The effect of flash-freezing temperature on stallion sperm DNA structure.
Abstract: The effect of flash-freezing storage temperature on stallion sperm DNA has not been evaluated. Commonly, sperm are flash-frozen at various temperatures to preserve sperm DNA prior to analysis. It is unclear whether the temperature at which sperm are frozen and stored may affect the results of DNA assays. In this study, the neutral comet assay was used to evaluate the effect of flash-freezing storage temperature (freezer [-60 °C], dry ice [-78.5 °C], liquid nitrogen [-196 °C]) compared to fresh sperm DNA structure. In addition, intra- and inter-assay and intra- and inter-stallion variabilities were determined. All comet tail measures were higher following any flash-freezing method, as compared to fresh sperm DNA (P 0.05). For most comet variables, intra- and inter-assay variabilities were <10%. Intra- and inter-stallion variabilities revealed that comet head length (HL) and width (CW) were less variable as compared to comet tail values, i.e., % comet tail DNA (T-DNA), tail length (TL), tail moment (OTM), and tail migration (TM). Certain comet tail values in fresh (% T-DNA, and OTM) and flash-frozen sperm (OTM, % T-DNA, TL, and TM) were correlated to the Sperm Chromatin Structure Assay (SCSA) variable, COMP-α. The comet tail measures were negatively correlated to % morphologically normal sperm (P < 0.05) and positively correlated to % abnormal heads and premature germ cells (P 0.05). While significant differences in the structure of the sperm DNA were identified in the flash-frozen as compared to the fresh sperm DNA with the neutral comet assay, it cannot be assumed that these changes are fertility limiting.
Copyright © 2017. Published by Elsevier Inc.
Publication Date: 2017-03-16 PubMed ID: 28460664DOI: 10.1016/j.theriogenology.2017.03.005Google Scholar: Lookup
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- Journal Article
Summary
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This research aims to determine the effect of different flash-freezing temperatures on the DNA of stallion sperm. Results show while there are clear structural changes in the DNA following flash-freezing, it is unclear if these changes have an impact on fertility.
Study Overview
- The study investigated the effect of different flash-freezing storage temperatures on the structure of stallion sperm DNA.
- The test temperatures were specified at -60 °C (freezer), -78.5 °C (dry ice), and -196 °C (liquid nitrogen).
- A neutral comet assay was used as a method of evaluating the DNA damage in sperm after freezing.
- Stallion sperm samples were collected and each was divided into four portions. One portion was left unfrozen, as the control, while the others were flash-frozen using the mentioned temperatures.
Key Findings
- The results showed that all measures of comet tail (indicator of DNA damage) were higher in the flash-frozen samples than the fresh ones.
- Regardless of the freezing-storage temperature, the degree of DNA damage was similar across all flash-frozen methods.
- For most comet variables, intra- and inter-assay variabilities were low (<10%).
- Variables depicting comet head measurements showed less variability in comparison to comet tail measurements.
- Certain comet tail values showed correlation with the Sperm Chromatin Structure Assay (SCSA) variables. Additionally, there was a negative correlation with the percentage of morphologically normal sperm and a positive correlation with the percentage of abnormal heads and premature germ cells.
- No correlation was found between the SCSA variable COMP-α, the percentage of total sperm motility, and any morphologic sperm feature.
Implications
- The study shed light on the fact that the process of flash-freezing does indeed cause DNA structure changes in stallion sperm.
- The findings suggested that the temperature at which the sperm are flash-frozen does not appear to impact this level of DNA change.
- Further research is needed to investigate the clinical significance of these findings, as it is not clear if these structural changes to the sperm DNA affect fertility.
Cite This Article
APA
Serafini R, Varner DD, Bissett W, Blanchard TL, Teague SR, Love CC.
(2017).
The effect of flash-freezing temperature on stallion sperm DNA structure.
Theriogenology, 95, 113-117.
https://doi.org/10.1016/j.theriogenology.2017.03.005 Publication
Researcher Affiliations
- Department of Large Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, Texas, USA. Electronic address: rosanna.serafini@gmail.com.
- Department of Large Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, Texas, USA.
- Department of Large Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, Texas, USA.
- Department of Large Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, Texas, USA.
- Department of Large Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, Texas, USA.
- Department of Large Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, Texas, USA.
MeSH Terms
- Animals
- Comet Assay / veterinary
- Cryopreservation / veterinary
- DNA Damage
- Freezing
- Horses
- Male
- Spermatozoa / cytology
- Temperature
Citations
This article has been cited 1 times.- Strassner FM, Demattio L, Siuda M, Malama E, Muffels G, Bollwein H. Relationships Between Metabolism of Cryopreserved Equine Sperm Determined by the Seahorse Analyzer and Sperm Characteristics Measured by Flow Cytometry and Computer-Assisted Analysis of Motility. Vet Sci 2025 Nov 21;12(12).
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