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Selection of developmentally competent immature equine oocytes with brilliant cresyl blue stain prior to in vitro maturation with equine growth hormone

Published online by Cambridge University Press:  01 February 2013

Gabriel R. Pereira ,
Pedro L. Lorenzo ,
Gustavo F. Carneiro ,
Sylvie Bilodeau-Goeseels ,
John P. Kastelic ,
Alejandro Esteller-Vico ,
Manel Lopez-Bejar  and
Irwin K.M. Liu
Gabriel R. Pereira*
Affiliation:
Federal University of Pelotas – UFPEL, School of Veterinary Medicine, Campus Capão do Leão, s/n° – Mailbox 354, Zip 96010–900, Pelotas, RS, Brasil. Department of Population Health and Reproduction, School of Veterinary Medicine, University of California, Davis, CA 95616, USA.
Pedro L. Lorenzo
Affiliation:
Animal Physiology Department, Veterinary School, Universidad Complutense de Madrid, Madrid 28040, Spain.
Gustavo F. Carneiro
Affiliation:
Animal Reproduction Department, Federal Rural University of Pernambuco, Garanhuns, PE 52296–901, Brazil.
Sylvie Bilodeau-Goeseels
Affiliation:
Lethbridge Research Centre, P.O. Box 3000, 5403 1st Avenue S., Lethbridge, AB T1J4B1, Canada.
John P. Kastelic
Affiliation:
Lethbridge Research Centre, P.O. Box 3000, 5403 1st Avenue S., Lethbridge, AB T1J4B1, Canada.
Alejandro Esteller-Vico
Affiliation:
Department of Population Health and Reproduction, School of Veterinary Medicine, University of California, Davis, CA 95616, USA.
Manel Lopez-Bejar
Affiliation:
Departament de Sanitat i d'Anatomia Animals, Autonomous University of Barcelona, Edifici V 08193 Bellaterra, Barcelona, Spain.
Irwin K.M. Liu
Affiliation:
Department of Population Health and Reproduction, School of Veterinary Medicine, University of California, Davis, CA 95616, USA.
*
All correspondence to: Gabriel Ribas Pereira. Federal University of Pelotas – UFPEL, School of Veterinary Medicine, Campus Capão do Leão, s/n° – Mailbox 354, Zip 96010–900, Pelotas, RS, Brasil. Tel: +55 55 99972240 (Mobile) or +55 53 3275 7188 (Laboratory). E-mail: gpereira@ucdavis.edu or gabriel.pereira@ufpel.edu.br
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Summary

Immature oocytes synthesize a variety of proteins that include the enzyme glucose-6-phosphate dehydrogenase (G6PDH). Brilliant cresyl blue (BCB) is a vital blue dye that assesses intracellular activity of G6PDH, an indirect measure of oocyte maturation. The objective was to evaluate the BCB test as a criterion to assess developmental competence of equine oocytes and to determine if equine growth hormone (eGH) enhanced in vitro maturation (IVM) of equine oocyte. Cumulus–oocytes complexes (COCs) were recovered by aspirating follicles <30 mm in diameter from abattoir-derived ovaries and were evaluated morphologically. Thereafter, COCs were exposed to BCB (26 μM) for 90 min at 39°C and selected based on the colour of their cytoplasm (BCB positive/BCB+ or BCB negative/BCB–). The COCs were allocated as follows: (a) IVM medium; (b) eGH group; (c) BCB–/IVM; (d) BCB+/IVM; (e) BCB–/eGH; and (f) BCB+/eGH. Then, COCs were cultured in vitro for 30 h, at 39°C in a 5%CO2 humidified air atmosphere. Cumulus-free oocytes were incubated in 10 μg/ml of bis-benzamide for 20 min at 39°C and nuclear maturation was evaluated with epifluorescence microscopy. Of the 39 COCs selected morphologically and subjected to BCB staining, 18/39 (46.2%) were classified as BCB+ and 21/39 (53.8%) as BCB– (P > 0.05). Maturation was not affected significantly by BCB classification, but the maturation rate was higher for oocytes that had been exposed to exogenous eGH versus controls (16/28, 57.1% versus 8/26, 30.8%, P < 0.05). In the present study, the BCB test was not useful for predicting competent equine oocytes prior to IVM. However, eGH enhanced equine oocyte maturation in vitro.

Keywords

Brilliant cresyl blueEquine growth hormoneHorseIn vitro maturationOocyte competence
Type
Research Article
Information
Zygote , Volume 22 , Issue 4 , November 2014 , pp. 500 - 504
Copyright
Copyright © Cambridge University Press 2013 

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