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Scientific reports2026; 16(1); 24646; doi: 10.1038/s41598-026-54284-8

Water and cryoprotectant permeability of mature equine oocytes: experimental measurements and in silico predictions.

Abstract: Vitrification of equine oocytes is an essential practice for advancing assisted reproductive technologies however, its efficiency remains limited due to the lack of stage and species-specific information on membrane permeability parameters. In this study, water (L) and CPA permeability (P) for dimethyl sulfoxide (Me₂SO) and ethylene glycol (EG) were measured in in vitro matured (MII) equine oocytes. Cumulus oocyte complexes were obtained from abattoir ovaries or by ovum pick-up and matured in vitro for 30 h at 6% CO. Oocytes followed ideal osmometer behavior principles, with an osmotically inactive volume of 27%. L increased with temperature from 0.941 ± 0.082 µm min atm at 25 °C to 1.462 ± 0.084 µm min atm at 38.5 °C in Me₂SO, and from 0.889 ± 0.094 to 1.613 ± 0.066 µm min atm in EG. P also increased significantly with temperature: P rose from 0.175 ± 0.024 µm s to 0.353 ± 0.022 µm s and P from 0.138 ± 0.020 µm s to 0.349 ± 0.014 µm s. Activation energies (E) for L were 6.03 and 8.15 kcal mol, and for P were 9.60 and 12.69 kcal mol for Me₂SO and EG, respectively, measured at 25 °C and 38.5 °C. In silico predictions closely matched in vitro observations. Simulations predicted that oocytes recovered their original volume after 7 min 42 s at 38.5 °C and at 25 °C after 17 min 8 s. This study provides the first stage and species-specific permeability values for MII equine oocytes, supporting improved vitrification modeling.
© 2026. The Author(s).
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Publication Date: 2026-05-29 PubMed ID: 42215553PubMed Central: PMC13454351DOI: 10.1038/s41598-026-54284-8Google 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 measured and predicted how water and cryoprotectant substances move through the membranes of mature horse egg cells (equine oocytes) to improve freezing techniques for horse reproduction.

Background and Purpose

  • Vitrification, a rapid freezing process, is essential for preserving equine oocytes in assisted reproductive technologies.
  • The efficiency of vitrification is currently limited because membrane permeability properties vary with species and cell stage, and specific data for mature equine oocytes were lacking.
  • This research aimed to experimentally measure water and cryoprotectant permeability in mature (MII-stage) horse oocytes and use computer simulations to predict their behavior during vitrification.

Experimental Design and Methods

  • Oocytes were collected either from horse ovaries at abattoirs or via ovum pick-up and matured in vitro for 30 hours under controlled conditions (6% CO2).
  • The study focused on two cryoprotectants widely used in vitrification: dimethyl sulfoxide (Me₂SO) and ethylene glycol (EG).
  • Membrane permeability parameters measured included:
    • Water permeability (L) – the rate at which water passes through the oocyte membrane.
    • Cryoprotectant permeability (P) – the rate at which Me₂SO and EG pass through the membrane.
  • Measurements were taken at two temperatures: room temperature (25 °C) and physiological temperature (38.5 °C), to understand the effect of temperature on permeability.
  • Oocyte volume changes under osmotic stress were monitored, confirming they obeyed ideal osmometer behavior with 27% osmotically inactive volume (volume that does not exchange water).

Key Results

  • Water permeability (L) increased significantly with temperature:
    • From approximately 0.941 to 1.462 µm/min/atm with Me₂SO.
    • From approximately 0.889 to 1.613 µm/min/atm with EG.
  • Cryoprotectant permeability (P) also rose with temperature for both Me₂SO and EG:
    • Me₂SO: from about 0.175 to 0.353 µm/s.
    • EG: from about 0.138 to 0.349 µm/s.
  • Activation energies (E) were calculated to describe the energy needed for permeability processes:
    • For water transport: 6.03 kcal/mol (Me₂SO) and 8.15 kcal/mol (EG).
    • For cryoprotectant transport: 9.60 kcal/mol (Me₂SO) and 12.69 kcal/mol (EG).
  • Computer simulations based on these permeability measurements closely matched actual lab observations, validating the experimental data.
  • Simulations predicted that:
    • At 38.5 °C, oocytes would return to their original volume approximately 7 minutes 42 seconds after osmotic challenge.
    • At 25 °C, recovery time extends to about 17 minutes 8 seconds.

