its embryonic genome activation (EGA) and associated epigenetic modifications are close to the human embryo. We compared DNA methylation and hydroxymethylation in in vivo developed embryos to those of in vitro cultured embryos using either a single medium (Global (Life global)) or a sequential medium (G1/G2 (Vitrolife)). Both of these media are currently used in human ART, and the choice between single or sequential media for embryo long-culture remains a puzzling question. In vivo, DNA methylation progressively decreases from the early 4-cell stage until the late 8-cell stage, while hydroxymethylation decreases during the 4 and 8-cell stages, then transiently increases at the 16-32-cell stage. Interestingly, our results evidenced culture condition-specific alterations in both demethylation and hydroxymethylation kinetics. We thus wondered whether the expression of genes encoding DNMT and TET enzymes, responsible for DNA methylation and hydroxymethylation respectively, were affected by in vitro culture conditions. TET1 and TET2 were underexpressed in in vitro cultured embryos after EGA. Whether these alterations in enzyme expression and in DNA demethylation/hydroxymethylation impact embryonic gene expression and fetal phenotype is currently under investigation.