17 Effect of the zona-free aggregation on the developmental competence of kodkod (Leopardus guigna) embryos generated by interspecies somatic cell nuclear transfer

Abstract

The kodkod is considered a vulnerable species by the International Union for Conservation of Nature. Phylogenetically, the kodkod is classified in the Leopardus genus, which has only 36 chromosome pairs compared with the domestic cat, which has 38. The proposed hypothesis was that domestic cat oocytes are capable of reprogramming somatic cells from kodkod after interspecies somatic cell NT (SCNT), allowing the in vitro embryo development up to blastocyst stage. Five experimental groups were made based on the technology and culture system: (1) cat embryos generated by IVF (IVF), (2) cat embryos generated by SCNT (Ca1x), (3) aggregated cat embryos generated by SCNT (Ca2x), (4) kodkod embryos generated by interspecies SCNT (K1x), and (5) aggregated kodkod embryos generated by interspecies SCNT (K2x). Interspecies SCNT was performed using a zona-free method. Reconstructed embryos were activated by 2 electrical pulses of 140 kV cm⁻¹ for 40 µs and then incubated for 5 h in 10 μg mL⁻¹ of cycloheximide and 5 μg mL⁻¹ of cytochalasin B. Embryos were cultured in SOF media using the well of the well system in a 5% O₂, 5% CO₂, and 90% N₂ atmosphere at 38.5°C for 8 days. The morulae and blastocysts rates were estimated, and diameter of cloned blastocysts was measured. The relative expression of OCT4, SOX2, and NANOG was evaluated in blastocysts by RT-qPCR using the standard curve method; SDHA was used for normalization. The Kruskal-Wallis test was used to evaluate the developmental parameters and gene expression. The t-test was used to evaluate blastocyst diameter. Statistical differences were considered at P < 0.05. The number of replicates was IVF = 10, Ca1x = 8, Ca2x = 6, K1x = 3, and K2x = 8. The morulae rate was lower when clone embryos were cultured individually (IVF = 97/153, 63.4%; Ca2x = 28/51, 54.9%; K2x = 63/110, 57.3%; Ca1x = 48/126, 38.1%; K1x = 22/87, 25.3%; P < 0.05). In the domestic cat, blastocysts rate was higher in IVF (58/153, 37.9%) and Ca2x (28/51, 29.4%) groups than in the Ca1x group (21/126, 16.7%; P < 0.05). No blastocysts were generated in the K1x group (0/87), whereas 5.5% of blastocysts were obtained from the K2x (6/110; 5.5%); this was not statistically different compared with the K1x group (P > 0.05). No differences were found in blastocyst diameter between the Ca1x (220.4 µm) and Ca2x (251.2 µm) groups (P > 0.05). However, the diameter of the blastocysts from the K2x group (172.8 µm) tended to be lower than that of the blastocysts from the Ca2x group (P = 0.05). Regarding gene expression, only 1 of the 6 kodkod blastocysts expressed OCT4, and none expressed SOX2 and NANOG. On the other hand, the relative expression of OCT4 tended to decrease in blastocysts from the Ca1x and Ca2x groups compared with the IVF group (P = 0.09), but no differences were found in the expression of SOX2 and NANOG among groups (P > 0.05). In conclusion, after interspecies SCNT, domestic cat oocytes support the development of kodkod embryos until the morula stage. However, the embryo aggregation did not significantly improve the blastocyst rate and gene expression.