Cholesterol addition protects membrane intactness during cryopreservation of stallion sperm.
Abstract: Addition of cholesterol to sperm membranes improved equine sperm stability during semen cryopreservation; however, it also reduced in vivo fertility. The objective of the present study was to determine the effects of adding cholesterol to stallion sperm prior to freezing, and subsequently removing it from frozen-thawed sperm. Semen from 12 stallions was subjected to four treatments: (T1) control, semen was diluted with Kenney extender, centrifuged, and resuspended to 100 x 10(6)spermatozoa/mL in INRA 82 freezing extender, packaged into 0.5-mL straws, cooled to 5 degrees C, and cryopreserved in liquid nitrogen; (T2) T1 with the addition of cholesterol before cooling (the cholesterol was incorporated to the sperm membranes with the methyl-beta-cyclodextrin-cholesterol complex); (T3) T2 with post-thaw removal of the cholesterol with 0.052 mg methyl-beta-cyclodextrin/50 x 10(6) sperm; and (T4) T3 with 0.156 mg methyl-beta-cyclodextrin/50 x 10(6) sperm. Sperm progressive motility and functional integrity of sperm plasma membranes were evaluated microscopically and by the hyposmotic swelling test, respectively. Using flow cytometry, physical integrity of sperm plasma membranes was assessed with propidium iodide, acrosomal integrity with fluoresceinated lectin peanut agglutinin, and rate of sperm acrosome reaction induced with of the calcium ionophore A23187. Cholesterol inclusion (T2) increased the proportion of frozen-thawed sperm with intact plasma membrane. Nevertheless, sperm from T2 (9.3+/-5.9%) had a lower rate of acrosome reaction after induction, compared to the control group (16.5+/-11.0%). After cholesterol removal, there was no increase in the induced acrosome reaction rate (T3: 11.3+/-7.1% and T4: 11.8+/-9.9%). Perhaps the cyclodextrin concentrations used were too low to remove sufficient cholesterol from sperm membranes to restore the ability of cryopreserved sperm to undergo an acrosome reaction. Regardless, the addition of cholesterol to improve post-thaw sperm integrity, and its subsequent removal, still has potential for cryopreservation of stallion sperm.
Copyright 2009 Elsevier B.V. All rights reserved.
Publication Date: 2009-08-25 PubMed ID: 19758774DOI: 10.1016/j.anireprosci.2009.08.011Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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The study explores the effects of adding cholesterol to stallion sperm prior to freezing it, then removing the cholesterol after thawing. The process improved sperm stability during freezing but reduced fertility levels and ability to undergo an acrosome reaction (an essential step in fertilization).
Methods
- Semen samples were taken from 12 stallions and divided into four different treatment groups.
- Control group (T1) had the semen diluted and cryopreserved in the standard way.
- Treatment 2 (T2) added cholesterol to the sperm before cooling.
- Treatment 3 (T3) replicated T2 but removed the cholesterol after thawing it.
- Treatment 4 (T4) followed the same procedure as T3 but increased the concentration of methyl-beta-cyclodextrin, used to remove the cholesterol.
Results
- Sperm movement and the functional and physical integrity of sperm plasma membranes were evaluated.
- When cholesterol was added (T2), more thawed sperm had intact plasma membranes than the control group. This indicated that the addition of cholesterol increased the sperm’s stability through the freezing process.
- However, T2’s rate of acrosome reaction was lower than the control group, suggesting a cholesterol-induced reduction in fertility levels.
- Upon removing the cholesterol (T3 and T4), no significant increase in rates of induced acrosome reaction was observed, suggesting the concentrations used for removal may have been too low to sufficiently extract the cholesterol from the sperm membranes to restore their ability to undergo this reaction.
Conclusion
- The study concludes that adding cholesterol to sperm prior to freezing and then removing it post-thaw could potentially enhance the process of sperm cryopreservation.
- However, the lower rates of acrosome reaction indicate challenges in restoring post-thaw fertility levels, as the presence of cholesterol reduces this crucial ability of the sperm.
- Adjustments in cholesterol removal process, possibly by increasing the concentration of cyclodextrin used, might be needed to properly restore the ability of the sperm for acrosome reaction.
Cite This Article
APA
Oliveira CH, Vasconcelos AB, Souza FA, Martins-Filho OA, Silva MX, Varago FC, Lagares MA.
(2009).
Cholesterol addition protects membrane intactness during cryopreservation of stallion sperm.
Anim Reprod Sci, 118(2-4), 194-200.
https://doi.org/10.1016/j.anireprosci.2009.08.011 Publication
Researcher Affiliations
- Department of Veterinary Clinics and Surgery, Veterinary School of the Federal University of Minas Gerais, Av. Antonio Carlos 6627, CEP: 31270-901, Belo Horizonte, MG, Brazil.
MeSH Terms
- Acrosome Reaction
- Animals
- Cell Membrane / chemistry
- Cell Membrane / physiology
- Cell Membrane / ultrastructure
- Cholesterol / administration & dosage
- Cholesterol / analysis
- Cryopreservation / methods
- Cryopreservation / veterinary
- Fertilization
- Horses
- Hot Temperature
- Male
- Semen Preservation / methods
- Semen Preservation / veterinary
- Sperm Motility
- Spermatozoa / physiology
- Spermatozoa / ultrastructure
- beta-Cyclodextrins / administration & dosage
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