Evaluation of cryopreserved stallion semen from Tori and Estonian breeds using CASA and flow cytometry.
Abstract: Methods to evaluate the quality of frozen-thawed stallion semen are still needed, particularly those considering the sperm function. The present study evaluated sperm motility, membrane and acrosome integrity and the capacitation status of frozen-thawed spermatozoa from seven Tori and six Estonian breed stallions by way of computer assisted sperm analysis (CASA), a triple fluorophore stain combination and Merocyanine 540, respectively, the latter ones using flow cytometry. Two ejaculates from each stallion were cryopreserved using the Hannover method in 0.5 ml plastic straws. Two straws per ejaculate per stallion were thawed at 37 degrees C for 30s. Motility was analysed with CASA immediately after thawing, while for flow cytometry spermatozoa were cleansed by 70:40% Percoll discontinuous density gradient separation before analysed for sperm viability, acrosome integrity (stained with SNARF, PI and FITC-PSA) and capacitation status (stained with Merocyanine 540/Yo-Pro-1). Results (as least square means) were as follows: the motility of frozen-thawed semen was 43.4% for Tori stallions and 42.3% for Estonian stallions (P>0.05). After Percoll separation 79.3% of the spermatozoa from Tori stallions had intact acrosomes and 1.7% of them showed early signs of capacitation. The same parameters for Estonian stallions were 84.5 and 2.3%, respectively. There were no statistically significant differences between breeds or ejaculates within breed for any evaluated parameter. We conclude that triple staining and flow cytometry are valuable techniques to evaluate frozen-thawed stallion spermatozoa, and that no differences in quality of frozen semen were registered between Tori and Estonian breed stallions, allowing implementation of this technology in the Estonian horse population.
Publication Date: 2003-02-15 PubMed ID: 12586493DOI: 10.1016/s0378-4320(02)00247-6Google Scholar: Lookup
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- Comparative Study
- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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This research article investigates the effectiveness of several scientific methods in assessing the quality of frozen, then thawed, semen from Tori and Estonian stallion breeds.
Overview of the Study
- The researchers conducted a study to evaluate the quality of frozen-then-thawed semen from two horse breeds, the Tori and the Estonian.
- The quality was gauged on the basis of sperm motility, membrane and acrosome integrity, and the capacitation status of the spermatozoa.
- The objective of the study was to refine the methods for appraising the quality of stallion sperm after cryopreservation and to identify any possible differences between the two breeds.
Methods and Techniques Used in the Study
- Techniques like Computer Assisted Sperm Analysis (CASA), triple fluorophore stain combination, and Merocyanine 540 were used for evaluation.
- Each stallion provided two ejaculates, which were frozen or cryopreserved using a method known as the Hannover method.
- Thawing was done at a carefully regulated temperature of 37 degrees Celsius for 30 seconds.
- CASA was employed immediately after thawing to analyze motility, whereas flow cytometry—as a part of which spermatozoa were cleansed with Percoll—was carried out to assess viability, acrosome integrity, and capacitation status.
Results of the Study
- The motility of the frozen-thawed semen was found to be similar (43.4% for Tori and 42.3% for Estonian stallions, with no statistically significant difference).
- On performing the Percoll separation, 79.3% of the Tori sperm and 84.5% of the Estonian sperm had intact acrosomes.
- Early signs of capacitation were seen in 1.7% and 2.3% of Tori and Estonian stallions’ sperm, respectively.
- The results did not show any significant differences between the two breeds or within the ejaculates from the same breed.
Conclusion
- The study concluded that CASA, triple staining, and flow cytometry are valuable techniques to evaluate the quality of frozen-thawed stallion spermatozoa.
- No significant variation was noted in the quality of frozen semen of Tori and Estonian breeds, thereby making it possible to use this technology in the Estonian horse population.
Cite This Article
APA
Kavak A, Johannisson A, Lundeheim N, Rodriguez-Martinez H, Aidnik M, Einarsson S.
(2003).
Evaluation of cryopreserved stallion semen from Tori and Estonian breeds using CASA and flow cytometry.
Anim Reprod Sci, 76(3-4), 205-216.
https://doi.org/10.1016/s0378-4320(02)00247-6 Publication
Researcher Affiliations
- Department of Obstetrics and Gynaecology, Faculty of Veterinary Medicine, Swedish University of Agricultural Sciences (SLU), P.O. Box 7039, S-750 07, Uppsala, Sweden. ants.kavak@og.slu.se
MeSH Terms
- Acrosome / physiology
- Animals
- Cell Membrane / physiology
- Centrifugation, Density Gradient
- Computers
- Cryopreservation / veterinary
- Flow Cytometry
- Horses
- Hot Temperature
- Male
- Semen / physiology
- Semen Preservation / veterinary
- Species Specificity
- Sperm Capacitation
- Sperm Motility
- Spermatozoa / ultrastructure
- Staining and Labeling
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