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Activated caspases are present in frozen–thawed canine sperm and may be related to post thaw sperm quality

Published online by Cambridge University Press:  06 May 2009

A. Sokolowska ,
B. Macías García ,
L. González Fernández ,
C. Ortega-Ferrusola ,
J. A. Tapia  and
F. J. Peña
A. Sokolowska
Affiliation:
Veterinary Teaching Hospital, University of Extremadura, Avd de la Universidad, s/n10071Cáceres, Spain.
B. Macías García
Affiliation:
Veterinary Teaching Hospital, University of Extremadura, Avd de la Universidad, s/n10071Cáceres, Spain.
L. González Fernández
Affiliation:
Veterinary Teaching Hospital, University of Extremadura, Avd de la Universidad, s/n10071Cáceres, Spain.
C. Ortega-Ferrusola
Affiliation:
Veterinary Teaching Hospital, University of Extremadura, Avd de la Universidad, s/n10071Cáceres, Spain.
J. A. Tapia
Affiliation:
Veterinary Teaching Hospital, University of Extremadura, Avd de la Universidad, s/n10071Cáceres, Spain.
F. J. Peña*
Affiliation:
Section of Reproduction and Obstetrics, Department of Medicine, Faculty of Veterinary Medicine, Avd de la Universidad, s/n10071Cáceres, Spain. Veterinary Teaching Hospital, University of Extremadura, Avd de la Universidad, s/n10071Cáceres, Spain.
*
All correspondence to: F.J. Peña. Section of Reproduction and Obstetrics, Department of Medicine, Faculty of Veterinary Medicine, Avd de la Universidad, s/n10071Cáceres, Spain. e-mail. fjuanpvega@unex.es
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Summary

The identification of early changes in the sperm plasmalemma is currently a factor in the improvement of freezing protocols. We analysed the presence of active caspases in freeze–thawed (FT) dog spermatozoa, and evaluated straws from eight dogs using flow cytometry and fluorescence microscopy with fluorescein isothyocyanate–Val–Ala–Asp–fluoromethylketone (FITC–VAD–fmk) combined with ethidum homodimers. Apoptotic-like changes were evaluated using the YO–PRO-1/ethidium homodimer combination, and changes in mitochondrial membrane potential were monitored with JC-1. Sperm motility post-thaw was evaluated using a CASA system. FITC–VAD–fmk stained sperm cells in situ and the subcellular labelling pattern was consistent with known localization of caspases. On average, a high proportion of FT canine sperm showed caspase activity, ranging from 30.2 to 70.7% of the live sperm compared with 7.3 to 24.0% in dead spermatozoa. This observed differentiation between caspase activity in dead and live spermatozoa may be a simple method to disclose subtle differences in sperm quality, since this staining allowed us to find statistically significant differences among dogs. Notably, the sperm sample with overall better results in all sperm parameters studied after thawing had a lower percentage of active caspases in both dead and live spermatozoa.

Keywords

ApoptosisCanineCaspasesFlow cytometryFreezingMitochondriaSperm
Type
Research Article
Information
Zygote , Volume 17 , Issue 4 , November 2009 , pp. 297 - 305
Copyright
Copyright © Cambridge University Press 2009

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