Comparison between different maturation systems acting trough the manipulation of cAMP dependent pathways in bovine oocytes

Recent studies reported that delaying meiotic resumption of bovine oocytes through the modulation of cAMP mediated pathways can increase in vitro developmental competence (1). Aim of this study was to compare different maturation systems in which oocytes were treated with modulators of intracellular cAMP levels for different times and concentrations. In the first group (G1), cumulus-­‐oocyte complexes (COCs) were collected, treated with an adenylate cyclase activator (forskolin) and a non selective phosphodiesterase (PDE) inhibitor (IBMX) for 2 h, then cultured in maturation medium (MM: TCM199+0.4%BSA, 0.2mM Na-­‐Pyruvate, 2 mM Glutamine, 0.1mM Cysteamine, 0.1 IU/ml rhFSH), supplemented with 20 μM cilostamide, a type 3-­‐specific PDE inhibitor (1). In the second group (G2), COCs were collected in IBMX supplemented medium, cultured for 6 h in MM with 10 μM cilostamide followed by 20 h culture in inhibitor-­‐free MM (2, 3). In the control group (G3) COCs were collected and cultured in MM without any inhibitor. Oocytes were fertilized with the same bull at 26 h post IVM for G1 and G2 and at 20 h for G3 group. Presumptive zygotes were cultured in mSOF+BSA+aa for 7 day in 5%CO2 5%O2 at 38.5 °C. Maturation rate after 18h and 30h of IVM was similar while at 24 h it was significantly lower in G1 vs G2 and G3 (17 vs 48 vs 64) (p<0.05 Student’s t test), while no differences were observed between G2 and G3. Fertilization rates were not different but a high polyspermy rate was observed in G1 compared to G2 and G3 (38 vs 15 vs 7). The percentages of cleavage and blastocyst development to D+7 were lower in G1 (47 and 13) vs G2 and G3 (p<0.05) respectively, while no differences were observed between G2 (61 and 19) and G3 (68 and 25). Embryo quality and cell number was significantly lower in G1 (G1: 130, G2: 159, G3: 150). Our results indicate that treatment of bovine oocytes with high concentration of cilostamide during the whole IVM phase together with a pre-­‐IVM with IBMX and forskolin is detrimental for fertilization and embryo development . On the other hand, short time treatment with cilostamide slightly increases embryo quality but not blastocyst rate. In conclusion, the definition of an optimal maturation system needs further studies in order to modulate more accurately the intracellular cAMP levels especially during the first hours of culture. 1. Albuz FK et al., Hum Reprod 2010; 25:2999; 2. Luciano AM, et al., Biol Reprod 2011; 85:1252; 3. Lodde V et al., Biol Reprod 2009; 81:281. Work supported by Grant n 26096200 (project Ex Ovo Omnia) from Regione Sardegna & Lombardia.