Suplementação do meio de transporte com antioxidantes e moduladores de AMP cíclico como estratégia para melhorar a qualidade de oócitos bovinos destinados à produção in vitro de embriões
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Data
2016-06-06
Autores
Ambrogi, Marcela [UNESP]
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
O objetivo desse estudo foi avaliar os efeitos da suplementação do meio com diferentes fontes de macromoléculas, com bloqueadores da meiose e com antioxidantes durante o transporte de oócitos bovinos por 6 horas sobre: 1) progressão da maturação nuclear; 2) maturação citoplasmática e 3) competência no desenvolvimento e criotolerância dos embriões produzidos. Para tanto, o meio de transporte de oócitos foi suplementado com bloqueadores da meiose (forscolina e IBMX; Experimento 1) ou com diferentes tipos de macromoléculas (SFB ou BSA; Experimento 2), sendo que estes tratamentos ainda receberam ou não a suplementação com antioxidantes (mistura de cisteína, cisteamina e catalase). Os oócitos foram incubados em incubadora portátil (Minitub®) para simulação de transporte. Posteriormente, foram submetidos à maturação in vitro (MIV) em incubadora a 5% de CO2 em ar até completar 24h e, em seguida, foram fecundados e os prováveis zigotos foram cultivados in vitro durante 7 dias. Foi feito um grupo controle adicional no experimento I: MIV em incubadora com 10% de SFB por 24h. No experimento II foram feitos dois grupos controle adicionais MIV em incubadora com 10% de SFB por 24h sem e com antioxidantes (cisteína, cisteamina e catalase). Nos experimentos 1 e 2 foi avaliada a cinética da maturação nuclear e a maturação citoplasmática (através do posicionamento de mitocôndrias, do potencial de membrana mitocondrial e do conteúdo intracelular de espécies reativas do oxigênio) após o transporte e após a MIV. Avaliou-se a taxa de clivagem (48 hpi) e de desenvolvimento embrionário até a fase de blastocistos (168 hpi) e re-expansão após 24 horas de re-cultivo. No experimento 1, a taxa de oócitos em GV foi maior nos oócitos (P<0,05) do grupo Transporte com bloqueador (37,9%) e menor no grupo Transporte sem bloqueador (10,1%), ambos estes grupos difeririam do grupo Controle (20,0%). Enquanto a taxa de GVBD foi maior (P<0,05) no grupo Transporte sem bloqueador (82,8%) e menor nos grupos Controle (77,0%) e Transporte com bloqueador (62,0%). Nos oócitos dos grupos tratados com antioxidantes, após 6h de transporte, a concentração intracelular de ROS foi maior (P<0,05) no grupo Transporte sem antioxidantes (1,5) e menor nos grupos Controle (1,0) e Transporte com antioxidantes (1,1). Com relação ao tratamento com bloqueadores, a contração de ROS foi maior (P<0,05) nos oócitos do grupo Transporte sem bloqueador (1,5) e menor nos grupos Controle (1,0) e Transporte com bloqueador (1,2). Após 24 horas de MIV houve interação entre os tratamentos (bloqueadores+antioxidantes), sendo que os oócitos dos grupos Transporte Pré-MIV com antioxidantes (1,0) e Controle (1,0) apresentaram a maior concentração de ROS (P<0,05), enquanto que os grupos Transporte controle (0,6) e Transporte Pré-MIV sem antioxidantes (0,5) exibiram as menores concentrações de ROS. Com relação ao potencial de membrana de mitocondrial (PMM), houve interação entre os tratamentos quando os oócitos foram valiados após 6 e 24 horas. Após 6 horas Transporte Pré-MIV sem antioxidantes (1,00) e Controle (0,9±0,08) apresentaram o maior PMM, que diferiu (P<0,05) dos grupos Transporte Pré-MIV com antioxidantes (0,6) e Transporte controle (0,6). Após 24 horas, os grupos Controle (1,00) e Transporte Pré-MIVcom antioxidantes (0,8) apresentaram o maior PMM, e diferiram (P<0,05) dos demais grupos (0,5-0,6). No experimento 2, a taxa de oócitos em GVBD foi maior (P<0,05) no grupo Transporte SFB (86,8%) e menor em Controle (77,0%), ambos os grupos foram semelhantes a Transporte BSA (82,8%). Nos oócitos dos grupos tratados com antioxidantes após 6 horas as concentrações de ROS foi maior (P<0,05) no grupo Transporte sem antioxidantes (1,6) e menor nos grupos Controle sem antioxidantes (1,0), Controle com antioxidantes (1,1) e Transporte com antioxidantes (1,2) e após a MIV foi maior no controle (1,0) diferindo (P<0,05) dos demais grupos (0,5-0,7). Com relação ao PMM, após 6 horas de transporte houve interação entre os tratamentos (macromoléculas+antioxidantes), o grupo Controle (1,0) apresentou maior (P<0,05) PMM, e nos grupos Controle com antioxidantes (0,6), Transporte SFB com antioxidantes (0,6) e Transporte BSA (0,6) foi observado menor PMM. Após 24 horas não foi verificado interação entre os tratamentos, nos oócitos tratados com antioxidantes, o grupo Controle (1,0) mostrou maior (P<0,05) PMM, os grupos Transporte sem antioxidantes (0,4) e Transporte com antioxidantes (0,5) apresentaram menor PMM. A adição de bloqueadores da meiose (IBMX associado à forscolina) ao meio de transporte de oócitos bovinos permite um transporte adequado por 6h, preservando a qualidade e integridade dos oócitos e permitindo uma produção embrionária semelhante à obtida in vitro a partir de oócitos que não foram submetidos ao transporte.
