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Scientific reports2025; 15(1); 43190; doi: 10.1038/s41598-025-24885-w

Quantitative holographic analysis in stallion spermatozoa following cryopreservation.

Abstract: This study employs Holographic tomography (HT) to examine structural and biophysical changes occurring during the cryopreservation of stallion sperm. HT is an advanced imaging technique that integrates digital holography with tomography to achieve three-dimensional, quantitative reconstructions of objects without the need for treatment or reporter dyes. By using refractive index (RI) intervals to represent specific structural regions of sperm cells, variations in optical density, surface area, volume, and dry mass across different cryopreservation extenders and donors have been quantified. Three main sperm components, (i) nuclear region, (ii) post-acrosomal region and midpiece and (iii) whole cell were identified and discriminated based on different RI. Our results revealed significant differences in volume of post-acrosomal region and midpiece among stallions as well as between fresh and frozen/thawed sperm, whereas no significant differences were observed between freezing extenders, aligning with our findings on sperm kinetics. A significant stallion-extender interaction underscores the need to personalize the sperm freezing process.
© 2025. The Author(s).
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Publication Date: 2025-12-04 PubMed ID: 41345140PubMed Central: PMC12680650DOI: 10.1038/s41598-025-24885-wGoogle 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 uses Holographic Tomography (HT), a cutting-edge imaging method, to analyze the detailed structural and biophysical changes that occur in stallion sperm cells during the process of cryopreservation (freezing and thawing).
  • It quantitatively measures variations in optical properties and morphological features of sperm cells from different stallions and freezing extenders, highlighting the importance of personalized approaches to sperm freezing.

Introduction to Holographic Tomography (HT)

  • HT combines digital holography with tomography to create three-dimensional, quantitative images without needing dyes or invasive treatments.
  • This allows non-destructive examination of living cells by measuring their refractive indices (RI), which correspond to cellular density and composition.
  • By using RI intervals, researchers can map specific sperm structural regions, such as the nucleus and midpiece, differentiating them based on optical density.

Objectives and Methods

  • To use HT for detailed, quantitative analysis of sperm cells before and after cryopreservation.
  • To identify changes in key parameters such as volume, surface area, dry mass, and refractive index distributions within sperm components.
  • To investigate the influence of different cryopreservation extenders and donors (different stallions) on sperm integrity and structure.

Key Components Analyzed

  • Nuclear region: The head part containing genetic material.
  • Post-acrosomal region and midpiece: Critical for energy production and fertilization processes.
  • Whole cell: The entire spermatozoon encompassing all subregions for comprehensive assessment.

Findings

  • Significant differences were observed in the volume of the post-acrosomal region and midpiece between fresh and frozen/thawed spermatozoa, indicating structural alterations due to cryopreservation.
  • Variability between different stallions was also noted, suggesting individual biological differences influence sperm response to freezing.
  • No significant differences were found between the different freezing extenders tested, which aligns with similar sperm kinetic (motility) results.
  • The interaction between stallion and extender was significant, pointing to the importance of personalized sperm freezing protocols tailored to individual stallions for optimal preservation.

Implications and Importance

  • The use of HT provides a powerful, label-free tool for monitoring the integrity of sperm cells during cryopreservation and assessing subtle structural changes that may impact fertility.
  • Findings emphasize that cryopreservation-induced damage is not uniform; it depends on both individual stallion characteristics and the preservation protocol used.
  • This suggests the necessity for customized cryopreservation approaches to improve post-thaw sperm quality and breeding success.
  • The methodology could be extended to other species or cell types where cryopreservation is essential.

Conclusions

  • Holographic Tomography effectively quantifies structural and biophysical changes in stallion sperm due to cryopreservation.
  • Distinct sperm regions can be discriminated using refractive index data, enhancing the understanding of cryoinjury location and degree.
  • Individual stallion variation and personalized extender choices both play a critical role in sperm preservation outcomes.
  • Future work should focus on optimizing cryopreservation protocols tailored to individual sperm characteristics to maximize fertility potential.

Cite This Article

APA
Ferrara MA, Preziosi G, Boni R, Ruggiero R, Gualandi SC. (2025). Quantitative holographic analysis in stallion spermatozoa following cryopreservation. Sci Rep, 15(1), 43190. https://doi.org/10.1038/s41598-025-24885-w

Publication

Scientific reports
ISSN: 2045-2322
NlmUniqueID: 101563288
Country: England
Language: English
Volume: 15
Issue: 1
Pages: 43190
PII: 43190

Researcher Affiliations

Ferrara, Maria Antonietta
  • Institute of Applied Sciences and Intelligent Systems , Unit of Naples Italian National Research Council (ISASI-CNR) , Via Pietro Castellino 111, 80131, Napoli, Italy. antonella.ferrara@na.isasi.cnr.it.
Preziosi, Graziano
  • Institute of Applied Sciences and Intelligent Systems , Unit of Naples Italian National Research Council (ISASI-CNR) , Via Pietro Castellino 111, 80131, Napoli, Italy.
Boni, Raffaele
  • Department of Basic and Applied Sciences (DiSBA), University of Basilicata, Via dell'Ateneo Lucano, 10, 85100, Potenza, Italy.
Ruggiero, Raffaella
  • Department of Basic and Applied Sciences (DiSBA), University of Basilicata, Via dell'Ateneo Lucano, 10, 85100, Potenza, Italy.
Gualandi, Stefano Cecchini
  • Department of Basic and Applied Sciences (DiSBA), University of Basilicata, Via dell'Ateneo Lucano, 10, 85100, Potenza, Italy.

MeSH Terms

  • Animals
  • Male
  • Cryopreservation / methods
  • Horses
  • Spermatozoa / cytology
  • Spermatozoa / physiology
  • Holography / methods
  • Semen Preservation / methods
  • Semen Preservation / veterinary

Grant Funding

  • PRIN EVIDENCE - code number: 2022ERKW4P - CUP MASTER: C53D23005260006 / Italian Ministry for University and Research (MUR)

Conflict of Interest Statement

Competing interests: The authors declare no competing interests.

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