The influence of oocyte and donor cell preparation conditions on the efficiency of somatic cloning in sheep (Ovis aries L.)

Obtaining embryos through somatic cell nuclear transfer (SCNT) is a reproductive biotechnology that can be applied in sheep farming to address issues related to the reproduction and preservation of valuable animals, as well as the creation of new genotypes through genome editing methods. However, the efficiency of cloning technology in Ovis aries remains comparatively low, necessitating the optimization of its various stages. In this context, the objective of this study was to assess the efficiency of SCNT based on the in vitro maturation (IVM) environment of oocytes, their age at the time of nuclear transfer (NT, procedure which includes enucleation of oocytes followed by transfer somatic cell into the perivitelline space of the obtained cytoplasts), as well as the preparation conditions of the somatic cells (duration of serum starvation in culture and storage time in suspension before NT). The impact of these factors on the formation of cytohybrids during the fusion of enucleated oocytes and somatic cells (fusion rate), as well as on the development of cloned embryos (cleavage rate of cytohybrids), was studied. Post mortem cumulus-oocyte complexes (COCs) were matured in TC-199 or in BO-IVM medium from IVF Bioscience (United Kingdom). Oocytes with the first polar body (PB1), aged between 20 to 23 hours (from the start of IVM), were used for NT, along with fetal fibroblasts (FFBs) as donor somatic cells. The latter were cultured to 80-100 % confluence, followed by incubation under serum starvation conditions (to synchronize the cell cycle) for 24 or 48 hours. FFBs were prepared for transfer into the enucleated oocyte in suspension, stored until NT for various durations: £ 30, 31-90, 91-150, and > 151 minutes. The cell complexes resulting from NT reconstruction underwent electrofusion, obtained cytohybrids were activated, and then cultured for 2 days for embryonic development. The fusion rate did not significantly differ between experimental groups, ranging from 31 to 42 %. The cleavage rate of cytohybrids also did not differ when oocyte maturation occurred in TCM-199 or BO-IVM medium (47.1±2.40 % and 50.9±3.30 %, respectively). When NT was performed using 22-hour-old oocytes, the yield of cloned embryos was 55.8±3.78 %. Earlier-stage oocytes showed comparable results, while older oocytes (23 hours) significantly reduced the yield (to 39.3±6.69 %, p function show_eabstract() { $('#eabstract1').hide(); $('#eabstract2').show(); $('#eabstract_expand').hide(); }