Detection of DNA damage in response to cooling injury in equine spermatozoa using single-cell gel electrophoresis.
Abstract: Single-cell gel electrophoresis (SCGE), or comet assay, has the ability to detect damage at the single cell level and has not been reported for equine sperm. The ability to detect nuclear damage at the single cell level could aid in the advancement of protocols for optimal semen preservation. The goals of these experiments were to adapt this assay for use with equine sperm and to utilize the assay for determining the integrity of equine sperm DNA following treatments with storage at various decreased temperatures (-20 degrees C and 5 degrees C). Results from experiments in which sperm were frozen (-20 degrees C) in the absence of cryoprotectants revealed that significantly more cells with fragmented tails of DNA, or comets, occurred among those exposed to 1, 3, and 5 freeze-thaw cycles (65% +/- 6%, 76% +/- 11%, 92% +/- 6%, respectively) compared with fresh, untreated sperm (19% +/- 16%, P < .05). In addition DNA damage was different (P < .05) between the three freeze-thaw treatments. Sensitivity of SCGE on equine sperm was further tested with known ratios of frozen-thawed and fresh cells. The amount of detectable DNA damage was positively correlated with the percentage of cryo-damaged cells in each treatment (r2 = 0.92, P < .05). Potential damage as a result of cooled storage was also investigated and results revealed that sperm stored for 48 hours (at 5 degrees C) had a higher percentage of comets than that of fresh sperm (63% +/- 13.9% and 28% +/- 15.6%, respectively, P < .05). The percentage of viable sperm also decreased linearly over time and was inversely correlated with percent of comets (r2 = 0.805, P < .001). Detection of sublethal and/or uncompensable fertility factors in semen, such as DNA fragmentation, could be useful for detecting male differences in semen for cooling or cryopreservation potential and could provide a tool for monitoring and preserving fertility for individual stallions.
Publication Date: 2002-01-10 PubMed ID: 11780916DOI: 10.1002/j.1939-4640.2002.tb02603.xGoogle Scholar: Lookup
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.
- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.
This research tests a method for assessing DNA damage in horse sperm caused by cooling the sperm and subsequent thawing. The method may be useful for developing better techniques for preserving sperm and monitoring fertility in horses.
Objective of the Study
- The purpose of this study was to apply the Single-cell gel electrophoresis (SCGE) or comet assay, which is known for detecting damage at single cell levels, on equine sperm.
- The researchers aimed to use this assay to determine the integrity of horse sperm DNA after various temperature storage treatments, specifically cooling at -20 degrees C and 5 degrees C.
Methods and Findings of the Study
- The researchers froze sperm at -20 degrees C without any cryoprotectants and subsequently thawed them for either 1, 3, or 5 cycles.
- The findings showed that significantly more cells with fragmented (damaged) DNA (referred to as “comets”) emerged from the sperm that had undergone the freeze-thaw cycles as compared to fresh, untreated sperm.
- The amount of detectable DNA damage was observed to increase with the percentage of damaged cells in each treatment.
Investigation of Cooled Storage Damage
- Another facet of the study was investigating potential damage from cooled storage. This involved storing sperm for 48 hours at 5 degrees C.
- Sperm stored in these conditions displayed a higher percentage of DNA damage (or comets) than fresh sperm.
- The percentage of viable (living) sperm reduced over time and was found to inversely correlate with the percentage of DNA-damaged cells.
Implications of the Study
- Understanding the extent of sublethal and uncompensated fertility factors such as DNA fragmentation in semen could be used to detect differences in semen cooling or cryopreservation potential. This would help to monitor and preserve fertility in individual stallions.
- The comet assay adapted for use with horse sperm in this research could provide a valuable tool in these investigations, potentially leading to advanced semen preservation protocols.
Cite This Article
APA
Linfor JJ, Meyers SA.
(2002).
Detection of DNA damage in response to cooling injury in equine spermatozoa using single-cell gel electrophoresis.
J Androl, 23(1), 107-113.
https://doi.org/10.1002/j.1939-4640.2002.tb02603.x Publication
Researcher Affiliations
- Department of Anatomy, Physiology, and Cell Biology, School of Veterinary Medicine, University of California, Davis 95616, USA.
MeSH Terms
- Animals
- Comet Assay
- Cryopreservation / veterinary
- DNA Damage
- Horses
- Male
- Semen Preservation / veterinary
- Spermatozoa / pathology
Citations
This article has been cited 5 times.- Sadeghi S, Del Gallego R, García-Colomer B, Gómez EA, Yániz JL, Gosálvez J, López-Fernández C, Silvestre MA. Effect of Sperm Concentration and Storage Temperature on Goat Spermatozoa during Liquid Storage.. Biology (Basel) 2020 Sep 19;9(9).
- Váradi É, Végi B, Liptói K, Barna J. Methods for cryopreservation of guinea fowl sperm.. PLoS One 2013;8(4):e62759.
- Gutiérrez-Cepeda L, Fernández A, Crespo F, Ramírez MÁ, Gosálvez J, Serres C. The effect of two pre-cryopreservation single layer colloidal centrifugation protocols in combination with different freezing extenders on the fragmentation dynamics of thawed equine sperm DNA.. Acta Vet Scand 2012 Dec 5;54(1):72.
- Li MW, Baridon B, Trainor A, Djan E, Koehne A, Griffey SM, Biggers JD, Toner M, Lloyd KC. Mutant mice derived by ICSI of evaporatively dried spermatozoa exhibit expected phenotype.. Reproduction 2012 Apr;143(4):449-53.
- Steif PS, Palastro M, Wan CR, Baicu S, Taylor MJ, Rabin Y. Cryomacroscopy of vitrification, Part II: Experimental observations and analysis of fracture formation in vitrified VS55 and DP6.. Cell Preserv Technol 2005 Sep;3(3):184-200.
Use Nutrition Calculator
Check if your horse's diet meets their nutrition requirements with our easy-to-use tool Check your horse's diet with our easy-to-use tool
Talk to a Nutritionist
Discuss your horse's feeding plan with our experts over a free phone consultation Discuss your horse's diet over a phone consultation
Submit Diet Evaluation
Get a customized feeding plan for your horse formulated by our equine nutritionists Get a custom feeding plan formulated by our nutritionists
