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Artificial activation of bovine and equine oocytes with cycloheximide, roscovitine, strontium, or 6-dimethylaminopurine in low or high calcium concentrations

Published online by Cambridge University Press:  23 January 2013

Claudia Barbosa Fernandes ,
Liani Gasparini Devito ,
Lilian Rigatto Martins ,
Ieda Dala Pria Blanco ,
João Ferreria de Lima Neto ,
Patricia Myakawa Tsuribe ,
Camila Gabriela Pereria Gonçalves  and
Fernanda da Cruz Landim-Alvarenga
Claudia Barbosa Fernandes*
Affiliation:
Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of São Paulo (USP), Prof. Dr Orlando Marques de Paiva, 87 Postal code: 05508 270–São Paulo/SP–Brazil.
Liani Gasparini Devito
Affiliation:
Department of Animal Reproduction and Veterinary Radiology, School of Veterinary Medicine and Animal Science, São Paulo State University (UNESP), Rubião Júnior District s/n.–Postal code: 18618–970–Botucatu–SP, Brazil.
Lilian Rigatto Martins
Affiliation:
Department of Animal Reproduction and Veterinary Radiology, School of Veterinary Medicine and Animal Science, São Paulo State University (UNESP), Rubião Júnior District s/n.–Postal code: 18618–970–Botucatu–SP, Brazil.
Ieda Dala Pria Blanco
Affiliation:
Department of Animal Reproduction and Veterinary Radiology, School of Veterinary Medicine and Animal Science, São Paulo State University (UNESP), Rubião Júnior District s/n.–Postal code: 18618–970–Botucatu–SP, Brazil.
João Ferreria de Lima Neto
Affiliation:
Department of Animal Reproduction and Veterinary Radiology, School of Veterinary Medicine and Animal Science, São Paulo State University (UNESP), Rubião Júnior District s/n.–Postal code: 18618–970–Botucatu–SP, Brazil.
Patricia Myakawa Tsuribe
Affiliation:
Endogin, Comandante João Ribeiro de Barros, Km 226, Bauru–SP, Brazil.
Camila Gabriela Pereria Gonçalves
Affiliation:
Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of São Paulo (USP), Prof. Dr Orlando Marques de Paiva, 87 Postal code: 05508 270–São Paulo/SP–Brazil.
Fernanda da Cruz Landim-Alvarenga
Affiliation:
Department of Animal Reproduction and Veterinary Radiology, School of Veterinary Medicine and Animal Science, São Paulo State University (UNESP), Rubião Júnior District s/n.–Postal code: 18618–970–Botucatu–SP, Brazil.
*
All correspondence to: Claudia Barbosa Fernandes. Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of São Paulo (USP), Prof. Dr Orlando Marques de Paiva, 87 Postal code: 05508 270–São Paulo/SP–Brazil. Tel: +55 113091 1296. Fax: +55 113091 7412. e-mail: fernandescb@usp.br
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Summary

Knowledge on parthenogenetic activation of oocytes is important to improve the efficiency of nuclear transfer (NT) and intracytoplasmic sperm injection (ICSI) because artificial activation of oocyte (AOA) is an essential step to achieve embryo production. Although different procedures for AOA have been established, the efficiency of in vitro production of embryos remains low, especially in equines and Bos taurus bovines. In an attempt to improve the techniques of NT and ICSI in bovine and equine species, we tested different combinations of drugs that had different mechanisms of action for the parthenogenetic activation of oocytes in these animals. The oocytes were collected, in vitro matured for 24 to 30 h and activated artificially, in the presence of low or high concentrations of calcium, with combinations of calcium ionophore (ionomycin) with cycloheximide, roscovitine, strontium, or 6-dimethylaminopurine (6-DMAP). For assessment of activation rates, oocytes were stained with Hoechst 33342 and observed under an inverted microscope. We showed that all combinations of drugs were equally efficient in activating bovine oocytes, with the best results obtained when high concentrations of calcium were adopted. For equine oocytes, high concentrations of calcium were not beneficial for the parthenogenetic activation and the combination of ionomycin with either 6-DMAP or roscovitine was effective in inducing artificial activation of oocytes. We believe that our preliminary findings provide some clues for the development of a better AOA protocol to be used with these species.

Keywords

CloningICSINuclear transferParthenogenetic activation
Type
Research Article
Information
Zygote , Volume 22 , Issue 3 , August 2014 , pp. 387 - 394
Copyright
Copyright © Cambridge University Press 2013 

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