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

Abstract

Fusion of 2-cell embryos generates 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 2-cell fused embryos; (2) to analyse the blastocyst cell number, distribution after synchronic (S) or asynchronic (AS) complementation, OCT4+ cells, DNA-fragmentation levels and CDX2 gene expression of IVF complemented 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 (n = 66), 2-cell embryos (2n) were exposed to two 30-ms DC pulses at 8 kV cm⁻¹ electric field in fusion media. Fused (4n) and nonfused embryos were cultured in vitro in 50-μL drops of modified Tyrode's medium. Karyotype analysis was performed at Day 4. For Experiment 2 (n = 24), zona-free IVF embryos were aggregated S (4-cell-2n/4-cell-2n) or AS (4-cell-2n/2-cell-2n and 4-cell-2n/1-cell-4n). For Experiment 3 (n = 36), blastocysts generated by AS complementation (4-cell-2n/2-cell-2n and 4-cell-2n/1-cell-4n) were either fixed with 4% paraformaldehyde for immunofluorescence and terminal deoxynucleotidyl transferase dUTP nick end labeling assay or saved in RNA-Later for RT-qPCR analysis. For this experiment, nonaggregated 2n and 4n blastocysts were used as a control. For Experiment 4 (n = 60), IVM oocytes were co-incubated with Leopardus geoffroyi and Felis catus (control) spermatozoa and then 4-cell-2n heterologous embryos were complemented with 1-cell-4n homologous IVF embryos. Data were analysed by Fisher's exact test. Our results showed that 67% of the 2-cell fused embryos were 4n. Moreover, 82% of nonfused embryos were aneuploids compared with 78% of 2n embryos in the control group. The AS complemented blastocysts (4-cell-2n/1-cell-4n and 4-cell-2n/2-cell-2n) had significantly higher cell number compared with S complemented (4-cell-2n/4-cell-2n) or noncomplemented embryos. The AS complementation also increased 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, noncomplemented 2n blastocysts showed a significantly lower expression compared with the others group. Finally, we observed that AS complementation of 2n hybrid embryos with 4n homologous embryos reached similar blastocyst rates, 70 and 88%, respectively. Our findings support the use of 2-cell fused embryos to generate 4n blastomeres and demonstrated that tetraploid complementation generates good quality embryos, providing evidences that encourage the use of this technology to improve the developmental competence of interspecific embryos after transfer.