Journal of Andrology
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Published-Ahead-of-Print November 1, 2006, DOI:10.2164/jandrol.106.000935
Journal of Andrology, Vol. 28, No. 2, March/April 2007
Copyright © American Society of Andrology
DOI: 10.2164/jandrol.106.000935

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Sperm Characteristics and DNA Integrity of Iberian Red Deer (Cervus elaphus hispanicus) Epididymal Spermatozoa Frozen in the Presence of Enzymatic and Nonenzymatic Antioxidants

MARÍA R. FERNÁNDEZ-SANTOS*,{dagger}, FELIPE MARTÍNEZ-PASTOR*,{ddagger}, VANESA GARCÍA-MACÍAS{ddagger}, MILAGROS C. ESTESO*,{dagger}, ANA J. SOLER*,{dagger}, P. PAZ{ddagger}, L. ANEL{ddagger} AND JOSÉ J. GARDE*,{dagger}

From the * Biology of Reproduction Group, Department of Game Resources (IDR), Castilla-La Mancha University (UCLM), Albacete, Spain; the {dagger} National Wildlife Research Institute (IREC), UCLM-CSIC-JCCM, Albacete, Spain; and the {ddagger} Animal Reproduction Group, Faculty of Veterinary Medicine, León University, León, Spain.

Correspondence to: José Julián Garde, Instituto de Investigación en Recursos Cinegéticos (IREC), Campus Universitario sn, 02071-Albacete, Spain (e-mail: Julian.Garde{at}uclm.es).


The main goal of this study was to investigate the potential protective effects of enzymatic and nonenzymatic antioxidants on cryopreservation injuries to red deer epididymal spermatozoa. In Experiment 1, the effects on sperm freezability of the enzymatic antioxidants catalase, superoxide dismutase, and a combination thereof were studied. In Experiment 2, sperm cryoresistance was evaluated when different nonenzymatic antioxidants, such as vitamin E, vitamin C, and butylated hydroxytoluene (BHT), were added to the freezing extender. Sperm quality was judged in vitro by microscopic assessments of individual sperm motility (SMI), viability, and acrosome (ie, spermatozoa with normal apical ridges; % NAR) and membrane (by means of the HOS test) integrity. To address fully these topics, we incorporated a new set of functional sperm tests for mitochondrial function, membrane phospholipid disorder, and sperm chromatin stability. Samples were evaluated after freezing and thawing, and after a 2-hour period of incubation at 37°C. The present study demonstrates that the addition of enzymatic antioxidants to freezing extenders improves sperm viability after cooling, and improves sperm motility, acrosome integrity, and mitochondrial status (P < .05) after thawing. After a 2-hour incubation period at 37°C in the presence of enzymatic antioxidants, an improvement in membrane integrity (P < .05) was observed. However, when nonenzymatic antioxidants were present in the freezing diluents, no positive effects on thawed sperm parameters were noted. The chromatin stability test did not show significant differences between the treatments. We conclude that enzymatic antioxidants should be present in the early steps of cryopreservation of epididymal spermatozoa from red deer, so as to improve motility and acrosome integrity.

     Key words: Cryopreservation, fertilization, gamete biology, reproductive technology






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