The effect of cytochalasin B during cytoplast preparation on the efficiency of somatic cloning in sheep (Ovis aries)

BACKGROUND: Somatic cloning of sheep is of great interest for genetic resource conservation, biomedicine, and biopharmaceuticals. However, its efficiency remains extremely low. One way to enhance the efficiency of this technology is to optimize its individual steps, particularly the procedure of oocyte enucleation and the transfer of somatic cell–derived cytoplasts into the perivitelline space — nuclear transfer. The chemical agent cytochalasin B enhances the oocyte’s resistance to external deformation and facilitates micromanipulation procedures. However, its application in cloning technology remains a subject of debate. AIM: To evaluate the efficiency of somatic cloning in sheep (Ovis aries) using cytochalasin B during the preparation of matured oocytes prior to nuclear transfer, depending on the duration of this procedure. MATERIALS AND METHODS: Nuclear transfer was performed using in vitro–matured oocytes containing a first polar body (PB1) in their perivitelline space. In the experimental group, oocytes with PB1 were incubated for 20 minutes in a medium containing 7.5 µg/mL cytochalasin B before nuclear transfer. The control group was maintained in an identical medium without cytochalasin B. Nuclear transfer was conducted using an inverted microscope equipped with a Narishige micromanipulation system (Narishige Scientific Inst. Lab., Japan). Oocyte chromosomes were removed blindly by aspirating the PB1 and the adjacent cytoplasm. Somatic cells were injected directly into the perivitelline space of the enucleated oocyte. Electrofusion was used to combine the oocyte–somatic cell complexes. The cytoplasmic hybrids formed as a result of electrical stimulation were activated using ionomycin, subjected to simultaneous treatment with 6-(dimethylamino)purine and cycloheximide, and then cultured for two days for embryonic development. RESULTS: The nuclear transfer efficiency (the number of oocyte–somatic cell complexes relative to the number of oocytes with PB1) and fusion rate (the number of cytoplasmic hybrids formed relative to the number of oocyte–somatic cell complexes) did not differ between the control and experimental groups, remaining at 96%–97% and 33%–35%, respectively. In the control group, the proportion of cytoplasmic hybrids undergoing cleavage after two days of culture was 35.7±7.7%. Pre-incubation of oocytes in cytochalasin B increased this parameter to 59.7±6.9% (p 0.0029). However, the effect of cytochalasin B treatment on cloning efficiency depended on the duration of nuclear transfer: when the procedure exceeded 40 minutes, a significant decrease in the fusion rate (p=0.002) and a reduction in the proportion of cloned embryos (relative to the number of oocyte–somatic cell complexes) (p=0.041) were observed. CONCLUSION: Short-term culture of matured oocytes in the presence of cytochalasin B before nuclear transfer increases the yield of cloned embryos, while prolonged nuclear transfer procedures negatively affect the efficiency of somatic cloning in sheep.