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Biology of reproduction2009; 81(6); 1106-1111; doi: 10.1095/biolreprod.109.078220

Effect of cryopreservation on nitric oxide production by stallion spermatozoa.

Abstract: 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.
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Publication Date: 2009-07-15 PubMed ID: 19605785DOI: 10.1095/biolreprod.109.078220Google Scholar: Lookup
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  • Journal Article
  • Research Support
  • Non-U.S. Gov't

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

This study investigates how the freezing and thawing process (cryopreservation) affects the ability of stallion sperm cells to produce nitric oxide (NO). It found that cryopreserved stallion sperm cells increase their production of NO, particularly when egg yolk is absent from the extender used in the preservation process. The presence of NO correlates positively with the sperm cells’ motility and velocity after thawing, implying a role for NO in the functionality of cryopreserved sperm, although this is not the case when egg yolk extenders are used.

Investigation Method

  • The researchers utilized flow cytometry to evaluate nitric oxide (NO) production by stallion sperm cells before and after freezing and thawing. To do this, they loaded the sperm suspension with the probe, 4,5-diaminofluorescein diacetate.
  • They further investigated the presence of nitric oxide synthase (NOS), the enzyme responsible for producing NO, using a technique called Western blotting. They used three different antibodies: anti-NOS1, anti-NOS3, and anti-universal NOS.

Findings

  • NO was detectable in both fresh (not frozen) and freeze-thawed sperm. However, they noted an increase in NO production after freezing and thawing, but only when the extender (a solution used to preserve sperm) did not contain egg yolk.
  • The anti-NOS1 antibody labeled a band with an approximate molecular mass of 83 kDa, while the antibody against NOS3 revealed a band of roughly 96 kDa in both fresh and frozen-thawed sperm samples. These findings confirm the presence of NOS in stallion sperm.
  • NO production exhibited a positive correlation with sperm motility and velocity after thawing. The suggests that NO plays a crucial role in the functioning of cryopreserved stallion sperm cells. However, the production of NO is compromised when an egg yolk-containing extender is used.

Implications

  • The findings of this research have important implications for processes involving the cryopreservation of stallion sperm. It suggests that the choice of extender could affect the functionality of the sperm after thawing due to its impact on NO production.
  • This research contributes to the understanding of how cryopreservation affects stallion sperm at a molecular level. Future studies can extend these findings to improve the outcomes of cryopreservation and promote the fertility of thawed sperm.

Cite This Article

APA
Ortega Ferrusola C, González Fernández L, Macías García B, Salazar-Sandoval C, Morillo Rodríguez A, Rodríguez Martinez H, Tapia JA, Peña FJ. (2009). Effect of cryopreservation on nitric oxide production by stallion spermatozoa. Biol Reprod, 81(6), 1106-1111. https://doi.org/10.1095/biolreprod.109.078220

Publication

Biology of reproduction
ISSN: 1529-7268
NlmUniqueID: 0207224
Country: United States
Language: English
Volume: 81
Issue: 6
Pages: 1106-1111

Researcher Affiliations

Ortega Ferrusola, C
  • Veterinary Teaching Hospital, Laboratory of Spermatology, University of Extremadura, Cáceres, Spain.
González Fernández, L
    Macías García, B
      Salazar-Sandoval, C
        Morillo Rodríguez, A
          Rodríguez Martinez, H
            Tapia, J A
              Peña, F J

                MeSH Terms

                • Animals
                • Blotting, Western
                • Cryopreservation
                • Cryoprotective Agents / pharmacology
                • Egg Yolk
                • Flow Cytometry
                • Horses / metabolism
                • Male
                • Microscopy, Confocal
                • Nitric Oxide / metabolism
                • Nitric Oxide Synthase / metabolism
                • Semen / metabolism
                • Semen Preservation
                • Sperm Motility
                • Spermatozoa / drug effects
                • Spermatozoa / metabolism
                • Statistics, Nonparametric

