X-Message-Number: 27650
Date: Thu, 23 Feb 2006 22:03:53 -0800 (PST)
From: Doug Skrecky <>
Subject: antioxidants useless for sperm cryopreservation

Am J Vet Res. 2005 May;66(5):772-9.
Assessment of the cryopreservation of equine spermatozoa in the
presence of enzyme scavengers and antioxidants.
  OBJECTIVE: To evaluate the effect of the addition of enzyme
scavengers and antioxidants to the cryopreservation extender on
characteristics of equine spermatozoa after freezing and thawing.
SAMPLE POPULATION: 2 ejaculates collected from each of 5 stallions.
PROCEDURE: Equine spermatozoa were cryopreserved in freezing
extender alone (control samples) or with the addition of catalase
(200 U/mL), superoxide dismutase (200 U/mL), reduced glutathione
(10 mM), ascorbic acid (10 mM), alpha-tocopherol (25, 50, 100, or
500 microM or 1 mM), or the vehicle for alpha-tocopherol
(0.5% ethanol). After thawing, spermatozoal motility was assessed
via computer-assisted analysis and DNA fragmentation was assessed
via the comet assay. Spermatozoal mitochondrial membrane potential,
acrosomal integrity, and viability were determined by use of various
specific staining techniques and flow cytometry. RESULTS: The
addition of enzyme scavengers or antioxidants to cryopreservation
extender did not improve spermatozoal motility, DNA fragmentation,
acrosomal integrity, viability, or mitochondrial membrane potential
after thawing. Superoxide dismutase increased DNA fragmentation,
likely because of the additional oxidative stress caused by the
generation of hydrogen peroxide by this enzyme. Interestingly, the
addition of the vehicle for alpha-tocopherol resulted in a
significant decrease in live acrosome-intact spermatozoa.
CONCLUSIONS AND CLINICAL RELEVANCE: The addition of antioxidants to
the cryopreservation extender did not improve the quality of equine
spermatozoa after thawing, which suggests that the role of oxidative
stress in cryopreservation-induced damage of equine spermatozoa
requires further investigation. Our data suggest that solubilizing
alpha-tocopherol in ethanol may affect spermatozoal viability;
consequently, water-soluble analogues of alpha-tocopherol may be
preferred for future investigations.

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