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SummaryThis 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 andin vitroembryo 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 ofin vitromaturation (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.

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 oocyte donors receive pasture-based feeding, since the method was effective in determining the effect of seasonality on the competence of bovine oocytes to reach nuclear maturation. Keywords: colorimetric test , cumulus-oophorus complexes , in vitro maturation , pasture , seasons. Resumen Antecedentes: La tincion con azul cresil brillante (BCB) es un metodo no invasivo para seleccionar ovocitos aptos para el desarrollo embrionario. Por tanto, es una herramienta util para selecionar los ovocitos de mejor calidad en temporadas de restriccion de forraje. Objetivo: Evaluar el efecto de la estacionalidad sobre la maduracion nuclear y calidad de los ovocitos seleccionados por el test BCB. Metodos: Los complejos cumulus-oophorus (CCOs) fueron obtenidos durante el verano y el invierno de 2010 y 2011. Los CCOs seleccionados se mantuvieron durante 90 min a 38,5 oC en una incubadora de CO 2 en un medio TCM 199 con 10% de suero fetal bovino y antibioticos, suplementado con 26 μM de azul cresil brillante. Luego se separaron segun el color del citoplasma (BCB+ —azul y BCB− —incoloro). Posteriormente, los CCOs se maduraron durante 22 h. La evaluacion de la maduracion nuclear se realizo a las 22 h de cultivo. Resultados: La proporcion de ovocitos BCB− fue mayor en el invierno de 2010, pero no hubo un aumento de ese grupo en el invierno de 2011. El porcentaje de ovocitos que alcanzaron la etapa de metafase II fue mayor en el grupo control y BCB+ con respecto al grupo BCB−. Conclusion: La estacion del ano influye en el porcentaje de ovocitos mas aptos para la produccion de embriones en situaciones donde las donadoras de ovocitos reciben alimentacion a base de pastos, ya que este metodo fue eficaz para determinar el efecto de la estacionalidad en la competencia de ovocitos bovinos en alcanzar la maduracion nuclear. Palabras clave: complejos de cumulus-oophorus , estaciones , maduracion in vitro , pastura , test colorimetrico. Resumo Antecedentes: O metodo do azul cresil brilhante (BCB) nao e invasivo e seleciona ovocitos mais aptos ao desenvolvimento embrionario. Portanto e ferramenta util para selecionar ovocitos de melhor qualidade em epocas do ano que ocorre restricao de pastagem. Objetivo: Avaliar o efeito da sazonalidade sobre a maturacao nuclear e a qualidade dos ovocitos selecionados pelo teste BCB. Metodos: Os complexos cumulus oophorus (CCOs) foram obtidos no verao e inverno de 2010 e 2011. Os CCOs selecionados foram mantidos por 90 min, a 38,5 °C, em incubadora de CO 2 , em meio TCM 199 contendo 10% de soro fetal bovino e antibioticos, e suplementado com 26 μM de azul cresil brilhante. Em seguida, estes foram divididos de acordo com a coloracao do citoplasma (BCB+ —azuis e BCB− —incolores). Entao os CCOs foram maturados durante 22 h. A avaliacao da maturacao nuclear foi realizada as 22 h de cultivo. Resultados: A proporcao dos ovocitos BCB− foi maior no inverno de 2010, mas nao houve aumento desse grupo no inverno de 2011. O percentual de ovocitos que atingiu o estagio de metafase II foi maior no controle e no grupo BCB+ em relacao ao grupo BCB−. Conclusao: A estacao do ano influencia o percentual de ovocitos mais aptos a producao de embrioes, em situacoes onde as doadoras de ovocitos recebem alimentacao baseada em pastagens, ja que o metodo se mostrou efetivo para determinacao do efeito da sazonalidade sobre a competencia de ovocitos bovinos em atingirem a maturacao nuclear. Palavras chave: complexos de cumulus-oophorus , estacoes do ano , maturacao in vitro , pastagem , teste colorimetrico.