20 Development and Oct4/Cdx2 gene expression of Puma concolor, Leopardus geoffroyi, and Panthera onca hybrid embryos produced using domestic cat oocytes

The development of assisted reproductive technologies (ARTs) in Felidae species has been proposed to guarantee the genetic viability of species that are on the verge of extinction. Investigation of sperm cryopreservation and IVF have been studied in several Felidae species, but the lack of viable homologous oocytes makes assessment of sperm fertilization capacity after cryopreservation difficult. We propose to evaluate the capability of Puma concolor (Pc), Leopardus geoffroyi (Lg), and Panthera onca (Po) cryopreserved sperm to induce domestic cat oocyte fertilization and subsequent pre-implantation embryo development. Additionally, we evaluated blastocyst quality by analysing the gene expression patterns of pluripotency (Oct4) and differentiation (Cdx2) related markers using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). After ovariectomy, domestic cat (Felis catus, Fc) cumulus–oocyte complexes were matured invitro at 38.5°C in a humidified atmosphere of 6.5% CO2 and 21% O2 for 22h. Cryopreserved sperm from Pc, Lg, and Po were used for heterologous IVF. The sperm was frozen using Tes-Tris–based diluent (TEST), and survival, sperm motility, and acrosome integrity were evaluated. The quality post-thaw was satisfactory. The survival was high, sperm motility (65%) did not decrease during incubation post-thaw, and sperm with intact acrosome was 62% for all species. Briefly, straws were thawed by exposing them to air for 10s and then immersing in a 37°C water bath for 30s. The contents of the straws were poured into prewarmed sterile 1.5-mL microtubes. The sperm suspension was diluted (1:3 vol/vol) by the slow (drop by drop) addition of a modified Tyrode’s solution (Pope et al. 2006 Methods Molec. Biol. 254, 227-244). For IVF, invitro matured oocytes (n=635) were co-incubated with 0.5×105 mL−1 motile spermatozoa at 38.5°C in a humidified atmosphere of 6.5% CO2 and 21% O2 for 18 to 20h. Presumptive zygotes were cultured invitro in 50-µL drops of modified Tyrode’s medium in a humidified gas mixture of 5% CO2, 5% O2, and 90% N2 at 38.5°C. Cleavage was determined 48h post-fertilization and blastocyst stage was evaluated at Day 8 and stored in RNA-Later at −20°C until RT-qPCR analysis. Data were analysed by Fisher’s exact test using GraphPad Prism 6.0 (GraphPad Inc.), and differences were considered significant at P<0.05. We observed significantly lower cleavage rates of Po hybrid (hPo) embryos compared with hPc, hLg, and control Fc embryos (41% vs. 57%, 68%, and 58%, respectively). Additionally, we found that the blastocyst rate was higher using Lg sperm compared with sperm of other groups [hLg (50%) vs. hPc (29%), hPo (21%), and Fc (27%)].