In vitro development of cloned embryo in cattle in relation with fusion and activation parameters

Embryo production through somatic cloning technology has the perspectives for application in reproductive biotechnologies in cattle in order to multiply the most productive and unique genotypes in livestock breeding and create new genotypes using genome editing. Success of somatic cloning depends on the ability of donor somatic cell nucleus (karioplast) to be reprogrammed to totipotent state. Relevant transformations of donor nucleus are mediated by oocyte cytoplasmic factors (cytoplasts) and start from the moment of their association (fusion). Effects of oocyte cytoplasm are direct and depend on many factors. The objective of the present study was to evaluate the cloning efficiency in terms of time of oocyte cytoplasm exposure to donor nucleus before activation, the time of oocyte maturation before their activation in the fused complexes (cytohybrids), and repeated electrofusion of the cytoplast and karyoplast. The effects of these factors on formation of cloned embryos and development to blastocyst stage were studied. Isolated oocyte-cumulus complexes (OCCs) were in vitro matured in TC-199 medium supplemented with 10 % fetal bovine serum, 10 μg/ml of FSH and 10 μg/ml of LH. After 20-24 h of maturation, OCCs were treated with a 0.1 % hyaluronidase, then cumulus cells were mechanically removed and the oocytes with the first polar body were selected. Long-time conserved fetal fibroblasts were in vitro cultured up to monolayer and maintained in contact inhibition during 2 days. Then, cell suspension was prepared for transferring into enucleated oocyte. Somatic cell was transferred to perivitelline space of the oocyte, and two consecutive rectangular 20 μs pulses at constant current with a voltage of 35 V were performed (once or twice if there were no signs of cell-oocyte fusion). The obtained cytohybrids were activated with the ionomycin 1 or 2 hours after fusion (recipient oocytes were matured either 23-25 hours or 26-28 hours). Activated cytohybrides were then cultured up to blastocyst stage. Oocyte cleavage rate were similar in all experimental groups (60.7 to 70.4 %). Blastocyst development rate did not differ between the groups where single or double fusions were performed (29.4±4.4 and 22.8±3.5 %, respectively). Blastocyst rate was 17.4±2.6 % at 1-hour interval between fusion and activation. Two-hour interval increased blastocyst rate to 31.1±3.8 % (p function show_eabstract() { $('#eabstract1').hide(); $('#eabstract2').show(); $('#eabstract_expand').hide(); }