Effect of a three-dimensional co-culture system on feline oocyte competence to develop into an embryo

Three-dimensional (3D) in vitro culture has been developed to improve the full competence of mammalian oocytes. These culture systems mimic the in vivo spatial arrangement and physiological conditions for the oocytes. In addition, the co-culture with companion cells, such as cumulus-denuded oocytes (CDOs) or cumulus cells (CCs) clumps, has been shown to improve the oocyte maturation and subsequent embryo development in different species, including domestic cat [1,2,3]. The aim of this study was to investigate the effect of an enriched culture system (3D barium alginate microcapsules associated with companion CDOs) on the in vitro embryo development of feline cumulus-oocyte complexes (COCs). Grade I COCs (n=545; 8 to 12 replicates) were collected from adult ovaries and 187 were mechanically deprived of CCs to obtain the CDOs. The COCs were co-cultured with CDOs in barium alginate (Sigma Chemical Co., MO, USA) microcapsules (3D system) in Quinn\u2019s Advantage Protein Plus Blastocyst medium (SBP, SAGE\uae In Vitro Fertilization, Trumbull, Connecticut, USA) with 75 UI FSH + 75 UI LH (Menogon\uae, Ferring Pharmaceuticals, Switzerland), 10 ng/ml of epidermal growth factor (EGF), antibiotics (AB) and 0.6 mM cysteine, in a controlled atmosphere (38.5\ub0C and 5% CO2 in air) for 24 h. Control groups of COCs alone were cultured separately. For in vitro fertilization, chilled epididymal spermatozoa were selected by swim-up treatment in SBP and the oocytes were inseminated with 0.75-1 x 106 motile spermatozoa/ml. At 18-24 h post-insemination, presumptive zygotes were in vitro cultured for 7 days in 3D microcapsules in SBP with 5% of FCS and AB (National Institutes of Health, Bethesda, MD, USA). The oocyte meiotic progression after IVM and embryo stages at the end of culture were determined by fixation and staining with bis-benzimide (Hoechst 33342; Sigma). Data were analyzed by Chi-square test (p<0.05). The meiosis resumption of COCs (in co-culture: 86.1% and cultured alone: 88.5%) was significantly higher than that of CDOs (40.2%; p<0.00001). The COCs cultured separately showed the highest full maturational (TI-MII) rates (p