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Home / Equine Research Bank / Reprod Domest Anim / Assessing the Sperm Head DNA Damage in Frozen/Thawed Horse S...
Reproduction in domestic animals = Zuchthygiene2026; 61(1); e70172; doi: 10.1111/rda.70172

Assessing the Sperm Head DNA Damage in Frozen/Thawed Horse Spermatozoa via Xenogeneic ICSI.

Abstract: In the mouse, spermatozoa are highly resistant to DNA damage, even when frozen without cryoprotectants, and can produce offspring when subsequently used for ICSI (intracytoplasmic sperm injection). It is not known whether the same applies to other mammals as well. For example, in the horse, even conventional sperm freezing is still very problematic and frequently leads to sperm immobility. It has, however, never been tested whether sperm immobility also mirrors sperm head DNA damage, and if so, to what extent. In our study, we evaluated the damage to DNA in horse frozen and thawed motile and immotile spermatozoa after their injection into ovulated mouse oocytes. In both groups, injected horse spermatozoa activated the mouse oocytes. This was followed by the extrusion of the second polar body (2 PB) and the formation of maternal pronuclei (Mo-fPN-mouse female pronucleus); in parallel, the horse sperm heads rapidly decondensed in the murine cytoplasm and formed paternal pronuclei (Ho-mPN-horse male pronucleus), which were larger than the female ones. With the exception of one stallion tested, DNA damage has been detected in almost all Ho-mPNs originating from immotile spermatozoa. DNA in motile (even sporadically) spermatozoa was mostly undamaged. Moreover, when the xenogeneic zygotes cleave to the two-cell stage, the incidence of micronuclei in blastomeres mirrors the extent of DNA damage in paternal pronuclei. In conclusion, and contrary to the mouse, where sperm DNA is very resistant to damage, we do not recommend the use of immotile horse spermatozoa for ICSI. On the other hand, even the sporadically motile mouse spermatozoa have no damaged DNA and can thus be used for intragenic ICSI.
© 2026 Wiley‐VCH GmbH. Published by John Wiley & Sons Ltd.
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Publication Date: 2026-01-09 PubMed ID: 41508721DOI: 10.1111/rda.70172Google Scholar: Lookup
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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.

Overview

  • This study investigates the extent of DNA damage in frozen and thawed horse sperm, specifically comparing motile and immotile spermatozoa, using a technique called xenogeneic ICSI where horse sperm is injected into mouse oocytes.
  • The findings indicate that immotile frozen/thawed horse sperm show significant DNA damage, unlike mouse sperm which is highly resistant, leading to recommendations against using immotile horse sperm for intracytoplasmic sperm injection (ICSI).

Background

  • Mouse sperm are known to be highly resistant to DNA damage, even after being frozen without protective chemicals (cryoprotectants), and can successfully fertilize eggs through ICSI.
  • In contrast, horse sperm freezing is problematic, often resulting in sperm immobility after thawing.
  • It was unknown whether immotile horse sperm also suffered from DNA damage, which could impact their fertilization potential and embryo development.

Research Objectives

  • To measure the DNA integrity of frozen and thawed horse spermatozoa, distinguishing between motile and immotile sperm.
  • To study how these sperm behave when injected into ovulated mouse oocytes (xenogeneic ICSI), assessing activation of the oocytes and subsequent embryo development events.
  • To analyze the relationship between sperm DNA damage and embryo developmental markers such as polar body extrusion, pronuclei formation, and micronuclei appearance in the resulting embryos.

Methods

  • Frozen/thawed horse spermatozoa were selected based on motility: motile and immotile groups.
  • These sperm were injected into ovulated mouse oocytes using xenogeneic ICSI, allowing the researchers to assess sperm DNA damage in a controlled environment.
  • Post-injection, several fertilization markers were monitored:
    • Activation of mouse oocytes by sperm injection
    • Extrusion of the second polar body (2 PB) from the oocytes
    • Formation of maternal pronuclei (female pronucleus) and paternal pronuclei (horse sperm-derived male pronucleus)
    • Observation of pronuclei size and sperm head decondensation
  • The presence of DNA damage in sperm was assessed by detecting micronuclei within blastomeres at the two-cell stage, which reflect chromosomal fragmentation or repair failures.

Key Findings

  • Both motile and immotile horse sperm could activate mouse oocytes and induce expected fertilization events such as polar body extrusion and pronuclei formation.
  • The paternal pronuclei derived from horse sperm heads decondensed in mouse cytoplasm and appeared larger than maternal pronuclei.
  • With one exception, almost all paternal pronuclei from immotile spermatozoa exhibited significant DNA damage.
  • Motile sperm showed mostly intact DNA, even when motility was only sporadic.
  • The extent of DNA damage seen in paternal pronuclei correlated with the incidence of micronuclei in two-cell stage embryos, confirming that DNA fragmentation in spermatozoa impacts early embryonic genome integrity.
  • This contrasts with mouse sperm, which generally maintain DNA integrity even when immotile or frozen without cryoprotectants.