                Citations

                This article has been cited 14 times.
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                2. Pintus E, Ros-Santaella JL. Impact of Oxidative Stress on Male Reproduction in Domestic and Wild Animals. Antioxidants (Basel) 2021 Jul 20;10(7).
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                4. Peña FJ, O'Flaherty C, Ortiz Rodríguez JM, Martín Cano FE, Gaitskell-Phillips GL, Gil MC, Ortega Ferrusola C. Redox Regulation and Oxidative Stress: The Particular Case of the Stallion Spermatozoa. Antioxidants (Basel) 2019 Nov 19;8(11).
                  doi: 10.3390/antiox8110567pubmed: 31752408google scholar: lookup
                5. Len JS, Koh WSD, Tan SX. The roles of reactive oxygen species and antioxidants in cryopreservation. Biosci Rep 2019 Aug 30;39(8).
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                6. Ortiz-Rodriguez JM, Balao da Silva C, Masot J, Redondo E, Gazquez A, Tapia JA, Gil C, Ortega-Ferrusola C, Peña FJ. Rosiglitazone in the thawing medium improves mitochondrial function in stallion spermatozoa through regulating Akt phosphorylation and reduction of caspase 3. PLoS One 2019;14(7):e0211994.
                  doi: 10.1371/journal.pone.0211994pubmed: 31276504google scholar: lookup
                7. Berlinguer F, Pasciu V, Succu S, Cossu I, Caggiu S, Addis D, Castagna A, Fontani V, Rinaldi S, Passino ES. REAC technology as optimizer of stallion spermatozoa liquid storage. Reprod Biol Endocrinol 2017 Feb 8;15(1):11.
                  doi: 10.1186/s12958-017-0229-6pubmed: 28179013google scholar: lookup
                8. Saeednia S, Shabani Nashtaei M, Bahadoran H, Aleyasin A, Amidi F. Effect of nerve growth factor on sperm quality in asthenozoosprmic men during cryopreservation. Reprod Biol Endocrinol 2016 May 27;14(1):29.
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                9. Plaza Davila M, Martin Muñoz P, Tapia JA, Ortega Ferrusola C, Balao da Silva C C, Peña FJ. Inhibition of Mitochondrial Complex I Leads to Decreased Motility and Membrane Integrity Related to Increased Hydrogen Peroxide and Reduced ATP Production, while the Inhibition of Glycolysis Has Less Impact on Sperm Motility. PLoS One 2015;10(9):e0138777.
                  doi: 10.1371/journal.pone.0138777pubmed: 26407142google scholar: lookup
                10. Reiter RJ, Rosales-Corral SA, Manchester LC, Tan DX. Peripheral reproductive organ health and melatonin: ready for prime time. Int J Mol Sci 2013 Apr 2;14(4):7231-72.
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                11. Gallardo Bolaños JM, Miró Morán Á, Balao da Silva CM, Morillo Rodríguez A, Plaza Dávila M, Aparicio IM, Tapia JA, Ortega Ferrusola C, Peña FJ. Autophagy and apoptosis have a role in the survival or death of stallion spermatozoa during conservation in refrigeration. PLoS One 2012;7(1):e30688.
                  doi: 10.1371/journal.pone.0030688pubmed: 22292020google scholar: lookup
                12. Catalán J, Yánez-Ortiz I, Torres-Garrido M, Ribas-Maynou J, Llavanera M, Barranco I, Yeste M, Miró J. Impact of Seminal Plasma Antioxidants on DNA Fragmentation and Lipid Peroxidation of Frozen-Thawed Horse Sperm. Antioxidants (Basel) 2024 Mar 6;13(3).
                  doi: 10.3390/antiox13030322pubmed: 38539855google scholar: lookup
                13. Palacin-Martinez C, Anel-Lopez L, Alvarez M, Neila-Montero M, Montes-Garrido R, Soriano-Úbeda C, de Paz P, Anel L, Riesco MF. The characterization of CellROX™ probes could be a crucial factor in ram sperm quality assessment. Front Vet Sci 2024;11:1342808.
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                14. Uribe P, Barra J, Painen K, Zambrano F, Schulz M, Moya C, Isachenko V, Isachenko E, Mallmann P, Sánchez R. FeTPPS, a Peroxynitrite Decomposition Catalyst, Ameliorates Nitrosative Stress in Human Spermatozoa. Antioxidants (Basel) 2023 Jun 14;12(6).
                  doi: 10.3390/antiox12061272pubmed: 37372002google scholar: lookup
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