title: Chemically induced enucleation of activated bovine oocytes: chromatin and microtubule organization and production of viable cytoplasts.
abstract: As the standard enucleation method in mammalian nuclear transfer is invasive and damaging to cytoplast spatial organization, alternative procedures have been developed over recent years. Among these techniques, chemically induced enucleation (IE) is especially interesting because it does not employ ultraviolet light and reduces the amount of cytoplasm eliminated during the procedure. The objective of this study was to optimize the culture conditions with demecolcine of pre-activated bovine oocytes for chemically IE, and to evaluate nuclear and microtubule organization in cytoplasts obtained by this technique and their viability. In the first experiment, a negative effect on oocyte activation was verified when demecolcine was added at the beginning of the process, reducing activation rates by approximately 30%. This effect was not observed when demecolcine was added to the medium after 1.5 h of activation. In the second experiment, although a reduction in the number of microtubules was observed in most oocytes, these structures did not disappear completely during assessment. Approximately 50% of treated oocytes presented microtubule reduction at the end of the evaluation period, while 23% of oocytes were observed to exhibit the complete disappearance of these structures and 28% exhibited visible microtubules. These findings indicated the lack of immediate microtubule repolymerization after culture in demecolcine-free medium, a fact that may negatively influence embryonic development. However, cleavage rates of 63.6-70.0% and blastocyst yield of 15.5-24.2% were obtained in the final experiment, without significant differences between techniques, indicating that chemically induced enucleation produces normal embryos.
authors.affiliation: Embrapa Amazônia Oriental,Trav. Dr. Enéas Pinheiro,s/no,Caixa Postal 48,CEP 66095-100,Belém,PA,Brazil.
authors.forename: Naiara Zoccal
authors.initials: NZ
authors.lastname: Saraiva
authors.affiliation: Embrapa Dairy Cattle,Valença,Brazil.
authors.forename: Clara Slade
authors.initials: CS
authors.lastname: Oliveira
authors.affiliation: Departamento de Medicina Veterinária,Faculdade de Zootecnia e Engenharia de Alimentos,Universidade de São Paulo,Pirassununga,Brazil.
authors.forename: Cláudia Lima Verde
authors.initials: CL
authors.lastname: Leal
authors.affiliation: Departamento de Medicina Veterinária Preventiva e Reprodução Animal,Universidade Estadual Paulista,Jaboticabal,Brazil.
authors.forename: Marina Ragagnin
authors.initials: MR
authors.lastname: de Lima
authors.affiliation: Departamento de Medicina Veterinária Preventiva e Reprodução Animal,Universidade Estadual Paulista,Jaboticabal,Brazil.
authors.forename: Maite
authors.initials: M
authors.lastname: Del Collado
authors.affiliation: Departamento de Medicina Veterinária Preventiva e Reprodução Animal,Universidade Estadual Paulista,Jaboticabal,Brazil.
authors.forename: Roberta
authors.initials: R
authors.lastname: Vantini
authors.affiliation: Centro APTA Bovinos de Corte,Instituto de Zootecnia,Sertãozinho,SP,Brazil.
authors.forename: Fabio Morato
authors.initials: FM
authors.lastname: Monteiro
authors.affiliation: Embrapa Southeast Livestock,São Carlos,Brazil.
authors.forename: Simone Cristina Méo
authors.initials: SC
authors.lastname: Niciura
authors.affiliation: Departamento de Medicina Veterinária Preventiva e Reprodução Animal,Universidade Estadual Paulista,Jaboticabal,Brazil.
authors.forename: Joaquim Mansano
authors.initials: JM
authors.lastname: Garcia
mesh_terms: D000818:Animals; D001755:Blastocyst / Q000502:physiology; D002417:Cattle; D018929:Cell Culture Techniques; D002843:Chromatin / Q000187:drug effects* / Q000648:ultrastructure; D003703:Demecolcine / Q000494:pharmacology*; D005260:Female; D059471:In Vitro Oocyte Maturation Techniques; D008297:Male; D008870:Microtubules / Q000187:drug effects*; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000187:drug effects* / Q000502:physiology*; D010312:Parthenogenesis / Q000187:drug effects; D050257:Tubulin Modulators / Q000494:pharmacology
chemical_list: D002843:Chromatin; D050257:Tubulin Modulators; D003703:Demecolcine
keywords: Bovine; Chemically induced enucleation; Chromatin; Microtubule; Nuclear transfer