Osmotic tolerance limits and membrane permeability characteristics of stallion spermatozoa treated with cholesterol.
Abstract: Stallion spermatozoa exhibit osmotic damage during the cryopreservation process. Recent studies have shown that the addition of cholesterol to spermatozoal membranes increases the cryosurvival of bull, ram and stallion spermatozoa, but the exact mechanism by which added cholesterol improves cryosurvival is not understood. The objectives of this study were to determine if adding cholesterol to stallion sperm membranes alters the osmotic tolerance limits and membrane permeability characteristics of the spermatozoa. In experiment one, stallion spermatozoa were treated with cholesterol-loaded cyclodextrin (CLC), subjected to anisotonic solutions and spermatozoal motility analyzed. The spermatozoa were then returned to isotonic conditions and the percentages of motile spermatozoa again determined. CLC treatment increased the osmotic tolerance limit of stallion spermatozoa in anisotonic solutions and when returned to isotonic conditions. The second and third experiments utilized an electronic particle counter to determine the plasma membrane characteristics of stallion spermatozoa. In experiment two, stallion spermatozoa were determined to behave as linear osmometers. In experiment three, spermatozoa were treated with CLC, incubated with different cryoprotectants (glycerol, ethylene glycol or dimethyl formamide) and their volume excursions measured during cryoprotectant removal at 5 degrees and 22 degrees C. Stallion spermatozoa were less permeable to the cryoprotectants at 5 degrees C than 22 degrees C. Glycerol was the least permeable cryoprotectant in control cells. The addition of CLC's to spermatozoa increased the permeability of stallion spermatozoa to the cryoprotectants. Therefore, adding cholesterol to spermatozoal membranes reduces the amount of osmotic stress endured by stallion spermatozoa during cryopreservation.
Publication Date: 2009-07-29 PubMed ID: 19646432DOI: 10.1016/j.cryobiol.2009.07.009Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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This research investigates how adding cholesterol to spermatozoal membranes of stallion spermatozoa may affect its osmotic tolerance limits and membrane permeability characteristics. Their findings suggest that this treatment could potentially reduce osmotic stress during cryopreservation, improving the survival rate of the spermatozoa.
Cholesterol Treatment of Stallion Spermatozoa
- The study seeks to understand the potential effects of adding cholesterol to stallion spermatozoa during the cryopreservation process, a domain still filled with many unknown aspects. Previous research has acknowledged that osmotic damage is a common occurrence for spermatozoa in this process. Adding cholesterol to these membranes has shown increased cryosurvival in bull, ram, and stallion spermatozoa. However, the precise mechanics behind this improvement are currently unknown.
Experiment One: Analysis of Osmotic Tolerance
- The first experiment treated stallion spermatozoa with cholesterol-loaded cyclodextrin (CLC), subjected them to anisotonic solutions, and analyzed spermatozoal motility. After treatment, the spermatozoa were returned to isotonic conditions and the percentages of motile spermatozoa assessed. The results showed that CLC treatment increased the osmotic tolerance limit of the spermatozoa both in anisotonic solutions and upon returning to isotonic conditions.
Experiment Two and Three: Examination of Membrane Permeability
- The two subsequent experiments utilized an electronic particle counter to further understand the changing properties of stallion spermatozoa with cholesterol treatment. It was determined through the second experiment that without cholesterol treatment, stallion spermatozoa behave as linear osmometers.
- In the third experiment, spermatozoa were treated with CLC, exposed to different cryoprotectants, and their volume excursions were measured during cryoprotectant removal at two temperatures. Researchers discovered that the permeability of the spermatozoa in regards to the cryoprotectants was lower at 5 degrees Celsius than at 22 degrees Celsius. Among control cells, glycerol proved to be the least permeable cryoprotectant.
Conclusion and Implications
- The addition of CLC to the spermatozoa substantively increased its permeability to cryoprotectants, suggesting that including cholesterol in spermatozoal membranes could diminish the amount of osmotic stress experienced by stallion spermatozoa during cryopreservation. These findings present an improvement in the cryosurvival rate, which could have meaningful applications in the realm of livestock production and animal conservation.
