Toxicity of glycerol for the stallion spermatozoa: effects on membrane integrity and cytoskeleton, lipid peroxidation and mitochondrial membrane potential.
Abstract: Glycerol is, to date, the most widely used cryoprotectant to freeze stallion spermatozoa at concentrations between 2% and 5%. Cryoprotectant toxicity has been claimed to be the single most limiting factor for the success of cryopreservation. In order to evaluate the toxic effects of the concentrations of glycerol used in practice, stallion spermatozoa were incubated in Biggers Whitten and Whittingham (BWW) media supplemented with 0%, 0.5%, 1.5%, 2.5%, 3.5%, and 5% glycerol. In two additional experiments, a hyposmotic (75 mOsm/kg) and a hyperosmotic (900 mOsm/kg) control media were included. Sperm parameters evaluated included cell volume, membrane integrity, lipid peroxidation, caspase 3, 7, and 8 activation, mitochondrial membrane potential, and integrity of the cytoskeleton. Glycerol exerted toxicity at concentrations ≥ 3.5% and the maximal toxicity was observed at 5%. The actin cytoskeleton was especially sensitive to glycerol presence, inducing rapid F actin depolymerization at concentrations over 1.5%. The sperm membrane and the mitochondria were other structures affected. The toxicity of glycerol is apparently related to osmotic and nonosmotic effects. In view of our results the concentration of glycerol in the freezing media for stallion spermatozoa should not surpass 2.5%.
Copyright © 2012 Elsevier Inc. All rights reserved.
Publication Date: 2012-02-10 PubMed ID: 22326587DOI: 10.1016/j.theriogenology.2011.10.033Google 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.
- 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.
The research tests the effects of glycerol, a common substance used to freeze stallion sperm, on the sperm’s structures and parameters, recommending that the concentration used should not be more than 2.5%.
Research Background
- The study is centered around the use of glycerol as a cryoprotectant to freeze stallion sperm. Cryoprotectants are substances used to protect biological tissue during freezing. In the case of glycerol, it is currently the most widely used cryoprotectant for such sperm, typically at concentrations between 2% and 5%.
- The purpose of the research is to ascertain the potential toxic effects of these glycerol concentrations impacting the integrity and functionality of the sperm. The impact of cryoprotectant toxicity is regarded as the most significant factor influencing the success of cryopreservation procedures.
Research Methodology
- To identify toxicity levels, sperm were incubated in BWW media with various concentrations of glycerol (0%, 0.5%, 1.5%, 2.5%, 3.5%, 5%).
- In addition, two tests were carried out with hyposmotic and hyperosmotic control media.
- Key sperm parameters were monitored, including cell volume, membrane integrity, lipid peroxidation, activation levels of caspase 3, 7 and 8, mitochondrial membrane potential, and cytoskeleton integrity.
Research Findings
- The study found that glycerol exhibited toxicity at concentrations equal to and above 3.5%, peaking at 5% concentration. This toxicity affected several sperm structures, including the membrane and mitochondria.
- The actin cytoskeleton was particularly sensitive to glycerol. Concentrations above 1.5% rapidly induced depolymerization of F actin, a part of the cytoskeleton structure.
- The research proposed that glycerol’s toxicity could be attributed to both osmotic effects (the movement of water in and out of the cell, impacted by the glycerol concentration), and non-osmotic effects, though further research would be needed to specify these.
Research Implications
- In light of the results, the researchers recommend that glycerol concentrations in stallion sperm freezing procedures should not exceed 2.5%.
- This advice is based on the observed effects of higher concentrations on sperm structure and function, potentially impacting the success of cryopreservation.
Cite This Article
APA
Macías García B, Ortega Ferrusola C, Aparicio IM, Miró-Morán A, Morillo Rodriguez A, Gallardo Bolaños JM, González Fernández L, Balao da Silva CM, Rodríguez Martínez H, Tapia JA, Peña FJ.
(2012).
Toxicity of glycerol for the stallion spermatozoa: effects on membrane integrity and cytoskeleton, lipid peroxidation and mitochondrial membrane potential.
