Cytosine methylation of sperm DNA in horse semen after cryopreservation.

Aurich, C
·
Schreiner, B
·
Ille, N
·
Alvarenga, M
·
Scarlet, D

Abstract: Semen processing may contribute to epigenetic changes in spermatozoa. We have therefore addressed changes in sperm DNA cytosine methylation induced by cryopreservation of stallion semen. The relative amount of 5-methylcytosine relative to the genomic cytosine content of sperm DNA was analyzed by ELISA. In experiment 1, raw semen (n = 6 stallions, one ejaculate each) was shock-frozen. Postthaw semen motility and membrane integrity were completely absent, whereas DNA methylation was similar in raw (0.4 ± 0.2%) and shock-frozen (0.3 ± 0.1%) semen (not significant). In experiment 2, three ejaculates per stallion (n = 6) were included. Semen quality and DNA methylation was assessed before addition of the freezing extender and after freezing-thawing with either Ghent (G) or BotuCrio (BC) extender. Semen motility, morphology, and membrane integrity were significantly reduced by cryopreservation but not influenced by the extender (e.g., total motility: G 69.5 ± 2.0, BC 68.4 ± 2.2%; P < 0.001 vs. centrifugation). Cryopreservation significantly (P < 0.01) increased the level of DNA methylation (before freezing 0.6 ± 0.1%, postthaw G 6.4 ± 3.7, BC 4.4 ± 1.5%; P < 0.01), but no differences between the freezing extenders were seen. The level of DNA methylation was not correlated to semen motility, morphology, or membrane integrity. The results demonstrate that semen processing for cryopreservation increases the DNA methylation level in stallion semen. We conclude that assessment of sperm DNA methylation allows for evaluation of an additional parameter characterizing semen quality. The lower fertility rates of mares after insemination with frozen-thawed semen may at least in part be explained by cytosine methylation of sperm-DNA induced by the cryopreservation procedure.

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Publication Date: 2016-05-06 PubMed ID: 27242182DOI: 10.1016/j.theriogenology.2016.04.077Google Scholar: Lookup

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Summary

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This study examines the changes in sperm DNA cytosine methylation induced by freezing horse semen, with the finding that the amount of DNA methylation increases with cryopreservation, though not influenced by the freezing extender. This increase in DNA methylation may contribute to the lower fertility rates observed after insemination with frozen-thawed semen.

Study Overview

The study was done in two experiments with horse semen to examine if cytosine DNA methylation changes due to freezing. Cytosine methylation is a type of DNA modification that can influence gene activity without changing the DNA sequence, and shifts in this process can have biological implications.

In the first experiment, raw semen from six stallions was flash-frozen. After thawing, sperm motility and membrane integrity were examined and found to be absent. The researchers found no significant change in DNA methylation compared to raw semen.
In the second experiment, semen samples from six stallions were assessed for quality and DNA methylation before and after freezing using either Ghent or BotuCrio as extenders. The extenders had no significant impact on the DNA methylation, but freezing did increase the DNA methylation level drastically.

Results and Conclusions

Freezing stallion semen significantly increases the level of DNA methylation.
Neither the Ghent nor BotuCrio extenders used during the freezing process influenced the level of DNA methylation.
While freezing significantly reduces semen quality (measured by sperm motility, morphology, and membrane integrity), these factors did not correlate with the level of DNA methylation.
The researchers suggest the increased DNA methylation may explain, at least in part, the lower fertility rates in mares inseminated with frozen-thawed semen.

This research adds insight into the complex ways semen processing can alter sperm on the epigenetic level. It also presents DNA methylation as an additional parameter to consider when evaluating semen quality, with potential implications for artificial insemination practices in veterinary science.

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APA

Aurich C, Schreiner B, Ille N, Alvarenga M, Scarlet D. (2016). Cytosine methylation of sperm DNA in horse semen after cryopreservation. Theriogenology, 86(5), 1347-1352. https://doi.org/10.1016/j.theriogenology.2016.04.077

Publication

Theriogenology

ISSN: 1879-3231

NlmUniqueID: 0421510

Country: United States

Language: English

Volume: 86

Issue: 5

Pages: 1347-1352

PII: S0093-691X(16)30120-0

Researcher Affiliations

Aurich, Christine

Artificial Insemination and Embryo Transfer, Department for Small Animals and Horses, Vetmeduni Vienna, Vienna, Austria. Electronic address: christine.aurich@vetmeduni.ac.at.

Schreiner, Bettina

Artificial Insemination and Embryo Transfer, Department for Small Animals and Horses, Vetmeduni Vienna, Vienna, Austria.

Ille, Natascha

Artificial Insemination and Embryo Transfer, Department for Small Animals and Horses, Vetmeduni Vienna, Vienna, Austria.

Alvarenga, Marco

Department of Animal Reproduction & Veterinary Radiology, Faculty of Veterinary Medicine, FMVZ - Sao Paulo State University UNESP, Botucatu, Brazil.

Scarlet, Dragos

Obstetrics, Gynecology, Andrology, Department for Small Animals and Horses, Vetmeduni Vienna, Vienna, Austria.

MeSH Terms

Animals
Cell Membrane
Cryopreservation / veterinary
Cytosine / chemistry
DNA Methylation
Horses / physiology
Male
Semen Analysis
Semen Preservation / veterinary
Sperm Motility
Spermatozoa / physiology

Citations

This article has been cited 16 times.

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