The formation of the zygote triggers a series of biological processes involved in mammalian embryogenesis. Cell lineages are specified after the embryonic genome is activated, differentiating into inner cell mass (ICM) and trophectoderm (TE), which develop into the fetus and placenta, respectively. In mice, these processes have been well studied and mainly involve cell polarization and the Hippo signaling pathway [1].

In the mouse, glucose is essential for cell differentiation and controls the fate of TE cells, as embryos undergo development block in the absence of glucose [2]. Mouse embryos exhibit progressive glucose consumption, with low use of glucose for energy metabolism in their early stages and a preference for the use of pyruvate for energy generation [3,4]. Glucose plays a crucial role in directing metabolic processes. These processes include the pentose phosphate pathway and the hexosamine biosynthetic pathway [2,5], which exert influence on the trophectoderm differentiation process. This influence occurs through post-transcriptional action in the Hippo pathway [2,5]. Glucose glycosylates the YAP1 (Yes-associated protein 1) transcription co-factor and this glycosylation leads to the expression of the transcription factors CDX2 (Caudal type homeobox 2) and GATA3 (GATA binding protein 3) [2,5]. These transcription factors are key markers of the trophectoderm.

Oxygen tension (O2) also interferes with cell differentiation, as 5 % tension is more beneficial for embryonic development than 20 % tension, resulting in increased rates of three-to four-cell embryos, morulas, and blastocysts, as well as the total number of cells [6]. Few studies show the molecular mechanisms involved in this improvement in the condition under low tension, but it is believed to be related to the decrease of reactive oxygen species (ROS) or oxidative stress conditions [7]. Nevertheless, the addition of antioxidants provides inconsistent results, presenting negative effects [8] no effects, or beneficial effects when there is a higher glucose concentration [9]. In mouse embryos, trophectoderm cells exhibit higher oxygen consumption and ATP production compared with ICM [10]. Interestingly, reducing oxygen levels from 20 % to 5 % during in vitro culture (IVC) did not impact the quantity of TE cells, but led to an increase in both total and ICM cell numbers [11].

Differently than in the mouse, bovine embryos cultured in the absence of glucose developed to the blastocyst stage [12]. Other studies show that when glucose supplementation occurs 120 h after fertilization, the blastocyst rate increases compared to the absence of glucose [13]. On the other hand, glucose was shown to be harmful when added at an early stage of embryo culture [13] or at higher doses [9,13]. In addition, bovine TE cells have greater pyruvate consumption, while ICM cells consume more glucose [14]. In one study, reducing oxygen levels from 20 % to either 2 % or 7 % resulted in a higher proportion of ICM cells [15]. Conversely, in another study, reducing oxygen levels from 20 % to 5 % led to an increase in the total number of cells [16]. Still, the influence of glucose and oxygen on cell differentiation in bovine embryos is not completely elucidated, especially because previous studies did not use lineage markers to determine TE or ICM cells.

Based on the above, we hypothesized that the absence of glucose and a higher O2 tension negatively impact TE cell allocation in the bovine embryo. This study aimed to examine how glucose and oxygen levels affect initial cell differentiation processes, including differential cell count using a TE marker, YAP1 cellular location, and the expression of genes related to this process. The experiment was conducted in a 2x2 factorial design, considering the addition or not of glucose and high or low oxygen tension.