Conclusions and Implications

  • The study demonstrates that immotile frozen/thawed horse spermatozoa suffer considerable DNA damage, raising concerns about their use in ICSI for reproductive purposes.
  • It recommends against using immotile horse sperm in ICSI procedures due to likely compromised DNA integrity and potential impacts on embryo development.
  • Conversely, even sporadically motile mouse sperm retain DNA integrity and can be safely used for intragenic intracytoplasmic sperm injection.
  • These findings highlight species differences in sperm DNA resistance to freeze-thaw damage and emphasize the need for species-specific evaluation when using frozen sperm in assisted reproduction.

Cite This Article

APA
Rychtarova J, Fulka H, Loi P, Fulka J. (2026). Assessing the Sperm Head DNA Damage in Frozen/Thawed Horse Spermatozoa via Xenogeneic ICSI. Reprod Domest Anim, 61(1), e70172. https://doi.org/10.1111/rda.70172

Publication

Reproduction in domestic animals = Zuchthygiene
ISSN: 1439-0531
NlmUniqueID: 9015668
Country: Germany
Language: English
Volume: 61
Issue: 1
Pages: e70172

Researcher Affiliations

Rychtarova, Jana
  • Institute of Animal Science, Prague 10, Czech Republic.
Fulka, Helena
  • Institute of Animal Science, Prague 10, Czech Republic.
  • Institute of Experimental Medicine CAS, Prague 4, Czech Republic.
Loi, Pasqualino
  • University of Teramo, Teramo, Italy.
Fulka, Josef
  • Institute of Animal Science, Prague 10, Czech Republic.

MeSH Terms

  • Animals
  • Male
  • Horses
  • Sperm Injections, Intracytoplasmic / veterinary
  • DNA Damage
  • Female
  • Mice
  • Cryopreservation / veterinary
  • Semen Preservation / veterinary
  • Sperm Head / physiology
  • Sperm Motility
  • Spermatozoa / physiology

Grant Funding

  • QK1910156 / National Agency for Agricultural Research
  • TN02000017 / National Agency for Agricultural Research
  • 0723 / National Agency for Agricultural Research
  • MUR Prin 2022CSHPAS / National Agency for Agricultural Research
  • Prin P2022FA79R / National Agency for Agricultural Research
  • GACR 21-42225L / Grantová Agentura České Republiky
  • Ministry of Agriculture
  • Technology Agency of the Czech Republic

