Efficiency of sheep somatic cloning depending on fusion conditions and karyoplast preparation

The aim of this study was to evaluate the impact of the frequency of electrofusion on the efficiency of sheep cloning and to assess the dependence of this effect on the duration of serum starvation of somatic cells (SC).
Materials and Methods. Fetal fibroblasts at passages IV-V were used as donor cells for somatic cloning. To arrest the SCs in the G0/G1 phase of the cell cycle, after reaching near-confluence (approximately 90%), the cells were subjected to serum starvation (SS) for 24 and 48 hours. Recipient oocytes were collected from post-mortem sheep ovaries, matured in vitro, and reconstructed through enucleation followed by the transfer of SC into their perivitelline space. Electrofusion was used to combine the oocyte/SC complexes. The oocyte/SC complexes were fused using electrofusion in a buffer containing 270 mM mannitol, which involved the application of two sequential direct current pulses at 40 V for 20 µs. Complexes that did not fuse after the first treatment were subjected to a second round of electrofusion. The obtained cytohybrids were activated using ionomycin, followed by a 4-hour incubation in the presence of 6-dimethylaminopurine and cycloheximide, and subsequently cultured for 48 hours to assess embryonic development.
Results. In this study, a total of 504 oocytes were reconstructed. A statistically significant higher number of cleaved cytohybrids was observed from the first fusion when fetal fibroblasts subjected to 48-hour SS were used as karyoplasts. The number of embryos obtained from the second fusion was not dependent on the duration of SS in SCs. When SCs subjected to 48-hour SS were used, statistically significant differences were found between the proportion of cleaved cytohybrids from the first and second fusions: 62,2±21,48 % and 31,4±26,43%, respectively (p=0?010). The proportion of cleaved cytohybrids obtained from the second fusion was 35,2 % and 28,6 % of the total number of cloned embryos for 24-hour and 48-hour SS, respectively, which significantly contributed to the total number of early-stage embryos. The results presented in this article should be considered preliminary, as further research is necessary to obtain embryos at later developmental stages and to assess their quality.

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