Oocytes vitribanking in the domestic cat model: developmental competence in 3D culture

In the domestic cat model, vitrified oocytes (VOs) produced promising results, however their potential to develop into late stage embryos is often impaired. Instead of culturing VOs in two-dimensional (2D) microdrops of medium (conventional system), the use of three-dimensional (3D) scaffolds could better maintain their functional integrity, contributing to oocyte in vitro outcomes. Therefore, the aim of this study was to investigate survival and embryo development (oocyte competence) of feline VOs cultured in 3D barium alginate microcapsules. Morphologically selected immature cumulus-oocyte complexes (COCs, n=250) were vitrified by Cryotop method, and viable oocytes after warming were in vitro matured for 24 hours in 2D (2D VOs) or 3D system, with fresh companion COCs in 1:1 ratio [3D VOs(+)] or without [3D VOs(-)]. After in vitro fertilization, presumptive zygotes were cultured for 7 days in 3D or 2D system, according to the maturation conditions. Viability of VOs after warming and number of blastomeres of embryos were assessed by fluorescent stainings (fluorescein diacetate/propidium iodide and bis-benzimide, respectively). Arcsin transformed data were analysed by one-way ANOVA followed by Tukey test, and the level of significance was set at p<0.05. After warming, 91.2% VOs were viable. Embryos were successfully obtained at similar proportions in each group [mean ± SD: 3D VOs(+) 0.18 ± 0.17; 3D VOs(-) 0.17 ± 0.12; 2D VOs 0.15 ± 0.09; p>0.05], but the use of the 3D system together with companion COCs was determinant for their development, since 3D VOs(+) were the only group to reach the blastocyst stage (mean ± SD: 0.01 ± 0.03, day 7). Degeneration rates did not differ among groups. These positive results prompt the use of 3D enriched culture systems for VOs to exploit this valuable germplasm and widen the potential of vitribanking.