References

This article includes 23 references
  1. Aurich J, Kuhl J, Tichy A, Aurich C. Efficiency of Semen Cryopreservation in Stallions. Animals 2020;10(6):1033.
    doi: 10.3390/ani10061033google scholar: lookup
  2. Fulka H, Barnetova I, Mosko T, Fulka JJ. Epigenetic Analysis of Human Spermatozoa After Their Injection Into Ovulated Mouse Oocytes. Human Reproduction 2008;23(3):627–634.
    doi: 10.1093/humrep/dem406google scholar: lookup
  3. Gonzales‐Castro RA, Trentin JM, Carnevale EM. Effects of Extender, Cryoprotectants and Thawing Protocol on Motility of Frozen‐Thawed Stallion Sperm That Were Refrozen for Intracytoplasmic Sperm Injection Doses. Theriogenology 2019;136:36–42.
    doi: 10.1016/j.theriogenology.2019.06.030google scholar: lookup
  4. Grenier L, Robaire B, Hales BF. Paternal Cyclophosphamide Exposure Induces the Formation of Functional Micronuclei During the First Mitotic Division. PLoS One 2015;6:e27600.
    doi: 10.1371/journal.pone.0027600google scholar: lookup
  5. Hildebrandt TB, Hermes R, Colleoni S. Embryos and Embryonic Stem Cells From the White Rhinoceros. Nature Communications 2018;9:2589.
    doi: 10.1038/s41467‐018‐04959‐2google scholar: lookup
  6. Kaneko T, Ito H, Sakamoto H, Onuma M, Inoue‐Murayama M. Sperm Preservation by Freeze‐Drying for the Conservation of Wild Animals. PLoS One 2014;9(11):e113381.
    doi: 10.1371/journal.pone.0113381google scholar: lookup
  7. Kimura Y, Yanagimachi R. Intracytoplasmic Sperm Injection in the Mouse. Biology of Reproduction 1995;52(4):709–720.
  8. Li C, Mizutani E, Ono T. Intracytoplasmic Sperm Injection With Mouse Spermatozoa Preserved Without Freezing for Six Months Can Lead to Full‐Term Development. Biology of Reproduction 2011;85(6):1183–1190.
    doi: 10.1095/biolreprod.111.091827google scholar: lookup
  9. Musson R, Gasior L, Bisogno S, Ptak GE. DNA Damage in Preimplantation Embryos and Gametes: Specification, Clinical Relevance and Repair Strategies. Human Reproduction Update 2022;28(3):376–399.
    doi: 10.1093/humupd/dmab046google scholar: lookup
  10. Newman H, Catt S, Vining B, Vollenhoven B, Horta F. DNA Repair and Response to Sperm DNA Damage in Oocytes and Embryos, and the Potential Consequences in ART: A Systematic Review. Molecular Human Reproduction 2022;28(1):gaab071.
    doi: 10.1093/molehr/gaab071google scholar: lookup
  11. Ogonuki N, Mochida K, Miki H. Spermatozoa and Spermatids Retrieved From Frozen Reproductive Organs or Frozen Whole Bodies of Male Mice Can Produce Normal Offspring. Proceedings of the National Academy of Sciences of the United States of America 2006;103(35):13098–13103.
    doi: 10.1073/pnas.0605755103google scholar: lookup
  12. Ohta H, Sakaide Y, Wakayama T. Long‐Term Preservation of Mouse Spermatozoa as Frozen Testicular Sections. Journal of Reproduction and Development 2008;54(4):295–298.
    doi: 10.1262/jrd.20027google scholar: lookup
  13. Prien S, Iacovides S. Cryoprotectants & Cryopreservation of Equine Semen: A Review of Industry Cryoprotectants and the Effects of Cryopreservation on Equine Semen Membrane. Journal of Dairy & Veterinary Sciences 2016;3(1):1–8.
    doi: 10.15406/jdvar.2016.03.00063google scholar: lookup
  14. Rychtarova J, Langerova A, Fulka H, Loi P, Benc M, Fulka J. Interspecific ICSI for the Assessment of Sperm DNA Damage: Technology Report. Animals 2021;11(5):1250.
    doi: 10.3390/ani11051250google scholar: lookup
  15. Salamone DF, Canel NG, Rodriguez MB. Intracytoplasmic Sperm Injection in Domestic and Wild Mammals. Reproduction 2017;154(6):F111–F124.
    doi: 10.1530/rep‐17‐0357google scholar: lookup
  16. Shamsi, M. B., S. N. Imam, and R. Dada. 2011. “Sperm DNA Integrity Assays: Diagnostic and Prognostic Challenges and Implications in Management of Fertility.” Journal of Assisted Reproduction and Genetics 28, no. 11: 1073–1085. https://doi.org/10.1007/s10815‐011‐9631‐8.
  17. Vazquez‐Diez, C., and G. FitzHarris. 2018. “Causes and Consequences of Chromosome Segregation Error in Preimplantation Embryos.” Reproduction 155, no. 1: R63–R76. https://doi.org/10.1530/REP‐17‐0569.
  18. Wakayama, T., T. Uehara, Y. Hayashi, and R. Yanagimachi. 1997. “The Response of Mouse Oocytes Injected With Sea Urchin Spermatozoa.” Zygote 5, no. 3: 229–234. https://doi.org/10.1017/s096719940000366x.
  19. Wakayama, T., D. G. Whittingham, and R. Yanagimachi. 1998. “Production of Normal Offspring From Mouse Oocytes Injected With Spermatozoa Cryopreserved With and Without Cryoprotection.” Journal of Reproduction and Fertility 112, no. 1: 11–17. https://doi.org/10.1530/jrf.0.1120011.
  20. Wakayama, T., and R. Yanagimachi. 1998. “Development of Normal Mice From Oocytes Injected With Freeze‐Dried Spermatozoa.” Nature Biotechnology 16, no. 7: 639–641. https://doi.org/10.1038/nbt0798‐639.
  21. Watanabe, S. 2022. “DNA Damage in Human Sperm: The Sperm Chromosome Assay.” Reproductive Medicine and Biology 21: e12461. https://doi.org/10.1002/rmb2.12461.
  22. Wu, Z., X. Zheng, Y. Luo, et al. 2015. “Cryopreservation of Stallion Spermatozoa Using Different Cryoprotectants and Combinations of Cryoprotectants.” Animal Reproduction Science 163: 75–81. https://doi.org/10.1016/j.anireprosci.2015.09.020.
  23. Yanez‐Ortiz, I., J. Catalan, J. E. Rodriguez‐Gil, J. Miro, and M. Yeste. 2021. “Advances in Sperm Cryopreservation in Farm Animals: Cattle, Horse, Pig and Sheep.” Animal Reproduction Science 246: 106904. https://doi.org/10.1016/j.animreprosci.2021.106904.

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