Developing 3D vitrification for cat oocytes: viability, maturation and actin pattern

Three-dimensional (3D) culture systems can improve viability, development and architecture of cultured cells, including oocytes. Cryopreservation of ovarian follicles in 3D has already proved successful, thus we hypothesized its applicability on cumulus-oocyte complexes (COCs) to combine the advantages of 3D preservation and 3D culture. The aim of this study was to assess the effect of 3D vitrification of domestic cat oocytes on their viability, maturation ability and actin pattern. Immature grade I COCs were collected from spaying-derived ovaries. Vitrification was performed following a standard Cryotop protocol (equilibration solution (ES) – 15 minutes; vitrification solution (VS) – 90 seconds). Oocytes were vitrified as isolated COCs (2D vitrification) or after encapsulation in 1% alginate dropped into CaCl2 100 mM (3D vitrification). Following unsuccessful trials of 3D vitrification with standard times in ES and VS, these times were increased 2.25 or 2.5-fold (2.25 or 2.5-fold 3D vitrification). After warming, COCs were cultured in in vitro maturation medium, followed by denuding, staining with fluorescein diacetate for viability, ReadyProbes™ Reagent for actin and Hoechst for nuclear status. At least 3 replicates were performed for each. Data were analyzed by chi-square test; p<0.05. Viability (2D: 71%, 2.25-fold 3D: 38%, 2.5-fold 3D: 32%) and normal actin patterns (66%, 25%, 27%) were significantly higher in 2D than in 3D vitrification (n=26-32 COCs/group; p=0.002), with no differences between 2.25 and 2.5-fold. However, maturation rates of viable oocytes were significantly higher in 2.5-fold 3D (72%) than in 2D (41%; p=0.003), with no differences compared to 2.25-fold 3D (53%; p=0.238). In conclusion, although 3D vitrification decreased COCs viability, it allowed a higher proportion of viable oocytes to mature in vitro and thus become fertilizable. Further investigations will warrant information on the improved maturational competence of viable 3D-preserved oocytes and suggest whether embryonic development may benefit from this oocyte preservation technique. Funding: We acknowledge financial support under the National Recovery and Resilience Plan (PNRR), M4.C2.1.1, Call N. 1409 published on 14.9.2022 by the Italian Ministry of University and Research (MUR), funded by the European Union – NextGenerationEU– Project “Bio3versity” (P2022PRFM7, CUP G53D23007780001) Conflict of Interest: None to disclose Corresponding Author: martina.colombo@unimi.it