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Normal zygotic maturation entails significant chromatin remodelling and epigenetic reprogramming of parental genomes to provide the framework for embryonic genome activation during the second cell cycle. Embryonic genome activation is required for proper embryonic development and involves a shift from maternal to embryonic regulation of gene transcription. Activation of the embryonic genome necessitates the transformation of chromatin from a transcriptionally repressed to active state. This event is disrupted by environmental factors such as culture in vitro, resulting in decreased embryonic viability. The biochemical reason for this is, however, unknown. Dynamic alterations in histone 3 acetylation on lysine 9 (H3K9ac) occur throughout preimplantation embryo development, because they are undetectable in oocytes and embryos that have just been fertilised. H3K9ac is detected around the time of first DNA replication (pronuclear stage 3) and increases in amount during preimplantation growth. H3K9ac is frequently connected with changing chromatin conformation to an active state. An increasing body of research suggests that modifications to an early embryo’s development environment, such as those that occur in assisted reproduction technologies (ART), can disrupt the usual processes of epigenetic reprogramming, which can lead to maladaptive lifetime changes in an organism’s homoeostasis. Aim This research aims to evaluate the influence of different in vitro conditions on zygotes, mimicking some ART steps, on the level of histone modification H3K9ac and chromatin configuration under different treatment conditions, and to determine if DNA is cross-linked to H3K9ac in preimplantation embryos using immune-localisation studies. Results Embryos obtained through IVF have greater H3K9ac levels than embryos obtained through natural mating. The opposite occurs when the culture time between IVF and embryos acquired by natural mating is the same. In embryos obtained through natural mating, shortening the insemination duration increases levels of H3K9ac. Embryos generated by IVF have lower levels of H3K9ac disturbance when cultured in an optimal medium. Additionally, the use of sequential media (placing a zygote in a fresh drop of medium after a period of culture) results in lower levels of H3K9ac, compared with a single step (where a zygote remains in a single drop of culture medium for the duration of culture) when an optimal medium (kSOM+AA) is used. A suboptimal single step medium (mod-HTF) manifests lower levels of H3K9ac than when using the same medium in a sequential manner. Using a suboptimal medium (mod-HTF) in the first 4 hours (h) of culture results in higher levels of H3K9ac than when using it in the second 4 h of culture, when combined with an optimal medium (kSOM+AA). Conversely, maturity alone did not cause any H3K9ac disturbance. However, when combining superovulation and age, younger mice showed higher levels of H3K9ac when compared with older mice that had not been superovulated. Zygotes from older superovulated females had lower levels of H3K9ac. Additionally, culturing in a low oxygen tension produced a similar effect to freshly obtained zygotes on H3K9ac. However, zygote density, whether 1 or 10 zygotes per drop of culture medium, had no significant effect on levels of H3K9ac. Additionally, changing the zygote environment and mode of fertilisation caused changes in chromatin configuration demonstrated by different DNA staining (DAPI) levels. A link is also demonstrated between DNA synthesis and H3K9ac in zygotes, using a DNA synthesis inhibitor. Conclusions Results demonstrate that in vitro culture is the main adverse stressor on H3K9ac irrespective of method of fertilisation, and that the impact is exacerbated when suboptimal media are used. Maternal age affects H3K9ac when combined with superovulation methods. While oxygen tension affects epigenetic reprogramming, embryo density does not. Chromatin configuration is affected by different manipulations. A new insight into the relationship between DNA replication and H3k9ac is demonstrated. The usefulness of H3K9ac measurements as a biomarker of stress in the early embryo in ART is confirmed. Implications H3K9ac is shown to have a powerful influence on transcription, chromatin conformation, and DNA replication. If ART manipulation can change this, then ART might indicate bigger epigenome aberrations that are critical for embryonic growth and development. While this research programme has contributed novel information that if applied might improve the quality of zygotes by using an optimising culture medium, decreasing the time of culture and using a low oxygen environment in vitro, it also identifies the need for further research in this discipline.