Conclusions and Implications

  • This study provides the first set of mature equine oocyte-specific data for water and cryoprotectant membrane permeability.
  • Understanding these parameters is critical for refining vitrification protocols to improve oocyte survival and reproductive outcomes in horses.
  • The results enable more accurate in silico vitrification modeling tailored to equine oocytes, which can guide optimized freezing and thawing procedures.
  • The temperature dependence of permeability highlights that warming and cooling rates must be carefully controlled in preservation protocols.
  • Overall, this research fills an important knowledge gap, supporting advances in equine assisted reproductive technologies by enabling more effective cryopreservation strategies.

Cite This Article

APA
Gago S, García-Martínez T, Diaz-Muñoz J, Acacio M, Catalán J, Miró J, Higgins AZ, Costa-Borges N, Mogas T. (2026). Water and cryoprotectant permeability of mature equine oocytes: experimental measurements and in silico predictions. Sci Rep, 16(1), 24646. https://doi.org/10.1038/s41598-026-54284-8

Publication

Scientific reports
ISSN: 2045-2322
NlmUniqueID: 101563288
Country: England
Language: English
Volume: 16
Issue: 1
PII: 24646

Researcher Affiliations

Gago, Sonia
  • Autonomous University of Barcelona, Barcelona, Spain.
  • Embryotools R&D Centre, Parc Científic de Barcelona, S08028, Barcelona, Spain.
García-Martínez, Tania
  • Neurobiology Laboratory, Research Unit, Hospital Universitari Son Espases (HSUE), Health Research Institute of Balearic Islands (IdISBa), Palma, Spain.
Diaz-Muñoz, Judith
  • Autonomous University of Barcelona, Barcelona, Spain.
Acacio, Mònica
  • Embryotools R&D Centre, Parc Científic de Barcelona, S08028, Barcelona, Spain.
Catalán, Jaime
  • Biotechnology of Animal and Human Reproduction (TechnoSperm), Institute of Food and Agricultural Technology, University of Girona, Girona, Spain.
  • Unit of Cell Biology, Department of Biology, Faculty of Sciences, University of Girona, Girona, Spain.
Miró, Jordi
  • Autonomous University of Barcelona, Barcelona, Spain.
Higgins, Adam Z
  • School of Chemical, Biological and Environmental Engineering, Oregon State University, Corvallis, USA.
Costa-Borges, Nuno
  • Embryotools R&D Centre, Parc Científic de Barcelona, S08028, Barcelona, Spain.
Mogas, Teresa
  • Autonomous University of Barcelona, Barcelona, Spain. teresa.mogas@uab.cat.

MeSH Terms

  • Animals
  • Oocytes / metabolism
  • Oocytes / cytology
  • Oocytes / drug effects
  • Cryoprotective Agents / metabolism
  • Horses
  • Water / metabolism
  • Female
  • Ethylene Glycol / metabolism
  • Cell Membrane Permeability
  • Vitrification
  • Computer Simulation
  • Dimethyl Sulfoxide / metabolism
  • Cryopreservation / methods
  • Permeability
  • Temperature

Grant Funding

  • DI00002 / Generalitat de Catalunya
  • 2021 SGR 00900 / Generalitat de Catalunya
  • PID2024-160962OB-I00 / Ministerio de Ciencia, Innovación y Universidades

Conflict of Interest Statement

Declarations. Competing interests: The authors declare no competing interests.

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