Alternative Culture Systems for Bovine Oocyte In Vitro Maturation: Liquid Marbles and Differentially Shaped 96-Well Plates

Abstract

In 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.

Keywords: assisted reproduction technologies; bovine; culture systems; in vitro maturation; oocyte.

Figure 1

Schematic representation of the experimental design.

Figure 2

Oocyte encapsulation in liquid marbles. (A) One droplet of maturation medium containing the oocytes was placed in treated fumed silica powder on a Petri dish. The Petri dish was gently shaken to form the liquid marble. (B) A modified 1000 µL micropipette tip was used to manipulate the liquid marbles. (C) Liquid marbles were placed individually in the wells of a 24-well plate containing a small amount of silica powder. The central space of the plate was filled with 5 mL HEPES-TALP to prevent evaporation. (D) Resulting liquid marble drop. (E) Five COCs encapsulated in a liquid marble drop before IVM, observed under a stereomicroscope. (F) After maturation, liquid marbles were dissolved in maturation medium.

Figure 3

Representative images of Hoechst-stained oocytes in different nuclear maturation stages. (GV) Germinal vesicle. (GVBD) Germinal vesicle breakdown. (MI) Metaphase I. (MII) Metaphase II. The scale bar applies to all the images.

Figure 4

Cleavage, day 7, and day 8 blastocyst rates are expressed as a percentage of presumed zygotes. (A) Experiment 1. Oocytes were in vitro matured in liquid marbles (LM), 2D droplets, and a control group. (B) Experiment 2. Oocytes were in vitro matured in flat, v-shaped, and ultra-low attachment round-bottom 96-well plates, and a control group. Different superscripts (a and b) represent statistical differences (p < 0.05) among groups. Results are expressed as least square means ± standard error (LSM ± SE).