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Detection of "Apoptosis-Like" Changes During the Cryopreservation Process in Equine Sperm

L. GONZÁLEZ-FERNÁNDEZ

J. A. TAPIA

From the ^* Veterinary Teaching Hospital, Laboratory of Spermatology, and the Department of Physiology, Faculty of Veterinary Medicine, University of Extremadura, Cáceres, Spain.

Correspondence to: Dr F. J. Peña, Section of Reproduction and Obstetrics, Department of Herd Health and Medicine, Faculty of Veterinary Medicine, Avd de la Universidad, s/n 10071 Cáceres, Spain (e-mail: fjuanpvega{at}unex.es).

The kinematics of the appearance of apoptotic markers was studied by flow cytometry and immunoblot assays in equine spermatozoa subjected to freezing and thawing. Caspase activity, low mitochondrial membrane potential, and increases in sperm membrane permeability were observed in all of the phases of the cryopreservation procedure. Freezing and thawing caused an increase in membrane permeability and changes in the pattern of caspase activity; decreases in mitochondrial membrane potential were observed after centrifugation and cooling to 4°C and after freezing and thawing. It is proposed that sperm mitochondria may be directly involved in the subtle damage that is present in most spermatozoa surviving freezing and thawing.

     Key words: JC-1, YO-PRO-1, caspases, flow cytometry

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