In vitro and in vivo development of marbled cat and flat-headed cat embryos derived by somatic cell nuclear transfer

Abstract

EXP. I was conducted to find appropriate in vitro maturation (IVM), fusion and activation protocols for somatic cell nuclear transfer (SCNT) in felid species. The oocytes were obtained from ovaries of ovariohysterectomized domestic cats (DC). The donor cells were obtained from a marbled cat (MC) died in captivity. The developmental rates of MC-DC (donor cell-recipient oocyte) cloned embryos derived from oocytes cultured for 24 h were greater than those cultured for 36 and 42 h (morula stage; 9.2, 4.3 and 0%, respectively, P<0.05). The fusion efficiency of the MC-DC couplets using the 2 pulses of 2.1 and 2.4 kV/cm for 80 µs and 3 pulses of 2.4 kV/cm for 50 µs was 46, 48.5 and 67.7% (P > 0.05). The development of cloned embryos derived from MC and DC fibroblast cells was not different (morula rate; 8 vs. 8.3%, P>0.05). Addition of the cytochalasin B in the activation medium provided a grater blastocyst rate compared to that without the cytochalasin B (8.3 vs. 35%, P<0.05). In conclusion, DC oocyte matured at 24 h is suggested to be used as recipient ooplasm for cloned MC embryos. The MC-DC couplets are fused efficiently by the introducing of 3 pulses of 2.4 kV/cm for 50 [mu]s. The MC fibroblast cells demonstrate the possibility to be reprogrammed in the DC oocytes, resulting in the development to the morula stage. Exposure of mature DC oocytes to cycloheximide and cytochalasin B after electrical stimulation improves the rate of blastocyst formation. EXP. II was conducted to investigate 1) the development of MC and FC cloned embryos reconstructed from DC and rabbit (RB) oocytes and 2) the effect of individual cell line and gender on FC-DC cloned embryo production. Using DC oocytes, the FC-DC couplets yielded a greater percentage of morula (53%) than those of MC- (23%) and DC-DC couplets (11%, P<0.05). However, the number of couplets achieving blastocyst expansion did not differ (MC; 5, FC; 8.3 and DC; 8.5%, P>0.05). Using RB oocytes, The blastocyst rate of FC- (8.5%) and DC-RB embryos (7.7%) was lower than those of RB-RB (14.3%, P<0.05) but not MC-RB embryos (11.5%, P>0.05). The variations of fusion and developmental rate through morula of FC-DC couplets were observed among three cell lines. The development of FC-DC embryos derived from female and male donor cells was not different (P>0.05). These results indicated that the MC and FC embryos could be produced successfully by SCNT of either DC or RB oocytes. Individual cell line but not gender of donor cells influenced the development of cloned FC embryos. EXP. III was conducted to investigate 1) in vitro development of DC IVF embryos and their transfer to recipients and 2) pregnancy establishment of recipients receiving FC-DC cloned embryos. The IVM oocytes were co-cultured with sperm for 18 h. The blastocyst rate and cell number of blastocysts derived from cleaved embryos collected at 18-27 h were greater than those cleaved during >27-42 h (61.4 vs. 18.6% and 106 +- 43 vs. 60 +- 27 cells, P<0.05). Total 150 of cleaved IVF embryos were transferred to 6 recipients (mean 25 +- 9). All recipients became pregnant resulting in five kittens delivered from three recipients. The recipients receiving FC-DC cloned (n=5, 41.4 +- 13 embryos), FC- and DC-DC cloned (n=5, 29.8 +- 20.8 embryos) and FC-DC cloned and DC IVF embryos (n=4, 55 +- 15 embryos) were observed the pregnancy establishment. One of the recipients receiving FC-DC cloned and DC IVF embryos became pregnant and delivered IVF kittens. In conclusion, the pregnancy could be established after DC IVF embryo transfers, and embryos cleaved early at 18-27 h pi would rather be used for transfer than those cleaved later due to the greater development and quality. The FC- and DC-DC cloned offspring were not able to be produced which may be caused by the low development and quality of the embryos.