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 30 h 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 µm min −1 atm −1 at 25 °C to 1.462 ± 0.084 µm min −1 atm −1 at 38.5 °C in Me₂SO, and from 0.889 ± 0.094 to 1.613 ± 0.066 µm min −1 atm −1 in EG. P s also increased significantly with temperature: P sMe₂SO rose from 0.175 ± 0.024 µm s −1 to 0.353 ± 0.022 µm s −1 and P sEG from 0.138 ± 0.020 µm s −1 to 0.349 ± 0.014 µm s −1 . Activation energies (E a ) for L p were 6.03 and 8.15 kcal mol −1 , and for P s were 9.60 and 12.69 kcal mol -1 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.