title: Blastocele fluid from in vitro- and in vivo-produced equine embryos contains nuclear DNA.
abstract: Normal mammalian early embryonic development involves apoptosis of blastomeres as a remodeling process during differentiation, starting at the blastocyst stage. Genomic DNA has been recently detected in the blastocele fluid of human embryos and has been amplified by real-time polymerase chain reaction (PCR) to diagnose the sex of in vitro-produced human embryos. This new approach varies from conventional preimplantation genetic diagnosis in that no cells are extracted from the embryo and only the blastocele fluid is aspirated and used as a DNA sample for diagnosis. In the present work, we investigated whether the blastocele fluid of equine preimplantation embryos contains nuclear DNA and whether this DNA could be used to diagnose the sex of the embryos by conventional PCR, using specific primers that target the TSPY and AMEL equine genes. The sex of 11 of 13 in vivo-produced embryos and of four of five in vitro-produced embryos was successfully diagnosed. The PCR amplification product was analyzed using genetic sequencing reporting that the DNA present in blastocele fluid was genomic. Additionally, after polyacrylamide gel electrophoresis and silver staining, the blastocele fluid from three different embryos produced a ladder pattern characteristic of DNA fragmented during apoptosis. Therefore, the results presented in this work report that blastocele fluid from in vivo- and in vitro-produced equine embryos contains nuclear DNA which is probably originated by apoptosis of embryonic cells, and this DNA could be used to diagnose the sex of preimlpantation embryos by conventional PCR.
authors.affiliation: Instituto de Investigaciones Biotecnológicas IIB-INTECH Dr. Rodolfo Ugalde, Universidad Nacional de San Martín, San Martín, Buenos Aires, Argentina; Cría Tanoira Embriones, Pilar, Buenos Aires, Argentina. Electronic address: caroherr@gmail.com.
authors.forename: C
authors.initials: C
authors.lastname: Herrera
authors.affiliation: Cría Tanoira Embriones, Pilar, Buenos Aires, Argentina.
authors.forename: M I
authors.initials: MI
authors.lastname: Morikawa
authors.affiliation: Cátedra de Teriogenología, Instituto de Investigación y Tecnología en Reproducción Animal (INITRA), Facultad de Ciencias Veterinarias, Universidad de Buenos Aires, Buenos Aires, Argentina.
authors.forename: C Baca
authors.initials: CB
authors.lastname: Castex
authors.affiliation: Cátedra de Teriogenología, Instituto de Investigación y Tecnología en Reproducción Animal (INITRA), Facultad de Ciencias Veterinarias, Universidad de Buenos Aires, Buenos Aires, Argentina.
authors.forename: M R
authors.initials: MR
authors.lastname: Pinto
authors.affiliation: Instituto de Investigaciones Biotecnológicas IIB-INTECH Dr. Rodolfo Ugalde, Universidad Nacional de San Martín, San Martín, Buenos Aires, Argentina.
authors.forename: N
authors.initials: N
authors.lastname: Ortega
authors.affiliation: Instituto de Investigaciones Biotecnológicas IIB-INTECH Dr. Rodolfo Ugalde, Universidad Nacional de San Martín, San Martín, Buenos Aires, Argentina.
authors.forename: T
authors.initials: T
authors.lastname: Fanti
authors.affiliation: Instituto de Investigaciones Biotecnológicas IIB-INTECH Dr. Rodolfo Ugalde, Universidad Nacional de San Martín, San Martín, Buenos Aires, Argentina.
authors.forename: R
authors.initials: R
authors.lastname: Garaguso
authors.affiliation: Instituto de Investigaciones Biotecnológicas IIB-INTECH Dr. Rodolfo Ugalde, Universidad Nacional de San Martín, San Martín, Buenos Aires, Argentina.
authors.forename: M J
authors.initials: MJ
authors.lastname: Franco
authors.affiliation: Laboratorio de Producción Equina, Universidad Nacional de Río Cuarto, Córdoba, Argentina.
authors.forename: M
authors.initials: M
authors.lastname: Castañares
authors.affiliation: Laboratorio de Producción Equina, Universidad Nacional de Río Cuarto, Córdoba, Argentina.
authors.forename: C
authors.initials: C
authors.lastname: Castañeira
authors.affiliation: Laboratorio de Producción Equina, Universidad Nacional de Río Cuarto, Córdoba, Argentina.
authors.forename: L
authors.initials: L
authors.lastname: Losinno
authors.affiliation: Cátedra de Teriogenología, Instituto de Investigación y Tecnología en Reproducción Animal (INITRA), Facultad de Ciencias Veterinarias, Universidad de Buenos Aires, Buenos Aires, Argentina.
authors.forename: M H
authors.initials: MH
authors.lastname: Miragaya
authors.affiliation: Instituto de Investigaciones Biotecnológicas IIB-INTECH Dr. Rodolfo Ugalde, Universidad Nacional de San Martín, San Martín, Buenos Aires, Argentina.
authors.forename: A A
authors.initials: AA
authors.lastname: Mutto
mesh_terms: D000818:Animals; D004247:DNA / Q000378:metabolism*; D004622:Embryo, Mammalian / Q000378:metabolism*; D006736:Horses / Q000196:embryology*; D019836:Preimplantation Diagnosis / Q000379:methods / Q000662:veterinary; D017422:Sequence Analysis, DNA / Q000662:veterinary; D012732:Sex Determination Analysis / Q000379:methods / Q000662:veterinary*
chemical_list: D004247:DNA
keywords: Apoptosis; Equine; Preimplantation embryo; Preimplantation genetic diagnosis; Sex determination