Ooplasm transfer and interspecies somatic cell nuclear transfer: heteroplasmy, pattern of mitochondrial migration and effect on embryo development.

Although interspecies somatic cell nuclear transfer (iSCNT) has potential applications in the conservation of exotic species, an in vitro developmental block has been observed in embryos produced by this approach. It has been suggested that mitochondrial mismatch between donor cell and recipient oocyte could cause embryonic developmental arrest. A series of experiments was conducted to investigate the effect of mixed mitochondrial populations (heteroplasmy) on early development of iSCNT-derived cloned embryos. The effect of combining the techniques of ooplasm transfer (OT) and somatic cell nuclear transfer (SCNT) was examined by monitoring in vitro embryonic development; the presence and pattern of migration of foreign mitochondria after OT was analysed by MitoTracker staining. In addition, the effect of transferring caprine ooplasm (iOT) into the bovine enucleated oocytes used in iSCNT was analysed. There was no significant effect of the sequence of events (OT-SCNT or SCNT-OT) on the number of fused, cleaved, blastocyst or hatched blastocyst stage embryos. MitoTracker Green staining of donor oocytes used for OT confirmed the introduction of foreign mitochondria. The distribution pattern of transferred mitochondria most commonly remained in a distinct cluster after 12, 74 and 144 h of in vitro culture. When goat ooplasm was injected into bovine enucleated oocytes (iSCNT), there was a reduction (p < 0.05) in fusion (52 vs. 82%) and subsequent cleavage rates (55 vs. 78%). The procedure of iOT prior to iSCNT had no effect in overcoming the 8- to 16-cell in vitro developmental block, and only parthenogenetic cow and goat controls reached the blastocyst (36 and 32%) and hatched blastocyst (25 and 12%) stages, respectively. This study indicates that when foreign mitochondria are introduced at the time of OT, these organelles tend to remain as distinct clusters without relocation after a few mitotic divisions. Although the bovine cytoplast appears capable of supporting mitotic divisions after iOT-iSCNT, heteroplasmy or mitochondrial incompatibilities may affect nuclear-ooplasmic events occurring at the time of genomic activation.

Production of nuclear transfer llama (lama glama) embryos from in vitro matured llama oocytes.

To date, there have been no reports of somatic cell nuclear transfer in llamas. The application of this methodology to the camelid industry could be helpful in the propagation of genetically valuable animals. The objective of this study was to produce nuclear transfer llama embryos comparing the development of these llama embryos cultured in either CR1aa medium (treatment A) or G1.2 medium (treatment B) medium. Llamas were superstimulated by double dominant follicle reduction 12 days apart, followed by pFSH administered in daily descending doses over a 3-day interval (total dose of 200 mg). Animals were ovariectomized by flank laparotomy, follicles were aspirated from excised ovaries and oocytes were in vitro matured for a 30-h period. Adult female llama fibroblasts were used as donor karyoplasts and injected into enucleated llama oocytes. Embryo development was assessed after 2 days of culture. A total of 307 follicles were aspirated from nine treated females, resulting in 298 (97%) oocytes recovered. Of a total of 229 evaluated oocytes, 120 (52%) achieved nuclear maturation. Of a total of 80 reconstructed couplets, 50 (62.5%) were successfully fused. Subsequent cleavage rates were 32 and 40% for treatments A and B, respectively, with no significant difference (p < 0.05) detected between treatment groups. A total of 11 embryos (8-cell to morula stages) were transferred to synchronized recipient llamas. Ultrasonography at 14 days post-transfer indicated that no pregnancies were established. This study shows that nuclear transfer can be successfully applied to the production of llama embryos. Further research is needed to identify optimal parameters to improve efficiency of nuclear transfer in this species.