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.