Development of a three-dimensional vitrification protocol for domestic cat cumulus-oocyte complexes and comparison with standard vitrification

Three-dimensional (3D) systems may better mimic native tissue conditions and provide more physiologically relevant platforms for the study of cell function compared to traditional two-dimensional (2D) cultures. Cryopreservation of cells after encapsulation in 3D matrices is also likely to be a suitable alternative to traditional 2D approaches, as demonstrated with preantral ovarian follicles. Building on these findings, this study aimed to develop an alginate-based 3D vitrification protocol for domestic cat ( Felis catus ) cumulus-oocyte complexes (COCs). After determining the most suitable alginate concentration, encapsulated COCs were vitrified following a Cryotop-based protocol, with variations in exposure time to cryoprotectant (CPA) solutions. Permeation of dimethyl sulfoxide (DMSO) and ethylene glycol (EG) in COCs was quantified by gas chromatography-triple quadrupole mass spectrometry. Oocyte functional competence was assessed by in vitro maturation (IVM), viability, actin distribution, and embryo development rates after in vitro fertilization (IVF). The results showed that 1% alginate improved IVM of fresh oocytes (75% vs. 59.4% in 2D culture, p = 0.03), therefore was adopted for the development of the 3D vitrification protocol. Alginate-encapsulated COCs required longer exposure to CPA solutions (i.e., 2.5-fold increase compared to the standard protocol) to achieve intracellular concentrations of DMSO and EG comparable to non-encapsulated (2D) controls ( p = 0.8). While post-IVM viability was lower in 3D vitrified oocytes (35.9%) than in standard vitrified (2D) controls (75.6%, p < 0.00001), among viable vitrified oocytes there were no significant differences in maturation rates (range 44.6%−65.2%, p = 0.14) or actin distribution (intact pattern range 76.9%−93.3%, p = 0.31), regardless of the vitrification protocol (2D vs. 3D). Similarly, cleavage rates following IVF did not differ between 3D vitrified and standard 2D vitrified oocytes when the longest exposure time to CPA was used (i.e., 2.5-fold increase; 10.6% vs. 27.8%, p = 0.08). These findings demonstrate that 3D vitrification in alginate has the potential to be employed for the cryopreservation of domestic cat COCs, and provides a proof-of-concept for further optimization. Refining 3D cryopreservation techniques in domestic cats might contribute to the optimization of translational fertility preservation strategies.