Embryonic development and gene expression in oocytes cultured in vitro in supplemented pre-maturation and maturation media.
This study aimed at assessing the effect of the addition of brain-derived neurotrophic factor (10 ng/ml BDNF) and/or cysteamine (100 μm CYS) during pre-maturation and BDNF, CYS or leptin (10 ng/ml LEP) during maturation culture in vitro on embryo development and oocyte gene expression in cattle. Oocytes were obtained by the aspiration of 2- to 8-mm follicles from slaughtered cows. In Experiment 1, oocytes were pre-matured for 24 h with 10 μm butyrolactone I in the presence or not of BDNF and/or CYS followed by in vitro maturation (IVM), fertilization (IVF) and culture (IVC). In Experiment 2, oocytes were submitted to IVM with BDNF, CYS or LEP or no supplements followed by IVF and IVC. In Experiment 3, oocytes were pre-matured with BDNF and CYS followed by IVM or only in vitro matured with BDNF. Samples for quantitative PCR (qPCR) were collected after pre-maturation (BGV) and after IVM of pre-matured oocytes (BMII) or immediately after follicle aspiration (immature control = GV) and IVM (matured control = MII). Embryo production was not affected by the inclusion of the different factors either during pre-maturation or maturation culture (∼ 43% blastocysts, p>0.05). Transcripts analysis showed that most genes (NLRP5, ZAR1, GPX1, KEAP1, SPHK2, HSP70 and PSMP1) were downregulated (p<0.05) after IVM irrespective of being previously pre-matured. The relative abundance of BAX, BCL2, IGFBP3 and ARFRP1 transcripts was unaffected by pre-maturation or maturation (p>0.05). In conclusion, supplementation of in vitro pre-maturation (BDNF and/or CYS) or maturation media (BDNF, CYS or LEP) did not improve embryo development. Gene expression was not affected by pre-maturation treatment, but some genes were downregulated after maturation, probably related to selective and differential degradation.
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Departamento de Ciências Básicas, Faculdade de Zootecnia e Engenharia de Alimentos, Universidade de São Paulo, Pirassununga, SP, Brazil.
de Bem
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Mesquita
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Rochetti
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Leal
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D000818:Animals; D019208:Brain-Derived Neurotrophic Factor / Q000008:administration & dosage; D002417:Cattle / Q000196:embryology; D002478:Cells, Cultured; D003470:Culture Media; D003543:Cysteamine / Q000008:administration & dosage; D046149:Embryo Culture Techniques / Q000662:veterinary*; D047108:Embryonic Development / Q000187:drug effects; D005260:Female; D005307:Fertilization in Vitro / Q000662:veterinary; D015870:Gene Expression / Q000187:drug effects; D020738:Leptin / Q000008:administration & dosage; D009865:Oocytes / Q000187:drug effects / Q000254:growth & development / Q000378:metabolism*; D016133:Polymerase Chain Reaction
D019208:Brain-Derived Neurotrophic Factor; D003470:Culture Media; D020738:Leptin; D003543:Cysteamine