Cite This Article
APA
Glazar AI, Mullen SF, Liu J, Benson JD, Critser JK, Squires EL, Graham JK.
(2009).
Osmotic tolerance limits and membrane permeability characteristics of stallion spermatozoa treated with cholesterol.
Cryobiology, 59(2), 201-206.
https://doi.org/10.1016/j.cryobiol.2009.07.009 Publication
Researcher Affiliations
- Department of Biomedical Science, Colorado State University, Fort Collins, CO 80523, USA.
MeSH Terms
- Animals
- Cell Membrane Permeability / drug effects
- Cholesterol / pharmacology
- Cryopreservation / veterinary
- Cryoprotective Agents / pharmacology
- Cyclodextrins
- Dimethylformamide / pharmacology
- Horses
- Male
- Osmotic Pressure
- Semen Preservation / veterinary
- Sperm Motility / drug effects
- Spermatozoa / drug effects
- Spermatozoa / metabolism
- Temperature
Citations
This article has been cited 14 times.- Barbosa BB, Evangelista ITA, Soares ARB, Leão DL, Pereira RJG, Domingues SFS. Kinetic vitrification: concepts and perspectives in animal sperm cryopreservation. Anim Reprod 2023;20(2):e20220096.
- 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).
- Swelum AA, Ba-Awadh HA, Olarinre IO, Saadeldin IM, Alowaimer AN. Effects of adding mixed chicken and quail egg yolks to the cryodiluent on the quality of ram semen before and after cryopreservation. Front Vet Sci 2022;9:1013533.
- Warner RM, Ampo E, Nelson D, Benson JD, Eroglu A, Higgins AZ. Rapid quantification of multi-cryoprotectant toxicity using an automated liquid handling method. Cryobiology 2021 Feb;98:219-232.
- Benson JD. Mathematical Modeling and Optimization of Cryopreservation in Single Cells. Methods Mol Biol 2021;2180:129-172.
- Benson JD, Higgins AZ, Desai K, Eroglu A. A toxicity cost function approach to optimal CPA equilibration in tissues. Cryobiology 2018 Feb;80:144-155.
- Bera A, Sawant PB, Dasgupta S, Chadha NK, Sawant BT, Pal AK. Diel cyclic hypoxia alters plasma lipid dynamics and impairs reproduction in goldfish (Carassius auratus). Fish Physiol Biochem 2017 Dec;43(6):1677-1688.
- Zhao G, Fu J. Microfluidics for cryopreservation. Biotechnol Adv 2017 Mar-Apr;35(2):323-336.
- Benson JD, Benson CT, Critser JK. Mathematical model formulation and validation of water and solute transport in whole hamster pancreatic islets. Math Biosci 2014 Aug;254:64-75.
- Blanch E, Tomás C, Hernández M, Roca J, Martínez EA, Vázquez JM, Mocé E. Egg yolk and glycerol requirements for freezing boar spermatozoa treated with methyl β-cyclodextrin or cholesterol-loaded cyclodextrin. J Reprod Dev 2014 Apr 24;60(2):143-9.
- Kim JS, Park DS, Park JK, Lee JE, Moon JC, Do Y. Optimizing Sperm Cryopreservation from Four Endangered Korean Amphibian Species: Species-Specific Effects of Cryoprotectants and Cooling Regimes on Membrane-Integrity Viability. Animals (Basel) 2025 Oct 17;15(20).
- Ahmadkhani N, Sugden C, Benson JD, Eroglu A, Higgins AZ. High-throughput evaluation of cryoprotective agents for mixture effects that reduce toxicity. Cryobiology 2025 Dec;121:105316.
- Ahmadkhani N, Sugden C, Benson JD, Eroglu A, Higgins AZ. High-Throughput Evaluation of Cryoprotective Agents for Mixture Effects That Reduce Toxicity. bioRxiv 2025 May 8;.
- Ahmet E, Ramazan A, Selin Y, İzem SA, Nur E, Kamber D, Mithat E, Kemal A. The Effect of Cholesterol-Loaded Cyclodextrin and Resveratrol Compounds on Post-Thawing Quality of Ram Semen. Vet Med Sci 2025 Jan;11(1):e70172.
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