Background: The brilliant cresyl blue (BCB) staining is a non-invasive test to select the best-suited oocytes for embryonic development. This makes it a useful tool to select best-quality oocytes at the times of the year when there is forage restriction. Objective: To evaluate the effect of seasonality on the nuclear maturation and quality of oocytes selected by the BCB test. Methods: The cumulus-oophorus complexes (COCs) were obtained in summer and winter of 2010 and 2011. Selected COCs were maintained for 90 min at 38.5 °C in a CO 2 incubator, in TCM 199 medium containing 10% fetal bovine serum and antibiotics, and supplemented with 26 µM brilliant cresyl blue. Afterwards, they were divided according to the ooplasm staining (BCB + -blue; BCB − -unstained). Subsequently, COCs were matured for 22 h. Nuclear maturation was evaluated at 22 h of culture. Results: The proportion of BCB − oocytes was higher in the winter of 2010, but there was no increase in this group in the winter of 2011. The percentage of oocytes that reached metaphase II was higher in control and BCB + groups in relation to oocytes from BCB − group. Conclusion: The season of the year influences the percentage of oocytes best suited for embryonic production in situations in which

It has been shown that phosphatidylinositol 3 kinase (PI3K) participates in oocyte maturation by regulating the activity of important reactions related to the resumption of meiosis and energy metabolism. Changes in the enzyme activity caused by in vitro conditions may impair important events of oocyte maturation and consequently the production of blastocysts. The study aimed to verify the effect of the addition of wortmannin (a specific inhibitor of PI3K) to the in vitro maturation medium on nuclear and cytoplasmic maturation of bovine oocyte, as well as on the production of blastocysts. Cumulus-oocyte complexes (COCs) were matured in vitro in medium supplemented with FCS and 0 (control) or 20 nM of wortmannin and then fertilized and cultured in vitro in the absence of inhibitor. Twenty-two hours after in vitro maturation the determination of PI3K activity of oocytes by Western blot was performed using anti-PI3K subunit P85 antibody/peroxidase. The activity quantification was performed by densitometry of the bands using the Gel Perfect software (Bozzo and Retamal 1991 Arch. Biol. Med. Exp. 63, 510). To check the effect of treatment on energy metabolism, glucose and glycogen concentration of oocytes was quantified by Glucox 500 test (Doles Reagentes e Equipamentos Para Laboratórios Ltd., Goiânia, Brazil). The oocyte viability determination was performed by double labelling with propidium iodide and calcein AM. To determine the nuclear maturation, the oocytes were stained with 2% acetic orcein, being considered matured those with chromosomes in metaphase plate. To assess the meiotic spindle organisation, the oocytes were labelled with anti-α tubulin-FITC and propidium iodide. The distribution of actin filaments and mitochondria was determined by rhodamine-phalloidin labelling. The distribution of cortical granules was observed by labelling the oocytes with Lens culinaris–fluorescein isothiocyanate (FITC). The cleavage and blastocyst rates were determined at 48 and 168 h post-fertilization, respectively, both calculated on the number of oocytes placed to mature. The treatment means were compared by t-test (SAS Institute Inc., Cary, NC, USA, 2003). Wortmannin produced a reduction around 40% of PI3K activity; however, the levels of glucose and glycogen were not altered. No changes were found in the viability of in vitro matured oocytes or in nuclear maturation rate (P ≥ 0.05). Treatment did not promote any change in the organisation of meiotic spindle, distribution of actin filaments, and positioning of mitochondria. However, oocytes treated with wortmannin showed a significant increase (P ≤ 0.05) in the migration of cortical granules compared with controls (87.4% ± 11.4 and 72.8 ± 11.8%, respectively). The cleavage rate was not influenced by treatment, but the blastocyst rate was higher when oocytes were matured in presence of wortmannin (34.2 ± 6.4% v. 20.0 ± 5.0%, respectively; P ≤ 0.05). The results indicate that partial inhibition of PI3K activity in bovine oocytes treated with wortmannin during in vitro maturation did not affect nuclear maturation, but improved the migration of cortical granules, which seems to have contributed to the increased the blastocyst rate.

Summary This study aimed to evaluate the effect of regulating phosphatidylinositol 3-kinase (PI3K) activity on the kinetics of oocyte nuclear maturation and the blastocyst rate. To evaluate oocyte viability, nuclear maturation rate and in vitro embryo production, cumulus–oocyte complexes (COCs) were maintained for 0, 10 min, 6 h or 22 h in TCM 199 medium supplemented with 20 nM wortmannin, an inhibitor of PI3K. After each period, COCs were transferred to the same medium without wortmannin and kept under the same conditions until completion of 22 h of in vitro maturation (IVM). To evaluate the effect of time on progression of nuclear maturation, COCs cultivated with 20 nM wortmannin was maintained for 22, 28 or 34 h of IVM. To determine the effect of wortmannin on the activity of maturation-promoting factor (MPF), COCs were kept under IVM conditions in the presence of the inhibitor for 0, 1, 3, 6, or 8 h. Exposure of COCs to wortmannin decreased (P < 0.05) the percentage of oocytes that reached metaphase II (MII) up to 22 h, MPF activity and reduced PI3K activity by 30%. However, after 28 and 34 h, 70% of oocytes reached the MII stage in the presence of inhibitor Moreover, COCs matured in the presence of wortmannin showed an increase (P < 0.05) in the blastocyst rate. These findings suggested that the regulation of the PI3K activity during IVM of bovine COCs interfered with the meiotic progression due to control of MPF activity, positively affecting the blastocyst rate.