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Research article - Peer-reviewed, 2009

Effect of Cryopreservation on Nitric Oxide Production by Stallion Spermatozoa

Ortega Ferrusola, C.; Gonzalez Fernandez, L.; Macias Garcia, B.; Salazar-Sandoval, C.; Morillo Rodriguez, A.; Rodriguez Martinez, H.; Tapia, J. A.; Pena, F. J.


The ability of stallion spermatozoa to produce nitric oxide (NO) before (fresh) and after freezing and thawing (FT) was evaluated by means of flow cytometry after loading the sperm suspension with the probe, 4,5-diaminofluorescenin diacetate. The presence of NO synthase (NOS) was investigated by Western blotting using anti-NOS1, anti-NOS3, or anti-universal NOS antibodies (Abs). While NO was detected both in fresh and FT sperm suspensions, its production increased after cryopreservation only when egg yolk was removed from the extender. Anti-NOS1 Ab intensively labeled a single band with an apparent molecular mass of approximately 83 kDa. On the other hand, the Ab developed against the NOS3 showed a band of approximately 96 kDa in fresh and FT sperm lysates. NO production was positively correlated with sperm motility and velocity after thaw, suggesting an NO role for the functionality of cryopreserved stallion spermatozoa; but the production of NO is compromised in egg yolk-containing extenders.


assisted reproductive technology; cryopreservation; flow cytometry; gamete biology; nitric oxide; nitric oxide synthase; sperm; stallion

Published in

Biology of Reproduction
2009, Volume: 81, number: 6, pages: 1106-1111

    UKÄ Subject classification

    Animal and Dairy Science

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