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.
Copyright © 2015 Elsevier Inc. All rights reserved.
Publication Date: 2014-10-13 PubMed ID: 25459423DOI: 10.1016/j.theriogenology.2014.10.006Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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The research article explains an investigation conducted to verify if the fluid in equine preimplantation embryos (blastocele fluid) contains nuclear DNA, and whether this DNA can be used to determine the sex of the embryos. The results confirm that both in vivo and in vitro produced equine embryos have nuclear DNA in the blastocele fluid, which can be used for sex determination using conventional PCR.
Examination of Blastocele Fluid in Equine Embryos
- The research scrutinizes whether the blastocele fluid, the fluid-filled cavity in early embryonic development of horses, contains nuclear DNA.
- The researchers further explore if this DNA can be used to determine the sex of the embryos.
- The study shifts from traditional preimplantation genetic diagnosis, which requires the extraction of cells from the embryo, focusing solely on utilizing the blastocele fluid for DNA samples.
Sex Determination using Conventional PCR
- PCR (polymerase chain reaction) is a method used in the study to amplify and analyse the DNA segments to determine the sex of the embryos.
- Specific primers were used that targeted the TSPY and AMEL genes in the equine DNA for amplification.
- The research was successful in determining the sex of 11 out of 13 naturally conceived (in vivo) embryos and four out of five lab-conceived (in vitro) embryos.
Findings and Conclusions
- Further analysis of the PCR amplification product via genetic sequencing reported that the DNA present in the blastocele fluid was genomic.
- Following polyacrylamide gel electrophoresis and silver staining, the researchers noted a ladder pattern that is characteristic of DNA fragmented during apoptosis, a normal embryonic cell death process in mammals.
- These findings indicate that the nuclear DNA found in the blastocele fluid most likely originates from the apoptosis of embryonic cells.
- This innovative approach proposes a reliable method for preimplantation sex determination without disturbing the embryos.
Cite This Article
APA
Herrera C, Morikawa MI, Castex CB, Pinto MR, Ortega N, Fanti T, Garaguso R, Franco MJ, Castañares M, Castañeira C, Losinno L, Miragaya MH, Mutto AA.
(2014).
Blastocele fluid from in vitro- and in vivo-produced equine embryos contains nuclear DNA.
Theriogenology, 83(3), 415-420.
https://doi.org/10.1016/j.theriogenology.2014.10.006 Publication
Researcher Affiliations
- 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.
- Cría Tanoira Embriones, Pilar, Buenos Aires, Argentina.
- Cátedra de Teriogenología, Instituto de Investigación y Tecnología en Reproducción Animal (INITRA), Facultad de Ciencias Veterinarias, Universidad de Buenos Aires, Buenos Aires, Argentina.
- Cátedra de Teriogenología, Instituto de Investigación y Tecnología en Reproducción Animal (INITRA), Facultad de Ciencias Veterinarias, Universidad de Buenos Aires, Buenos Aires, Argentina.
- Instituto de Investigaciones Biotecnológicas IIB-INTECH Dr. Rodolfo Ugalde, Universidad Nacional de San Martín, San Martín, Buenos Aires, Argentina.
- Instituto de Investigaciones Biotecnológicas IIB-INTECH Dr. Rodolfo Ugalde, Universidad Nacional de San Martín, San Martín, Buenos Aires, Argentina.
- Instituto de Investigaciones Biotecnológicas IIB-INTECH Dr. Rodolfo Ugalde, Universidad Nacional de San Martín, San Martín, Buenos Aires, Argentina.
- Instituto de Investigaciones Biotecnológicas IIB-INTECH Dr. Rodolfo Ugalde, Universidad Nacional de San Martín, San Martín, Buenos Aires, Argentina.
- Laboratorio de Producción Equina, Universidad Nacional de Río Cuarto, Córdoba, Argentina.
- Laboratorio de Producción Equina, Universidad Nacional de Río Cuarto, Córdoba, Argentina.
- Laboratorio de Producción Equina, Universidad Nacional de Río Cuarto, Córdoba, Argentina.
- Cátedra de Teriogenología, Instituto de Investigación y Tecnología en Reproducción Animal (INITRA), Facultad de Ciencias Veterinarias, Universidad de Buenos Aires, Buenos Aires, Argentina.
- Instituto de Investigaciones Biotecnológicas IIB-INTECH Dr. Rodolfo Ugalde, Universidad Nacional de San Martín, San Martín, Buenos Aires, Argentina.
MeSH Terms
- Animals
- DNA / metabolism
- Embryo, Mammalian / metabolism
- Horses / embryology
- Preimplantation Diagnosis / methods
- Preimplantation Diagnosis / veterinary
- Sequence Analysis, DNA / veterinary
- Sex Determination Analysis / methods
- Sex Determination Analysis / veterinary
Citations
This article has been cited 2 times.- Benammar A, Derisoud E, Vialard F, Palmer E, Ayoubi JM, Poulain M, Chavatte-Palmer P. The Mare: A Pertinent Model for Human Assisted Reproductive Technologies?. Animals (Basel) 2021 Aug 4;11(8).
- Galluzzi L, Palini S, Stefani S, Andreoni F, Primiterra M, Diotallevi A, Bulletti C, Magnani M. Extracellular embryo genomic DNA and its potential for genotyping applications. Future Sci OA 2015 Nov;1(4):FSO62.
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