Asynchronic tetraploid complementation and embryo quality in domestic cat and Leopardus geoffroyi hybrid embryos

M DUQUE RODRIGUEZ; A GAMBINI; C. GUTNISKY; LD RATNER; S. RULLI; A. SESTELO; O. BRISKI; R. FERNANDEZ-MARTIN; P. D. CETICA; DF SALAMONE

Nueva York

Congreso; International Embryo Technology Society?s 46th Annual Meeting; 2021

International Embryo Technology Society

Fusion of 2-cell embryos generates tetraploid (4n) blastomeres with an increased commitment to trophectoderm. Complementation of embryos fromendangered species with 4n blastomeres derived from a phylogenetically related domestic species could improve healthy pregnancy establishmentafter embryo transfer in domestic females. However, generation, development, and quality of tetraploid complemented embryos in felids remainunstudied. Therefore, our objectives were (1) to evaluate tetraploidy of 2-cell fused embryos; (2) to analyse the blastocyst cell number, distributionafter synchronic (S) or asynchronic (AS) complementation, OCT4þ cells, DNA-fragmentation levels and CDX2 gene expression of IVFcomplemented embryos; and (3) to evaluate the developmental rates of tetraploid complemented Felis catus-Leopardus geoffroyi hybrid embryos.After ovariectomy, Felis catus oocytes were IVM and subjected to IVF. For Experiment 1 (n¼66), 2-cell embryos (2n) were exposed to two 30-msDC pulses at 8 kV cm1 electric field in fusion media. Fused (4n) and nonfused embryos were cultured in vitro in 50-mL drops of modified Tyrode?smedium. Karyotype analysis was performed at Day 4. For Experiment 2 (n¼24), zona-free IVF embryos were aggregated S (4-cell-2n/4-cell-2n) orAS (4-cell-2n/2-cell-2n and 4-cell-2n/1-cell-4n). For Experiment 3 (n¼36), blastocysts generated by AS complementation (4-cell-2n/2-cell-2n and4-cell-2n/1-cell-4n) were either fixed with 4% paraformaldehyde for immunofluorescence and terminal deoxynucleotidyl transferase dUTP nick endlabeling assay or saved in RNA-Later for RT-qPCR analysis. For this experiment, nonaggregated 2n and 4n blastocysts were used as a control. ForExperiment 4 (n¼60), IVM oocytes were co-incubated with Leopardus geoffroyi and Felis catus (control) spermatozoa and then 4-cell-2nheterologous embryos were complemented with 1-cell-4n homologous IVF embryos. Data were analysed by Fisher?s exact test. Our results showedthat 67% of the 2-cell fused embryos were 4n. Moreover, 82% of nonfused embryos were aneuploids compared with 78% of 2n embryos in the controlgroup. The AS complemented blastocysts (4-cell-2n/1-cell-4n and 4-cell-2n/2-cell-2n) had significantly higher cell number compared with Scomplemented (4-cell-2n/4-cell-2n) or noncomplemented embryos. The AS complementation also increased the number of OCT4þ cellsindependently of the ploidy of the embryos. Interestingly, AS tetraploid complemented embryos had significantly lower number of cells withfragmented DNA. No differences were found in CDX2 expression among complemented embryos; however, noncomplemented 2n blastocystsshowed a significantly lower expression compared with the others group. Finally, we observed that AS complementation of 2n hybrid embryos with4n homologous embryos reached similar blastocyst rates, 70 and 88%, respectively. Our findings support the use of 2-cell fused embryos to generate4n blastomeres and demonstrated that tetraploid complementation generates good quality embryos, providing evidences that encourage the use of thistechnology to improve the developmental competence of interspecific embryos after transfer.