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Post-hatching development of bovine embryos in vitro: the effects of tunnel preparation and gender

Published online by Cambridge University Press:  23 March 2011

Grazieli Marinheiro Machado ,
Ester Siqueira Caixeta ,
Carolina Madeira Lucci ,
Rodolfo Rumpf ,
Maurício Machaim Franco  and
Margot Alves Nunes Dode
Grazieli Marinheiro Machado
Affiliation:
Laboratory of Animal Reproduction, Embrapa Genetic Resources and Biotechnology, Parque Estação Biológica – PqEB – Av. W5 Norte (final), Caixa Postal 02372, Brasília, DF - Brazil – 70.770-917. College of Agriculture and Veterinary, University of Brasília, Campus Universitário Darcy Ribeiro – Asa Norte, Instituto Central de Ciências Ala Sul–Caixa Postal 4.508, Brasília, DF – Brazil, CEP: 70.910-970.
Ester Siqueira Caixeta
Affiliation:
Laboratory of Animal Reproduction, Embrapa Genetic Resources and Biotechnology, Parque Estação Biológica – PqEB – Av. W5 Norte (final), Caixa Postal 02372, Brasília, DF - Brazil – 70.770-917. College of Agriculture and Veterinary, University of Brasília, Campus Universitário Darcy Ribeiro – Asa Norte, Instituto Central de Ciências Ala Sul–Caixa Postal 4.508, Brasília, DF – Brazil, CEP: 70.910-970.
Carolina Madeira Lucci
Affiliation:
College of Agriculture and Veterinary, University of Brasília, Campus Universitário Darcy Ribeiro – Asa Norte, Instituto Central de Ciências Ala Sul–Caixa Postal 4.508, Brasília, DF – Brazil, CEP: 70.910-970.
Rodolfo Rumpf
Affiliation:
Laboratory of Animal Reproduction, Embrapa Genetic Resources and Biotechnology, Parque Estação Biológica – PqEB – Av. W5 Norte (final), Caixa Postal 02372, Brasília, DF - Brazil – 70.770-917. College of Agriculture and Veterinary, University of Brasília, Campus Universitário Darcy Ribeiro – Asa Norte, Instituto Central de Ciências Ala Sul–Caixa Postal 4.508, Brasília, DF – Brazil, CEP: 70.910-970.
Maurício Machaim Franco
Affiliation:
Laboratory of Animal Reproduction, Embrapa Genetic Resources and Biotechnology, Parque Estação Biológica – PqEB – Av. W5 Norte (final), Caixa Postal 02372, Brasília, DF - Brazil – 70.770-917. College of Agriculture and Veterinary, University of Brasília, Campus Universitário Darcy Ribeiro – Asa Norte, Instituto Central de Ciências Ala Sul–Caixa Postal 4.508, Brasília, DF – Brazil, CEP: 70.910-970.
Margot Alves Nunes Dode*
Affiliation:
Laboratory of Animal Reproduction, Embrapa Genetic Resources and Biotechnology, Parque Estação Biológica, PqEB, Av. W5 Norte (final), Caixa Postal 02372, Brasília, DF – Brazil – 70.770-917. College of Agriculture and Veterinary, University of Brasília, Campus Universitário Darcy Ribeiro – Asa Norte, Instituto Central de Ciências Ala Sul–Caixa Postal 4.508, Brasília, DF – Brazil, CEP: 70.910-970.
*
All correspondence to: Margot Alves Nunes Dode. Laboratory of Animal Reproduction, Embrapa Genetic Resources and Biotechnology, Parque Estação Biológica, PqEB, Av. W5 Norte (final), Caixa Postal 02372, Brasília, DF – Brazil – 70.770-917. Tel. +55 61 34484659. Fax: +55 61 33403658. e-mail: margot@cenargen.embrapa.br.
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Summary

The objective of this study was to compare morphological characteristics, kinetics of development, and gene expression of male and female IVP embryos that were cultured until day (D)15 (fertilization = D0), using either phosphate-buffered saline (PBS) or Milli-Q water (MQW) to dilute the agarose gel used for tunnel construction. On D11, embryos (n = 286) were placed in agarose gel tunnels diluted in PBS and MQW. Embryos were evaluated for morphology, and embryo size was recorded on D11, D12.5, D14 and D15. Then, embryos were sexed and used for gene expression analyses (G6PD, GLUT1, GLUT3, PGK1, PLAC8, KRT8, HSF1 and IFNT). The percentage of elongated embryos at D15 was higher (p < 0.05) in the PBS (54%) than in the MQW (42%) gel. However, embryos produced in MQW were bigger (p < 0.05) and had a lower expression of GLUT1 (p = 0.08) than those cultured in PBS. There was a higher proportion of male than female embryos at D15 in both treatments, MQW (65% vs. 35%; p < 0.05) and PBS (67% vs. 33%; p < 0.05); however, embryo size was not significantly different between genders. Moreover, D15 female embryos had greater expression of G6PD (p = 0.05) and KRT8 (p = 0.03) than male embryos. In conclusion, the diluent used for tunnel construction affected embryo development in the post-hatching development (PHD) system, and the use of MQW was the most indicative measure for the evaluation of embryo quality. Male and female embryos cultured from D11 to D15, either in an MQW or PBS agarose gel, demonstrated similar development but different gene expression.

Keywords

Agarose gelCattleEmbryo cultureGene expressionSexing
Type
Research Article
Information
Zygote , Volume 20 , Issue 2 , May 2012 , pp. 123 - 134
Copyright
Copyright © Cambridge University Press 2011

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