Freezing, Vitrification, and Freeze-Drying of Equine Spermatozoa: Impact on Mitochondrial Membrane Potential, Lipid Peroxidation, and DNA Integrity.
Abstract: Maintaining the integrity of equine sperm subjected to preservation protocols is essential for the successful development of assisted reproduction procedures. The aim of this study was to assess the mitochondrial membrane potential, lipid peroxidation, and DNA integrity of equine sperm subjected to freezing, vitrification, and freeze-drying. Eight ejaculates obtained from four Colombian Creole horses were subjected to programmable freezing, vitrification, and freeze-drying. After thawing or rehydration, sperm motility and kinetics were assessed through a CASA system. The mitochondrial membrane potential (ΔΨM), lipid peroxidation (LPO), and DNA fragmentation index (DFI) of the spermatozoa were assessed by flow cytometry using the DiOC6 (3), C11-Bodipy 581/591, and propidium iodide (PI) fluorescent dyes. The statistical analysis was conducted via generalized linear models, mean comparisons via the Duncan test, and a principal component analysis. A higher rate of spermatozoa with a high ΔΨM was found for freeze-drying (40.26 ± 7.79%) compared with freezing (21.82 ± 5.38%) and vitrification (5.32 ± 1.17%) (P < .05). Likewise, a higher rate of nonperoxidized viable spermatozoa (Bodipy-/PI-) was found for freeze-drying (35.98 ± 7.01%) in relation to frozen (10.34 ± 2.69%) and vitrified (7.07 ± 2.00%) sperm (P < .05). The DFI of vitrified spermatozoa (0.12 ± 0.04%) was higher when compared with the frozen (0.03 ± 0.01%) and freeze-dried (0.02 ± 0.01%) samples (P < .05). The researchers conclude that vitrification generates greater sperm alterations than freeze-drying and freezing, whereas freeze-drying produces lower LPO and higher ΔΨM for equine spermatozoa.
Copyright © 2018 Elsevier Inc. All rights reserved.
Publication Date: 2018-10-11 PubMed ID: 30929788DOI: 10.1016/j.jevs.2018.10.006Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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This research article investigates the effects of different sperm preservation methods on the health and vitality of equine sperm. The key finding is that freeze-drying preservation appears to result in less damage to the sperm than the other methods tested.
Objective and Methodology
The aim of the study was to evaluate the impact of three different preservation methods – freezing, vitrification, and freeze-drying – on various aspects of equine sperm health. These aspects were:
- the mitochondrial membrane potential (a measure of the health and functionality of the sperm cell’s mitochondria)
- the extent of lipid peroxidation (a process that can damage cell membranes)
- DNA integrity (preservation of the genetic material within the sperm cell).
The researchers obtained eight ejaculates from four Colombian Creole horses for the study. Each sample was subjected to the three preservation methods and then analyzed for the aspects listed above.
Results
The results showed that, compared to the other two methods, freeze-drying:
- resulted in a higher proportion of spermatozoa with a high mitochondrial membrane potential.
- yielded a higher rate of viable, non-peroxidized spermatozoa, suggesting that it caused less damage to the cell membrane.
However, all three methods resulted in some DNA fragmentation, with vitrified sperm having the highest DNA fragmentation index.
Conclusion
The study concluded that of the preservation methods tested, vitrification led to the most damage in the equine sperm, as evidenced by the higher DNA fragmentation. Freeze-drying, on the other hand, resulted in less lipid peroxidation and maintained higher mitochondrial membrane potential, suggesting it might be the least damaging of the three methods tested. These findings could have important implications for the field of equine assisted reproduction, providing valuable information for selecting the most appropriate preservation method.
Cite This Article
APA
Restrepo G, Varela E, Duque JE, Gómez JE, Rojas M.
(2018).
Freezing, Vitrification, and Freeze-Drying of Equine Spermatozoa: Impact on Mitochondrial Membrane Potential, Lipid Peroxidation, and DNA Integrity.
J Equine Vet Sci, 72, 8-15.
https://doi.org/10.1016/j.jevs.2018.10.006 Publication
Researcher Affiliations
- Department of Animal Sciences, Faculty of Agricultural Sciences, Universidad Nacional de Colombia, Medellín, Colombia. Electronic address: grestre0@unal.edu.co.
- Faculty of Agricultural Sciences, Politécnico Colombiano Jaime Isaza Cadavid, Medellín, Colombia.
- Faculty of Agricultural Sciences, Politécnico Colombiano Jaime Isaza Cadavid, Medellín, Colombia.
- Faculty of Agricultural Sciences, Politécnico Colombiano Jaime Isaza Cadavid, Medellín, Colombia.
- Institute of Medical Research, Universidad de Antioquia, Medellín, Colombia.
MeSH Terms
- Animals
- Cryopreservation / veterinary
- DNA
- Freezing
- Horses
- Humans
- Lipid Peroxidation
- Male
- Membrane Potential, Mitochondrial
- Semen Preservation / veterinary
- Sperm Motility
- Spermatozoa
- Vitrification
Citations
This article has been cited 9 times.- Levano G, Quispe J, Vargas D, García M, López A, Aguila L, Valdivia M. Effect of Atomized Black Maca (Lepidium meyenii) Supplementation in the Cryopreservation of Alpaca (Vicugna pacos) Epididymal Spermatozoa. Animals (Basel) 2023 Jun 21;13(13).
- Sharafi M, Borghei-Rad SM, Hezavehei M, Shahverdi A, Benson JD. Cryopreservation of Semen in Domestic Animals: A Review of Current Challenges, Applications, and Prospective Strategies. Animals (Basel) 2022 Nov 24;12(23).
- Bossi RL, Cabral M, Oliveira M, Lopes S, Hurtado R, Sampaio M, Geber S. Ultrastructural analysis of Lyophilized Human Spermatozoa. JBRA Assist Reprod 2021 Jul 21;25(3):473-479.
- Palazzese L, Anzalone DA, Turri F, Faieta M, Donnadio A, Pizzi F, Pittia P, Matsukawa K, Loi P. Whole genome integrity and enhanced developmental potential in ram freeze-dried spermatozoa at mild sub-zero temperature. Sci Rep 2020 Nov 2;10(1):18873.
- Strassner FM, Demattio L, Siuda M, Malama E, Muffels G, Bollwein H. Relationships Between Metabolism of Cryopreserved Equine Sperm Determined by the Seahorse Analyzer and Sperm Characteristics Measured by Flow Cytometry and Computer-Assisted Analysis of Motility. Vet Sci 2025 Nov 21;12(12).
- Onochie C, Evi K, O'Flaherty C. Role of Redox-Induced Protein Modifications in Spermatozoa in Health and Disease. Antioxidants (Basel) 2025 Jun 12;14(6).
- Esmaeili A, Esmaeili V, Shahverdi A, Eslaminejad MB. Engineered extracellular vesicles: a breakthrough approach to overcoming sperm cryopreservation challenges. Reprod Biol Endocrinol 2025 May 21;23(1):75.
- Zhang LX, Mao J, Zhou YD, Mao GY, Guo RF, Ge HS, Chen X. Evaluation of microRNA expression profiles in human sperm frozen using permeable cryoprotectant-free droplet vitrification and conventional methods. Asian J Androl 2024 Jul 1;26(4):366-376.
- Al-Kass Z, Morrell JM. Freezing Stallion Semen-What Do We Need to Focus on for the Future?. Vet Sci 2024 Feb 2;11(2).
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