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Assessment of the cryopreservation of equine spermatozoa in the presence of enzyme scavengers and antioxidants.
Abstract: 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. Methods: 2 ejaculates collected from each of 5 stallions. Methods: 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: 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.
Publication Date: 2005-06-07 PubMed ID: 15934604DOI: 10.2460/ajvr.2005.66.772Google Scholar: Lookup The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Comparative Study
- Evaluation Study
- Journal Article
- Research Support
- Non-U.S. Gov't
- Research Support
- U.S. Gov't
- Non-P.H.S.
Summary
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This study evaluated how the addition of enzyme scavengers and antioxidants to a sperm cryopreservation formula impacts equine sperm quality post-freezing and thawing. The researchers found that these added substances did not improve sperm health or functionality, suggesting that more research is necessary to understand how oxidative stress relates to sperm damage during cryopreservation.
Experiment Methodology
- The study was conducted with the sperm of five stallions, with two ejaculates taken from each animal.
- The collected sperm was cryopreserved in a freezing extender, either alone as control samples or with various amounts of catalase, superoxide dismutase, reduced glutathione, ascorbic acid, alpha-tocopherol, or ethanol (used as a vehicle for alpha-tocopherol).
- After thawing, various assessments were performed on the sperm: computer-assisted analysis for motility, the comet assay for DNA fragmentation, and specific staining techniques and flow cytometry for the detection of mitochondrial membrane potential, acrosomal integrity, and viability.
Results and Observations
- The researchers found that there was no notable improvement in the sperm quality in terms of motility, DNA fragmentation, mitochondrial membrane potential, viability, or acrosomal integrity from the addition of enzyme scavengers and antioxidants.
- In fact, the introduction of superoxide dismutase seemed to increase DNA fragmentation because of additional oxidative stress generated from the creation of hydrogen peroxide by the enzyme.
- Indeed, the surprising outcome was that adding ethanol, the vehicle for alpha-tocopherol, led to a significant decline in the quantity of live sperm with intact acrosomes.
Conclusions and Future Recommendations
- Despite the popular belief that antioxidants might help improve the quality of cryopreserved sperm, this study found no such benefits in the case of equine spermatozoa. This implies that a better understanding of the role of oxidative stress in sperm damage during cryopreservation is required.
- Interestingly, the study found that the application of ethanol, carrying alpha-tocopherol, had a negative effect on the viability of spermatozoa. This concludes that for future studies and potential improvements of the process, water-soluble analogues of alpha-tocopherol might be a preferable choice.
Cite This Article
APA
Baumber J, Ball BA, Linfor JJ.
(2005).
Assessment of the cryopreservation of equine spermatozoa in the presence of enzyme scavengers and antioxidants.
Am J Vet Res, 66(5), 772-779.
https://doi.org/10.2460/ajvr.2005.66.772 Publication
Researcher Affiliations
- Department of Population Health and Reproduction, School of Veterinary Medicine, University of California, Davis, CA 95616, USA.
MeSH Terms
- Animals
- Antioxidants
- Catalase
- Cryopreservation / veterinary
- Dose-Response Relationship, Drug
- Free Radical Scavengers
- Horses
- Male
- Oxidative Stress
- Semen Preservation / methods
- Semen Preservation / veterinary
- Sperm Motility
- Spermatozoa / physiology
- Superoxide Dismutase
Citations
This article has been cited 12 times.- Medica AJ, Aitken RJ, Nicolson GL, Sheridan AR, Swegen A, De Iuliis GN, Gibb Z. Glycerophospholipids protect stallion spermatozoa from oxidative damage in vitro. Reprod Fertil 2021 Jul;2(3):199-209.
- Asadpour R, Taravat M, Rahbar M, Khoshniyat M, Hamidian G. Effects of vitamin D supplementation in extender on sperm kinematics and apoptosis following the freeze-thaw process in normozoospermic and asthenozoospermic Holstein bulls. Basic Clin Androl 2021 Aug 5;31(1):20.
- Al-Khaldi K, Yimer N, Al-Bulushi S, Haron AW, Hiew M, Babji AS. A preliminary study on the effects of E-Z Mixin® and EquiPlus® extenders supplemented with Edible Bird's Nest on the quality of chilled Arabian stallion semen. Anim Reprod 2021 Jun 21;18(2):e20200027.
- Qamar AY, Fang X, Kim MJ, Cho J. Myoinositol Supplementation of Freezing Medium Improves the Quality-Related Parameters of Dog Sperm. Animals (Basel) 2019 Nov 27;9(12).
- Heidari Nasirabadi M, Shirazi A, Kadivar A, Shams-Esfandabadi N, Mohebbi A, Ahmadi E. Sericin Ameliorates the Capacitation State and Chromatin Integrity of Frozen-Thawed Stallion Spermatozoa by Reducing Oxidative Stress. Avicenna J Med Biotechnol 2019 Jul-Sep;11(3):245-252.
- Al-Otaibi NAS, Cassoli JS, Martins-de-Souza D, Slater NKH, Rahmoune H. Human leukemia cells (HL-60) proteomic and biological signatures underpinning cryo-damage are differentially modulated by novel cryo-additives. Gigascience 2019 Mar 1;8(3).
- Gibb Z, Aitken RJ. The Impact of Sperm Metabolism during In Vitro Storage: The Stallion as a Model. Biomed Res Int 2016;2016:9380609.
- Lohiya NK, Alam I, Hussain M, Khan SR, Ansari AS. RISUG: an intravasal injectable male contraceptive. Indian J Med Res 2014 Nov;140 Suppl(Suppl 1):S63-72.
- Martorana K, Klooster K, Meyers S. Suprazero cooling rate, rather than freezing rate, determines post thaw quality of rhesus macaque sperm. Theriogenology 2014 Feb;81(3):381-8.
- McCarthy MJ, Meyers SA. Antioxidant treatment in the absence of exogenous lipids and proteins protects rhesus macaque sperm from cryopreservation-induced cell membrane damage. Theriogenology 2011 Jul 1;76(1):168-76.
- Li SF, Liu HX, Zhang YB, Yan YC, Li YP. The protective effects of alpha-ketoacids against oxidative stress on rat spermatozoa in vitro. Asian J Androl 2010 Mar;12(2):247-56.
- McCarthy MJ, Baumber J, Kass PH, Meyers SA. Osmotic stress induces oxidative cell damage to rhesus macaque spermatozoa. Biol Reprod 2010 Mar;82(3):644-51.
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