Effects of phosphatidylserine and cholesterol liposomes on the viability, motility, and acrosomal integrity of stallion spermatozoa prior to and after cryopreservation.
Abstract: Computer-assisted motion analyses (CASA) and flow cytometry were used to evaluate stallion spermatozoa prior to and after cryopreservation. Spermatozoa were pretreated with: (1) Hepes-buffered medium (SHB); (2) phosphatidylserine (PS) liposomes; or (3) liposomes composed of both PS and cholesterol (PSCH) prior to dilution in either SHB or skim milk-egg yolk extender (SMEY). After cooling to 5 degrees C in SHB, PS and PSCH pretreatment (23%). Spermatozoal motion parameters were higher for spermatozoa diluted in SMEY than dilution in SHB. In Experiment 2, motion parameters were compared for spermatozoa pretreated with PSCH liposomes and cryopreserved in either SMEY or a high salt-skim milk-egg yolk extender (CO). Spermatozoal motion characteristics were similar for all spermatozoal treatments after cooling at 5 degrees C. After cryopreservation, PSCH liposome-treated samples had higher percentages of motile spermatozoa than untreated samples regardless of freezing extender. Samples frozen in CO medium had higher percentages of motile spermatozoa than samples frozen in SMEY (P < 0.05; 63% in CO + PSCH and 54% in CO vs 55% in SMEY + PSCH and 48% in SMEY, respectively). In Experiment 3, spermatozoa were treated with dilauroylphosphatidylcholine (PC12) to induce the acrosome reaction. The percentages of viable cells and viable acrosome-reacted spermatozoa were higher for fresh spermatozoa than for cryopreserved spermatozoa (P 0.05). Addition of PSCH liposomes improved recovery of motile spermatozoa after cryopreservation but did not affect the ability of spermatozoa to undergo a PC12-induced acrosome reaction.
Publication Date: 1996-06-01 PubMed ID: 8689889DOI: 10.1006/cryo.1996.0032Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
- Research Support
- U.S. Gov't
- Non-P.H.S.
Summary
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The study investigates the impact of lipid molecules treatment on the survival, activity, and acrosomal state of horse sperm cells before and after the freezing process. It specifically shows that application of liposomes containing phosphatidylserine (PS) and cholesterol (PSCH) improves sperm movement and viability, without impacting the acrosomal reaction–a vital step in fertilization.
Research Methodology
- The study used computer-assisted motion analyses (CASA) and flow cytometry to evaluate stallion sperm cells both before and after cryopreservation.
- Sperm samples were subjected to three types of pre-treatment: no treatment (labeled as Hepes-buffered medium or SHB), treatment with phosphatidylserine (PS) liposomes, and treatment with liposomes of both PS and cholesterol (PSCH).
- These samples were then diluted in either Hepes-buffered medium (SHB) or a mixture of skim milk and egg yolk (SMEY) and cooled to 5 degrees Celsius.
Findings from the First Experiment
- The first experiment revealed that following cooling to 5 degrees Celsius in SHB, sperm motion parameters were higher for those treated with PS and PSCH.
- Sperm motion parameters were also higher for samples that were diluted in SMEY as compared with those diluted in SHB, meaning, the medium of dilution played a role in preserving sperm motility.
Findings from the Second Experiment
- In a second experiment, the researchers compared motion parameters for sperm subjected to PSCH liposomes pre-treatment and then frozen in either SMEY or a different medium referred to as CO (high salt-skim milk-egg yolk extender).
- The motion characteristics were similar for all sperm samples after cooling to 5 degrees Celsius regardless of the treatment kind or the freezing medium.
- However, post-cryopreservation, the samples treated with PSCH liposomes showed higher percentages of moving sperm cells than untreated samples irrespective of the freezing extender.
- Along with the treatment, the freezing medium again seemed to play a significant role. Samples frozen in the CO medium had a larger percentage of moving sperm cells than those frozen in the SMEY medium.
Findings from the Third Experiment
- The third experiment further examined these treated and cryopreserved sperm cells under a different condition. Sperm cells were exposed to the molecule dilauroylphosphatidylcholine (PC12) to instigate the acrosome reaction, necessary for a sperm cell to successfully fertilize an egg.
- The scores of viable cells were higher for fresh sperm than for cryopreserved sperm, which can be expected given the harsh conditions of cryopreservation.
- However, there was no sign that the treatment with PSCH liposomes had any effect on the sperm’s capability to undergo the PC12 induced acrosome reaction.
Conclusion
- The overall conclusion of this research is that pre-treatment of stallion sperm cells with liposomes containing PS and cholesterol can indeed improve the recovery of active sperm cells after cryopreservation.
- However, this treatment doesn’t seem to influence the acrosome reaction, a necessary event for a sperm cell to fertilize an egg, suggesting that the PSCH liposome treatment specifically aids in preserving structural and motility aspects of the sperm cells during the freezing process without affecting their functionality.
Cite This Article
APA
Wilhelm KM, Graham JK, Squires EL.
(1996).
Effects of phosphatidylserine and cholesterol liposomes on the viability, motility, and acrosomal integrity of stallion spermatozoa prior to and after cryopreservation.
Cryobiology, 33(3), 320-329.
https://doi.org/10.1006/cryo.1996.0032 Publication
Researcher Affiliations
- Department of Physiology, Colorado State University, Fort Collins 80523, USA.
MeSH Terms
- Acrosome / drug effects
- Animals
- Cell Survival / drug effects
- Cholesterol / pharmacology
- Cryopreservation / methods
- Cryoprotective Agents / pharmacology
- Evaluation Studies as Topic
- Horses
- In Vitro Techniques
- Liposomes
- Male
- Phosphatidylserines / pharmacology
- Sperm Motility / drug effects
- Spermatozoa / cytology
- Spermatozoa / drug effects
Citations
This article has been cited 4 times.- Wysokińska A, Szablicka D. Integrity of Sperm Cell Membrane in the Semen of Crossbred and Purebred Boars during Storage at 17 °C: Heterosis Effects. Animals (Basel) 2021 Nov 25;11(12).
- de Almeida RA, da Silva LS, de Lima LGF, Lourencetti FA, Freitas-Dell Aqua C, Ferreira JCP, Oba E. Egg-yolk- and a liposome-based extenders: refrigeration time and effects on ram semen quality. Anim Reprod 2025;22(2):e20240052.
- Dcunha R, Mutalik SP, Reji RA, Mutalik S, Kalthur SG, Hegde P, Murari MS, Raghu SV, Banerjee S, Kumar A, Adiga SK, Zhao Y, Kannan N, Kalthur G. Liposome-based Freezing Medium Improves the Outcome of Mouse Prepubertal Testicular Tissue Cryopreservation. Reprod Sci 2024 Nov;31(11):3532-3548.
- Salama MS, Ashour MA, Taher ES, Rashed F, Ibrahim IM, El-Nablaway M, Ibrahim AM, Mihaela O, Olga R, Mohammed NA, Abdeen A, Shukry M. Effect of autologous platelet-rich plasma on the fertility and quality of cryopreserved buffalo bull semen: a comparative study using OptiXcell® and tris egg yolk extenders. BMC Vet Res 2024 Jun 7;20(1):250.
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