The objective of this study was to evaluate the effects of supplementation of the medium with different sources of macromolecules with blockers of meiosis and antioxidants during transport of bovine oocytes for 6 hours on: 1) progression of nuclear maturation; 2) cytoplasmic maturation and 3) competence in the development and cryotolerance of embryos produced. Therefore, the medium of transport oocytes was supplemented with blocking of meiosis (forskolin and IBMX; Experiment 1) or with different types of macromolecules (FCS or BSA; Experiment 2), and these treatments yet received or not antioxidant supplementation (mixture of cysteine, cysteamine and catalase). Oocytes were incubated in a portable incubator (Minitub®) for transport simulation. Posteriorly were submitted in vitro maturation (IVM) in incubator at 5% CO2 in air until to complete 24 hours and then were fertilized and presumptive zygotes were cultured in vitro for 7 days. Has been made an additional control group in the experiment I: MIV incubator with 10% FCS for 24 hours (Control). In the second experiment were performed two additional control groups: IVM incubator with 10% FCS for 24 hours (Control); and IVM in an incubator with 10% FCS and antioxidants (cysteine, cysteamine and catalase) for 24 hours (Contr+Atx). In Experiments 1 and 2 were evaluated after nuclear maturation kinetics and cytoplasmic maturation (made by positioning mitochondria, the mitochondrial membrane potential and intracellular content of reactive oxygen species) of transport and after IVM. We evaluated the cleavage (48 hpi), embryo development to the blastocyst stage (168 hpi), and the re-expansion rate after 24 hours of re-cultivation. In experiment 1, the GVBD rate was higher (P<0.05) in Transport group (82.8%) and lower in control group (77.0%) and Transport with blocker (62.0%). In oocytes treated groups antioxidants, after 6 hours of transport, the intracellular concentration of ROS was higher (P<0.05) in Transport group (1.5) and lower in group Control (1.0) and Transport with Antioxidants (1.1). Regarding the treatment with blockers, ROS contraction was higher (P <0.05) in the oocytes Transport group (1.5) and lower in groups Ccontrol (1.0) and Transport with Blocker (1.2). After 24 hours of IVM interaction was observed between treatments (blockers+antioxidants), and the groups of oocytes Transport Pre-IVM + Atx(1.0) and Control (1.0) had the highest concentration of ROS (P<0.05), while Transport Control (0.6) and Transport Pre-IVM (0.5) groups exhibited the lowest concentrations of ROS. Regarding the mitochondrial membrane potential (MMP), there was an interaction between treatments when the oocytes were evaluated after 6 and 24 hours. After 6 hours Transport Pre-IVM (1.00) and Control (0.9 ± 0.08) had the highest MMP, which differed (P<0.05) Transport Pre-IVM + Atx (0.6) and Transport Control (0.6) groups. After 24 hours, the Control (1.0) and Transport Pre-IVM + Atx (0.8) groups had the highest MMP and differ (P<0.05) from the others groups (0.5 to 0.6). In experiment 2, the GVBD oocyte rate was higher (P<0.05) in the Transport FCS group (86.8%) and lowest in Control FCS (77.0%), both groups were similar Transport BSA (82.8%). With respect to the concentration of ROS in the groups supplemented with macromolecules, after 6 hours was higher (P<0.05) in the TB group (1.5) and TS (1.3) and lower in CS (1.0). In oocytes groups treated with antioxidants after 6 hours ROS concentration was higher (P <0.05) in Transport group (1.6) and lower in Control (1.0), Control with Antioxidants (1.1) and Transport with Antioxidants (1.2) groups and after IVM was higher in Control (1,0) differ (P<0.05) from the other groups (0.5 to 0.7). Regarding PMM, after 6 hours of transport was no interaction between treatments (macromolecules+antioxidants), Control (1,0) group had higher (P<0.05) MMP, and Transport BSA (0.6) was observed less MMP. After 24 hours was not verified interaction between treatments, in oocytes treated with antioxidants, the Control (1.0) group demonstrate higher (P<0.05) MMP, Transport (0.4) and Transport with Antioxidants (0.5) groups had a lower MMP. The addition of blocking meiosis (IBMX associated with forskolin) in oocytes transport medium permits adequate transport for 6h, preserving the quality and integrity of the oocyte and allowing embryo production similar to that obtained in vitro from oocytes that were not subjected to transport.
