142 Mimicking the follicular environment: in vitro maturation of prepubertal ovine oocytes in a liquid marble bioreactor as a 3-dimensional culture system

In vitro oocyte maturation (IVM) is a well-established technique largely applied to in vitro embryo production in the livestock field. Despite the high IVM rates obtained in most mammalian species (60 to 90%), the developmental competence of IVM oocytes is still suboptimal, as indicated by the relatively low development to blastocyst stage and the poor viability to term after transfer into recipient animals. Gametes from prepubertal donors are competent to undergo normal embryo development and produce viable offspring, albeit with lower rates. This reduced developmental competence is linked to several morphological, biochemical, and molecular features that differ from the oocytes of adult donors. The aim of this work was to evaluate potential beneficial effects of using a liquid marble microbioreactor (LM) as a 3-dimensional culture system to mature in vitro prepubertal ovine oocytes. Cumulus–oocyte complexes (COCs) obtained from slaughterhouse prepubertal (30–40 days old) sheep ovaries underwent IVM in a LM system prepared by placing a drop (30 μL containing 10 COCs) suspended in TCM 199 supplemented with 10% (v/v) oestrus sheep serum (OSS) and 0.1 IU FSH and LH onto a hydrophobic fumed silica nanoparticle (Cab-O-Sil/Cabot) bed (LM group). As a control group (CTR), COCs underwent IVM in standard volume and conditions (600 μL of IVM medium in four-well dishes). After 24 h of culture at 38.5°C in 5% CO₂ in air, COCs were released from LM and the following parameters were evaluated: (a) percentage of MII oocytes; (b) developmental rates following IVF and embryo culture for 8 days in synthetic oviducal fluid (SOF) medium at 38.5°C in 5% O₂, 5% CO₂, and 90% N₂; (c) expression of a panel of genes by real-time PCR. The LM and CTR oocytes (n = 5 and n = 6 pools of 10 oocytes, respectively) were used for expression analysis of seven members of the subcortical maternal complex (SCMC), a maternally inherited biological structure crucial for the initial stages of embryogenesis in mammals. LM and CTR groups showed similar IVM (LM: 90% vs. CTR: 92.2%) and IVF (LM: 69.7% vs. CTR: 78.0%) rates, whereas in vitro development to blastocyst stage approached significance (LM: 14,1% vs. CTR: 28,3%; P = 0.066, chi-square test). All SCMC components (KHDC3, NLRP2, NLRP5, OOEP, PADI6, TLE6, and ZBED3) showed differences in expression between LM and CTR groups (P < 0.05). Results indicate that LM microbioreactors provide a suitable microenvironment to induce oocyte IVM and may be used to improve the developmental competence of prepubertal oocytes. Further application of LM bioreactors to mature oocytes with reduced developmental competence should be considered in other species, including humans.