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Cryopreservation reduces the ability of equine spermatozoa to attach to oviductal epithelial cells and zonae pellucidae in vitro.
Abstract: Two bioassays were used to evaluate the interaction of fresh and cryopreserved equine semen with oviductal epithelial cells (OEC) and with the zona pellucida (ZP). Split ejaculates were either stored at room temperature or frozen and thawed. In experiment 1, progressive motility and membrane integrity were evaluated for each treatment. Fluorescent labeled spermatozoa were cocultured with monolayers of OEC for 30 minutes, and the number of sperm attached to OEC was counted by fluorescence microscopy and analysis of digitized images. Motility of spermatozoa attached to OEC was observed at 0.5, 3, 6, 18, 24, and 48 hours after insemination. In experiment 2, progressive motility, membrane integrity, and acrosomal integrity were determined. Differential labeling with the fluorochromes fluorescein isothiocyanate (FITC) or tetramethylrhodamine isothiocyanate (TRITC) was used to distinguish fresh and frozen-thawed spermatozoa. Equal numbers of motile spermatozoa from each treatment were incubated with salt-stored equine oocytes for 4 hours, and the number of spermatozoa firmly bound to the ZP was counted using dual-wavelength epifluorescence microscopy. Fewer (P < 0.001) cryopreserved spermatozoa attached to OEC compared to spermatozoa stored at room temperature. The motility of spermatozoa attached to OEC decreased over time within each treatment group (P < 0.001), but this decrease was not different between treatments. The mean number of spermatozoa bound per ZP and percentage of acrosome-intact spermatozoa were lower (P < 0.05) for frozen-thawed than for fresh spermatozoa. There was no effect of stallion on acrosomal status of frozen-thawed spermatozoa; however, the number of spermatozoa bound per ZP was different between stallions within treatments (P < 0.05). These results indicate that the ability of cryopreserved equine spermatozoa to attach to equine OEC or ZP in vitro is reduced compared to fresh extended spermatozoa due to changes other than a reduction in post-thaw motility or membrane integrity. The decreased ability of frozen-thawed spermatozoa to attach to OEC or to ZP could explain, in part, the reduced fertility of cryopreserved compared to fresh spermatozoa in the horse.
Publication Date: 1995-11-01 PubMed ID: 8867602 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.
- Comparative Study
- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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The research paper investigates the influence of cryopreservation on the attachment capability of horse sperm to both the oviductal epithelial cells and the zona pellucida. Results show that freezing and thawing sperm reduce their ability to attach, which might partially explain decreased fertility rates in comparison to fresh sperm.
Research Methodology
- The study utilized two bioassays to observe the interaction of fresh and frozen-thawed horse sperm with oviductal epithelial cells (OEC) and zona pellucida (ZP), a membrane surrounding mammalian oocytes.
- Ejaculates were split into two; one was stored at room temperature while the other was frozen and thawed. The impact of these storage methods was then evaluated.
- In the first experiment, the researchers took measurements of the sperm’s progressive motility and membrane integrity. They stained the sperm cells with a fluorescent label and then co-cultured them with OEC monolayers.
- They counted the number of sperm cells attached to the OEC using fluorescence microscopy and digital image analysis.
Experimental Results
- In the second experiment, the team examined the sperm’s motility, membrane integrity, and the intactness of the acrosome – a cap-like structure in the head of the sperm cell which is essential for fertilization.
- Scoring was done by dual-wavelength epifluorescence microscopy to calculate the number of sperm cells attached to the ZP.
- The findings revealed that the count of cryopreserved sperm cells attached to the OEC was substantially fewer. Meanwhile, both types of sperm showed decreasing motility over time. However, the rate of this decrease was similar across both types.
- Additionally, frozen-thawed sperm showed a lower number of cells bound per ZP and a reduced percentage of acrosome-intact sperm.
- The results varied amongst different stallions, but not based on whether the sperm cells were fresh or frozen-thawed.
Conclusion and Implications
- The study shows that the cryopreservation process reduces the capability of horse sperm cells to attach to either OEC or ZP compared to fresh extended sperm cells.
- This decreased attachment capability is potentially a key reason why frozen-thawed sperm cells have lower fertility rates compared to fresh sperm cells.
Cite This Article
APA
Dobrinski I, Thomas PG, Ball BA.
(1995).
Cryopreservation reduces the ability of equine spermatozoa to attach to oviductal epithelial cells and zonae pellucidae in vitro.
J Androl, 16(6), 536-542.
Publication
Researcher Affiliations
- Department of Clinical Sciences, Cornell University, Ithaca, New York 14853, USA.
MeSH Terms
- Animals
- Biological Assay
- Coculture Techniques
- Cryopreservation
- Epithelial Cells
- Epithelium / physiology
- Fallopian Tubes / cytology
- Fallopian Tubes / physiology
- Female
- Horses / physiology
- Male
- Orchiectomy
- Sperm Motility
- Sperm-Ovum Interactions
- Spermatozoa / physiology
- Zona Pellucida / physiology
Citations
This article has been cited 4 times.- Soto-Heras S, Sakkas D, Miller DJ. Sperm selection by the oviduct: perspectives for male fertility and assisted reproductive technologies†. Biol Reprod 2023 Apr 11;108(4):538-552.
- Gimeno BF, Bariani MV, Laiz-Quiroga L, Martínez-León E, Von-Meyeren M, Rey O, Mutto AÁ, Osycka-Salut CE. Effects of In Vitro Interactions of Oviduct Epithelial Cells with Frozen-Thawed Stallion Spermatozoa on Their Motility, Viability and Capacitation Status. Animals (Basel) 2021 Jan 3;11(1).
- Zampini R, Castro-González XA, Sari LM, Martin A, Diaz AV, Argañaraz ME, Apichela SA. Effect of Cooling and Freezing on Llama (Lama glama) Sperm Ultrastructure. Front Vet Sci 2020;7:587596.
- Wang S, Wang Q, Min L, Cao H, Adetunji AO, Zhou K, Zhu Z. Pyrroloquinoline Quinone Improved Boar Sperm Quality via Maintaining Mitochondrial Function During Cryopreservation. Antioxidants (Basel) 2025 Jan 16;14(1).
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