Theriogenology, 77(7), 1280-1289.
https://doi.org/10.1016/j.theriogenology.2011.10.033 Publication
Researcher Affiliations
- Veterinary Teaching Hospital, Laboratory of Equine Reproduction, Faculty of Veterinary Medicine, University of Extremadura, Cáceres, Spain.
MeSH Terms
- Actins / metabolism
- Animals
- Caspase 3 / metabolism
- Caspase 7 / metabolism
- Caspase 8 / metabolism
- Cell Membrane / drug effects
- Cryopreservation / veterinary
- Cryoprotective Agents / toxicity
- Cytoskeleton / drug effects
- Cytoskeleton / ultrastructure
- Glycerol / toxicity
- Horses
- Lipid Peroxidation / drug effects
- Male
- Membrane Potential, Mitochondrial / drug effects
- Osmotic Pressure
- Spermatozoa / drug effects
Citations
This article has been cited 10 times.- Dziekońska A, Neuman NM, Burdal KK, Wiszniewska-Łaszczych A, Bogdaszewski M. The Effect of Different Extenders on the Quality Characteristics of European Red Deer Epididymal Sperm Stored at 5 °C.. Animals (Basel) 2022 Oct 4;12(19).
- Akhtar MF, Ma Q, Li Y, Chai W, Zhang Z, Li L, Wang C. Effect of Sperm Cryopreservation in Farm Animals Using Nanotechnology.. Animals (Basel) 2022 Sep 2;12(17).
- Ma L, Kim DH, Jung EJ, Lee WJ, Hwang JM, Bae JW, Jung DJ, Yi JK, Lee SM, Ha JJ, Kwon WS. Effect of glycerol addition time on the cryopreserved Korean native brindle cattle (Chikso) sperm quality.. Anim Reprod 2022;19(1):e20210058.
- Takeuchi H, Nishioka M, Maezawa T, Kitano Y, Terada-Yoshikawa K, Tachibana R, Kato M, Hyon SH, Gen Y, Tanaka K, Toriyabe K, Nii M, Kondo E, Ikeda T. Carboxylated Poly-l-Lysine as a Macromolecular Cryoprotective Agent Enables the Development of Defined and Xeno-Free Human Sperm Cryopreservation Reagents.. Cells 2021 Jun 8;10(6).
- Lançoni R, Celeghini ECC, Giuli V, de Carvalho CPT, Zoca GB, Garcia-Oliveros LN, Batissaco L, Oliveira LZ, de Arruda RP. Coenzyme Q-10 improves preservation of mitochondrial functionality and actin structure of cryopreserved stallion sperm.. Anim Reprod 2021 Mar 31;18(1):e20200218.
- Engel KM, Schiller J, Müller K, Dannenberger D, Jakop U. The Phospholipid Composition of Kangaroo Spermatozoa Verified by Mass Spectrometric Lipid Analysis.. Lipids 2017 Oct;52(10):857-869.
- Yoon SJ, Rahman MS, Kwon WS, Park YJ, Pang MG. Addition of Cryoprotectant Significantly Alters the Epididymal Sperm Proteome.. PLoS One 2016;11(3):e0152690.
- Best BP. Cryoprotectant Toxicity: Facts, Issues, and Questions.. Rejuvenation Res 2015 Oct;18(5):422-36.
- Ros-Santaella JL, Domínguez-Rebolledo AE, Garde JJ. Sperm flagellum volume determines freezability in red deer spermatozoa.. PLoS One 2014;9(11):e112382.
- Traudt CM, McPherson RJ, Studholme C, Millen KJ, Juul SE. Systemic glycerol decreases neonatal rabbit brain and cerebellar growth independent of intraventricular hemorrhage.. Pediatr Res 2014 Mar;75(3):389-94.
Use Nutrition Calculator
Check if your horse's diet meets their nutrition requirements with our easy-to-use tool Check your horse's diet with our easy-to-use tool
Talk to a Nutritionist
Discuss your horse's feeding plan with our experts over a free phone consultation Discuss your horse's diet over a phone consultation
Submit Diet Evaluation
Get a customized feeding plan for your horse formulated by our equine nutritionists Get a custom feeding plan formulated by our nutritionists
