41 EFFICIENT STRATEGY FOR INTERSPECIFIC CLONING IN FELIDS

Abstract

Most of the 36 species of wild felids are at a level of threat, and interspecific SCNT (iSCNT) comes as a strategy to contribute to these species conservation. The aim of this study was to evaluate the effect of embryo aggregation in cheetah (Ch, Acinonyx jubatus), bengal (Ben, a hybrid between Felis silvestris and Prionailurus bengalensis), and domestic cat (DC, Felis silvestris) embryos generated by cloning. DC oocytes were in vitro matured and zona-free SCNT (with DC fibroblasts) or iSCNT (with Ch or Ben fibroblasts) was performed. The reconstructed embryos were activated with 5 μM ionomycin and 1.9 mM 6-DMAP, and cultured in SOF using microwells. Cloned embryos were cultured individually or as 2-embryo aggregates. The experimental groups were Ch1X, Ch2X, Ben1X, Ben2X, and the control groups were DC1X and DC2X. Embryo development was compared by Fisher's exact test (P ≤ 0.05). Embryo aggregation improved cleavage (Day 2) and blastocyst (Day 7) rates per well in all the groups (87.2% v. 96.7%, 83.8% v. 93.3% and 87.6% v. 98.2% for cleavage; and 13.7% v. 28.6%, 33.3% v. 43.8% and 27.4% v. 47.7% for blastocyst, for Ch1X (n = 102), Ch2X (n = 91), Ben1X (n = 154), Ben2X (n = 105), DC1X (n = 113), and DC2X (n = 109), respectively. Moreover, the Ch2X blastocyst rate was statistically similar as the control group DC1X. The mean total cell numbers of the blastocysts obtained were 264 ± 211 and 400.8 ± 97 for Ch1X and Ch2X, 278 ± 62 and 517 ± 104 for Ben1X and Ben2X, 385 ± 127 and 625 ± 183 for DC1X and DC2X, respectively. Although no statistical differences were obtained between the 1X and 2X groups, the 2X groups nearly doubled the average number of cells compared with the 1X groups. Blastocysts were also classified as grade 1 (expanded blastocysts with a well-defined ICM), grade 2 (expanded blastocysts without a well-defined ICM), and grade 3 (not expanded blastocysts). This classification showed an increase in grade 1 DC2X blastocyst compared with DC1X blastocysts (36.7% v. 16.1%), but no differences were observed in the other species. Expression of OCT-4 was assessed by inmunocytochemistry. The cheetah blastocysts markedly over-expressed this protein: the percentage of cells that expressed OCT-4 in Ch1X, Ch2X, Ben1X, Ben2X, DC1X, and DC2X was 88.2, 80.2, 46.3, 45.4, 51, and 47.4%, respectively, with statistical differences among all the groups except Ben1X and Ben2X. The proportion of OCT-4 expressing cells over total cell numbers was analysed by the difference of proportions test (P ≤ 0.05). In conclusion, iSCNT resulted in high rates of blastocyst formation, especially when embryo aggregation was applied. This strategy has not been previously evaluated in felids or iSCNT procedures, and has been demonstrated to improve blastocyst formation, the number of cells in the 3 groups, and the blastocyst quality in the DC. Other pluripotent genes besides OCT-4 should be studied to determine whether the overexpression of this gene in cheetah embryos is the consequence of an inefficient nuclear reprogramming that prevents a correct regulation. Finally, the iSCNT and embryo aggregation could contribute to species conservation in felids.