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

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 _p ) and CPA permeability ( P _s ) 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 30h at 6% CO ₂ . Oocytes followed ideal osmometer behavior principles, with an osmotically inactive volume of 27%. L _p increased with temperature from 0.941 ± 0.082 µmmin⁻¹atm⁻¹ at 25°C to 1.462 ± 0.084 µmmin⁻¹atm⁻¹ in Me₂SO, and from 0.889 ± 0.094 to 1.613 ± 0.066 µmmin⁻¹ atm⁻¹ in EG. P _s also increased significantly with temperature: P _sMe₂SO rose from 0.175 ± 0.024 µm/s to 0.353 ± 0.022 µm/s and P _sEG from 0.138 ± 0.020 µm/s to 0.349 ± 0.014 µm/s. Activation energies (E _a ) were 6.03 and 8.15 kcal/mol for L _p , and 9.60 and 12.69 kcal/mol for P _s for Me₂SO and EG, respectively. In silico predictions closely matched in vitro observations. Simulations predicted that oocytes recovered their original volume after 7min 42s at 38.5°C and at 25°C after 17min 8s. This study provides the first stage and species-specific permeability values for MII equine oocytes, supporting improved vitrification modeling.