Simple SummaryOocyte maturation is a complex process in which the oocyte acquires the capacity to be fertilized and to support embryo development. Although oocyte maturation in vitro has undergone significant advancements, the systems that are currently in place still need to be further optimized. This study aimed to examine alternative culture systems for bovine oocyte in vitro maturation. Specifically, we used liquid marbles and differentially shaped 96-well plates (flat, v-shaped, and round-bottom). Both systems were able to support oocyte nuclear maturation, but embryo development was reduced when in vitro maturation was performed in liquid marbles. Differently shaped wells during maturation did not affect embryo yield but presented a reduction in blastocyst quality. Hence, 96-well plates might be an alternative culture system to mature oocytes in small groups, but further analyses to test possible toxic effects are needed.AbstractIn vivo-matured oocytes exhibit higher developmental competence than those matured in vitro but mimicking the in vivo environment by in vitro conditions has been challenging. Until now, conventional two-dimensional (2D) systems have been used for in vitro maturation of bovine cumulus-oocytes-complexes (COCs). However, using such systems present certain limitations. Therefore, alternative low-cost methodologies may help to optimize oocyte in vitro maturation. Here, we used two different systems to culture COCs and evaluate their potential influence on embryo development and quality. In the first system, we used treated fumed silica particles to create a 3D microenvironment (liquid marbles; LM) to mature COCs. In the second system, we cultured COCs in 96-well plates with different dimensions (flat, ultra-low attachment round-bottom, and v-shaped 96-well plates). In both systems, the nuclear maturation rate remained similar to the control in 2D, showing that most oocytes reached metaphase II. However, the subsequent blastocyst rate remained lower in the liquid marble system compared with the 96-well plates and control 2D systems. Interestingly, a lower total cell number was found in the resulting embryos from both systems (LM and 96-well plates) compared with the control. In conclusion, oocytes matured in liquid marbles or 96-well plates showed no remarkable change in terms of meiotic resumption. None of the surface geometries influenced embryo development while oocyte maturation in liquid marbles led to reduced embryo development. These findings show that different geometry during maturation did not have a large impact on oocyte and embryo development. Lower embryo production after in vitro maturation in liquid marbles was probably detected because in vitro maturation was performed in serum-free medium, which makes oocytes more sensitive to possible toxic effects from the environment.