Journal of Andrology
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Published-Ahead-of-Print November 1, 2006, 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 Non-Enzymatic Antioxidants

María Rocio Fernández-Santos , Felipe Martínez-Pastor , Vanesa García-Macías , Milagros C Esteso , Ana J Soler , Paulino de Paz , Luis Anel , and José J Garde *

* To whom correspondence should be addressed. E-mail: julian.garde{at}uclm.es.

The main goal of this study was to investigate the protective effect of enzymatic and non-enzymatic antioxidants against cryopreservation injuries to red deer epididymal spermatozoa. In experiment 1, the effects of two enzymatic antioxidants (Catalase, superoxide dismutase, and combination of them) on sperm freezability, were studied. In experiment 2, sperm cryoresistance was evaluated when different non-enzymatic antioxidants (Vitamin E, Vitamin C and butylated hydroxytoluene) were added to the freezing extender. Sperm quality was judged in vitro by microscopic assessments of individual sperm motility (SMI), viability, and acrosome (i.e., spermatozoa with normal apical ridges; % NAR) and membrane (by means of the HOS test) integrities. To fully address these questions, we incorporated a new set of functional sperm tests: tests of mitochondrial function, membrane fluidity and sperm chromatin stability. Sub-samples were evaluated after freezing and thawing, and after a 2 h incubation period at 37°C. Our study demonstrated that the addition of enzymatic antioxidants to the freezing extender improved sperm viability after cooling and also sperm motility, acrosome integrity and mitochondrial status (P < 0.05) after thawing. After a 2 h incubation period at 37°C, an improvement of membrane integrity (P < 0.05) was observed when enzymatic antioxidants where added to the diluent. However, when non-enzymatic antioxidants were presented in the freezing diluents, no differences in sperm parameters were found. Chromatin stability test did not show significant differences among treatments. We conclude that enzymatic antioxidants should be present in the earlier steps of the cryopreservation process for improving motility and acrosome integrity of cryopreserved red deer epididymal spermatozoa.


Key words: Assisted reproduction • Cryopreservation • Sperm • red deer






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