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Reliability of Hoechst 33342 Staining under Wide-Field Microscopy for Evaluation of the Nuclear Status of Living Dog Oocytes

Published online by Cambridge University Press:  12 April 2012

Martine Chebrout ,
Pierre-Gaël Adenot ,
Karine Reynaud  and
Sylvie Chastant-Maillard
Martine Chebrout
Affiliation:
INRA, UMR 1198 Biologie du Développement et Reproduction, F-78350 Jouy en Josas, France ENVA, UMR 1198 Biologie du Développement et Reproduction, 7 Avenue du Général de Gaulle, F-94700 Maisons-Alfort, France
Pierre-Gaël Adenot
Affiliation:
INRA, UMR 1198 Biologie du Développement et Reproduction, F-78350 Jouy en Josas, France
Karine Reynaud
Affiliation:
INRA, UMR 1198 Biologie du Développement et Reproduction, F-78350 Jouy en Josas, France ENVA, UMR 1198 Biologie du Développement et Reproduction, 7 Avenue du Général de Gaulle, F-94700 Maisons-Alfort, France
Sylvie Chastant-Maillard*
Affiliation:
INRA, UMR 1198 Biologie du Développement et Reproduction, F-78350 Jouy en Josas, France ENVA, UMR 1198 Biologie du Développement et Reproduction, 7 Avenue du Général de Gaulle, F-94700 Maisons-Alfort, France ENVA, Reproduction, 7 Avenue du Général de Gaulle, F-94700 Maisons-Alfort, France
*
Corresponding author. E-mail: [email protected]
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Abstract

Due to the marked cytoplasmic opacity of canine oocytes, the diagnosis of their nuclear status is difficult. The objective of the present study was to evaluate the accuracy of Hoechst staining observed under epifluorescence wide-field microscopy [living oocyte observation (LivOO)] by comparison to a reference technique [DNA staining with ethidium homodimer-2 under confocal microscopy; fixed oocyte observation (FixOO)]. Four Hoechst 33342 concentrations (200 ng, 500 ng, 1 μg, 2 μg/mL) were tested and 1 μg/mL was the lowest one with the lowest proportion of oocytes in which DNA was missed. At this concentration, LivOO procedure did not affect the degeneration rate. On 379 oocytes observed individually with the two techniques successively, diagnosis of meiosis resumption by LivOO was exact in 87.3% of the cases, but the meiosis resumption rate was underestimated (23.5% versus 34.3% with FixOO; p < 0.001). Diagnosis for metaphase II was exact in 80% of the cases, but LivOO detected only 72.7% of the metaphase II oocytes present. Metaphase rates did not differ between LivOO and FixOO. This study contributes to a better interpretation of in vitro maturation results. The developmental potential of metaphase II canine oocytes sorted after Hoechst staining is to be evaluated.

Keywords

confocal microscopywide-field microscopyfluorescenceHoechstmeiosisdog oocyte
Type
Biological Applications
Information
Microscopy and Microanalysis , Volume 18 , Issue 3 , June 2012 , pp. 483 - 492
Copyright
Copyright © Microscopy Society of America 2012

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