CONICET | Buscador de Institutos y Recursos Humanos

Asynchronic tetraploid complementation and embryo quality in domestic cat and Leopardus Geoffroyi hybrid embryosM Duque Rodriguez*1,3, A Gambini1,3, Cynthia Gutnisky5, LD Ratner1,3, SB Rulli4, A Sestelo2, O Briski1,3, R Fernandez Martin1,3, Pablo Daniel Cetica5, DF salamone1,3;1Facultad de Agronomia, Universidad de Buenos Aires (FAUBA), Buenos Aires, Argentina. 2Laboratory of Reproductive Biotechnology, Buenos Aires eco-park . Buenos Aires, Argentina, 3National Scientific and Technical Research Council (CONICET), Buenos Aires, Argentina, Buenos Aires, Argentina, 4Instituto de Biología y Medicina Experimental- Consejo Nacional de Investigaciones Científicas y Técnicas, Buenos Aires, Argentina, 5Universidad de Buenos Aires, Facultad de Ciencias Veterinarias, Instituto de Investigación y Tecnología en Reproducción Animal (INITRA). Fusion of 2cell embryos generate tetraploid (4n) blastomeres with an increased commitment to trophectoderm. Complementation of embryos from endangered species with 4n blastomeres derived from a phylogenetically related domestic species could improve healthy pregnancy establishment after embryo transfer in domestic females. However, generation, development and quality of tetraploid complemented embryos in felids remain unstudied. Therefore, our objectives were 1) to evaluate tetraploidy of 2cell fused embryos, 2) to assess blastocyst cell number and distribution after synchronic (S) or asynchronic (AS) complementation of IVF cat embryos, 3) to analyze the number of OCT4+ cells, DNA-fragmentation levels and CDX2 gene expression of IVF complemented embryos and 4) to evaluate the developmental rates of tetraploid complemented Felis catus (FC)-Leopardus Geoffroyi(LG) hybrid embryos.FC oocyte were in vitro matured and subjected to IVF. For experiment 1, 2-cell embryos (2n) were exposed to two 30 ms DC pulses at 8 kV/cm electric field in fusion media. Fused (4n) and non-fused embryos were cultured in vitro in 50 μl drops of modified Tyrode?s medium. Karyotype analysis was performed at day 4. For experiment 2, zona free IVF embryos were aggregated S (4cell-2n/4cell-2n) or AS (4cell-2n/2cell-2n and 4cell-2n/1cell-4n). For experiment 3, blastocyst generated by AS complementation (4cell-2n/2cell-2n and 4cell-2n/1cell-4n) were either fixed with 4% paraformaldehyde for inmunofluorescence and TUNEL assay or saved in RNA-Later for RT-qPCR analysis. For this experiment, non-aggregated 2n and 4n blastocyst were used as a control. For experiment 4, in vitro matured oocytes were co-incubated with LG and FC (control) spermatozoa and then 4cell-2n heterologous embryos were complemented with 1cell-4n homologous IVF embryos. Data were analyzed by Fisher?s exact test. Our results showed that 67% of the 2cell fused embryos were 4n. Moreover, 82% of non-fused embryos were aneuploids compared to 78% of 2n embryos in the control group. AS complemented blastocyst (4cell-2n/1cell-4n and 4cell-2n/2cell-2n) had significantly higher cell number compared to S complemented (4cell-2n/4cell-2n) or non-complemented embryos. AS complementation also showed to increase the number of OCT4+ cells independently of the ploidy of the embryos. Interestingly, AS tetraploid complemented embryos had significantly lower number of cells with fragmented DNA. No differences were found in CDX2 expression among complemented embryos, however, non-complemented 2n blastocyst showed a significantly lower expression compared to the others group. Finally, we observed that AS complementation of 2n hybrids embryos with 4n homologous embryos reached similar blastocysts rates, 70% and 88% respectively. Our findings support the use of 2cell fused embryos to generate 4n blastomeres, and demonstrated that tetraploid complementation generate good quality embryos providing evidences that encourage the use of this technology to improve the developmental competence of interspecific embryos after transfer. KEYWORDS Tetraploid complementation