The objective of this study was to evaluate the effects of supplementation of the medium with different sources of macromolecules with blockers of meiosis and antioxidants during transport of bovine oocytes for 6 hours on: 1) progression of nuclear maturation; 2) cytoplasmic maturation and 3) competence in the development and cryotolerance of embryos produced. Therefore, the medium of transport oocytes was supplemented with blocking of meiosis (forskolin and IBMX; Experiment 1) or with different types of macromolecules (FCS or BSA; Experiment 2), and these treatments yet received or not antioxidant supplementation (mixture of cysteine, cysteamine and catalase). Oocytes were incubated in a portable incubator (Minitub®) for transport simulation. Posteriorly were submitted in vitro maturation (IVM) in incubator at 5% CO2 in air until to complete 24 hours and then were fertilized and presumptive zygotes were cultured in vitro for 7 days. Has been made an additional control group in the experiment I: MIV incubator with 10% FCS for 24 hours (Control). In the second experiment were performed two additional control groups: IVM incubator with 10% FCS for 24 hours (Control); and IVM in an incubator with 10% FCS and antioxidants (cysteine, cysteamine and catalase) for 24 hours (Contr+Atx). In Experiments 1 and 2 were evaluated after nuclear maturation kinetics and cytoplasmic maturation (made by positioning mitochondria, the mitochondrial membrane potential and intracellular content of reactive oxygen species) of transport and after IVM. We evaluated the cleavage (48 hpi), embryo development to the blastocyst stage (168 hpi), and the re-expansion rate after 24 hours of re-cultivation. In experiment 1, the GVBD rate was higher (P<0.05) in Transport group (82.8%) and lower in control group (77.0%) and Transport with blocker (62.0%). In oocytes treated groups antioxidants, after 6 hours of transport, the intracellular concentration of ROS was higher (P<0.05) in Transport group (1.5) and lower in group Control (1.0) and Transport with Antioxidants (1.1). Regarding the treatment with blockers, ROS contraction was higher (P <0.05) in the oocytes Transport group (1.5) and lower in groups Ccontrol (1.0) and Transport with Blocker (1.2). After 24 hours of IVM interaction was observed between treatments (blockers+antioxidants), and the groups of oocytes Transport Pre-IVM + Atx(1.0) and Control (1.0) had the highest concentration of ROS (P<0.05), while Transport Control (0.6) and Transport Pre-IVM (0.5) groups exhibited the lowest concentrations of ROS. Regarding the mitochondrial membrane potential (MMP), there was an interaction between treatments when the oocytes were evaluated after 6 and 24 hours. After 6 hours Transport Pre-IVM (1.00) and Control (0.9 ± 0.08) had the highest MMP, which differed (P<0.05) Transport Pre-IVM + Atx (0.6) and Transport Control (0.6) groups. After 24 hours, the Control (1.0) and Transport Pre-IVM + Atx (0.8) groups had the highest MMP and differ (P<0.05) from the others groups (0.5 to 0.6). In experiment 2, the GVBD oocyte rate was higher (P<0.05) in the Transport FCS group (86.8%) and lowest in Control FCS (77.0%), both groups were similar Transport BSA (82.8%). With respect to the concentration of ROS in the groups supplemented with macromolecules, after 6 hours was higher (P<0.05) in the TB group (1.5) and TS (1.3) and lower in CS (1.0). In oocytes groups treated with antioxidants after 6 hours ROS concentration was higher (P <0.05) in Transport group (1.6) and lower in Control (1.0), Control with Antioxidants (1.1) and Transport with Antioxidants (1.2) groups and after IVM was higher in Control (1,0) differ (P<0.05) from the other groups (0.5 to 0.7). Regarding PMM, after 6 hours of transport was no interaction between treatments (macromolecules+antioxidants), Control (1,0) group had higher (P<0.05) MMP, and Transport BSA (0.6) was observed less MMP. After 24 hours was not verified interaction between treatments, in oocytes treated with antioxidants, the Control (1.0) group demonstrate higher (P<0.05) MMP, Transport (0.4) and Transport with Antioxidants (0.5) groups had a lower MMP. The addition of blocking meiosis (IBMX associated with forskolin) in oocytes transport medium permits adequate transport for 6h, preserving the quality and integrity of the oocyte and allowing embryo production similar to that obtained in vitro from oocytes that were not subjected to transport.
Descrição
Palavras-chave
Bloqueadores da meiose, Maturação in vitro, Antioxidantes, Macromoléculas, Transporte de oócitos, Blocking meiosis, In vitro maturation, Antioxidants, Macromolecules, Oocyte transport