81 Generation of presumptive domestic cat tetraploid embryos and its application for asynchronic complementation with diploid blastomeres

Abstract

Tetraploid complementation has been extensively used to verify the pluripotency of stem cells and also for improving placenta formation when tetraploid embryos are aggregated synchronously or asynchronously with diploid (2n) embryos. Generation of tetraploid embryos can be achieved by the electric fusion of a 2-cell embryo. However, the optimal electric intensity pulse to generate tetraploid embryos has not been studied in the feline. The aims of this study were to (1) evaluate the optimal fusion conditions to achieve the highest fusion rate without affecting embryo developmental competence, (2) compare the in vitro development of synchronic and asynchronic aggregated domestic cat IVF embryos, and (3) assess pre-implantation development of embryos generated by asynchronic complementation of presumptive 1-cell tetraploid embryos with diploid blastomeres. Domestic cat cumulus-oocyte complexes were matured in vitro on 21% O₂ in air at 38.5°C for 22 h. The IVF embryos were generated by co-incubation of in vitro-matured oocytes with 2 × 10⁶ motile spermatozoa mL⁻¹ on 21% O₂ in air at 38.5°C for 18 to 20 h. After 24 h of IVF, 2-cell embryos were selected. For Experiment 1, membrane fusion of 2-cell IVF embryos (n = 164) was performed with two 30-ms DC pulses at different electric field (0.8, 2, 4, and 8 kV/cm) in fusion media (Mannitol, MgSO₄, CaCl₂, and polyvinyl alcohol). Presumptive fused embryos and nonfused were cultured in vitro in 50-µL drops of modified Tyrode’s medium on 6.5% CO₂ in air at 38.5°C (Pope et al. 2006 Methods in Molecular Biology 254, 227-244). Cleavage was determined 24 h after pulse. For Experiment 2, zona pellucida-free IVF embryos (n = 110) were synchronically (two 4-cell embryos) or asynchronically (one 4-cell embryo and one 2-cell embryo) aggregated in 1 microwell. For Experiment 3, 1-cell presumptive tetraploid embryo (2-cell fused embryo) was asynchronically complemented with a 4-cell embryo (n = 38). For all experiments, blastocyst stage was evaluated at Day 8, and embryos presenting more than one structure per microwell were considered non-aggregated. Data were analysed by Fisher’s exact test using GraphPad Prism 6.0 (GraphPad Inc., San Diego, CA, USA), and differences were considered significant at P < 0.05. The highest fusion rates (30 and 46%) with the best developmental competence (31 and 46%) were observed with 4 and 8 Kv/cm electric pulses, respectively. Electric fusion did not affect the embryo developmental competence. We observed that synchronic and asynchronic complementation reached similar blastocysts rates (54 and 65%, respectively), indicating that both techniques are suitable for tetraploid embryo complementation. Finally, when presumptive tetraploid embryos were asynchronically complemented with diploid blastomeres, the high blastocyst rate (90%) was obtained from embryos that form only one structure (aggregated embryos). Further experiments will be performed to track the distribution of cells using mitotrackers after complementation using tetraploid IVF and diploid somatic cell nuclear transfer embryos.