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Histone deacetylase 1 (HDAC1) is one of the most conserved enzymes present in the nuclei of cells, including bovine oocytes and pre-implantation embryos. However, the biological functions of HDAC1 in supporting the growth and development of bovine oocytes and embryos are still not fully elucidates. In this study, three siRNAs (si299, si672, and si1272) targeting to HDAC1 mRNA sequence were designed. After transfection into bovine fibroblast cells, si299, the most effective one in HDAC1 knock-down, was selected. The selected siRNA was microinjected into bovine germinal vesicle (GV) stage oocytes to determine the functions of HDAC1 in the maturation of bovine oocytes. Finally, the siRNA was microinjected into mature oocytes, which were then parthenogenetically activated and cultured in vitro until the blastocyst stage. The rates of cleavage, blastocyst development and acetylation of lysine 14 of H3 (H3K14) state were checked. The results suggest that HDAC1 knock-down in oocytes did not influence the rates of maturation or cleavage of parthenogenetic embryos. However, the rates of blastocyst decreased after siRNA microinjection. Furthermore, histone H3K14 acetylation level increased after siRNA microinjection into parthenogenetic embryos.

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The objective of our study was to establish the feasibility of experimental protocols for cloning sika deer. We performed auxiliary enucleation to improve the efficiency of nuclear transfer operation by optimizing the demecolcine concentration to induce cytoplasmic protrusions in the sika deer oocytes. In the present study,we had studied the impact of different demecolcine concentrations on cytoplasmic protrusions and enucleation rates. We determined that 95.9% of the sika deer oocytes formed cytoplasmic protrusions when treated for 1 h with 0.8 μg/ml demecolcine. The lowest observed rate of protrusion was 19.3% after overnight treatment with demecolcine. When the oocytes aged or had a poor cumulus expansion, they exhibited a significant decrease in the ability to form cytoplasmic protrusions. The rates of enucleation (94.9% vs 85.8%, p < 0.05), cell fusion (84.6% vs 70.1%, p < 0.05) and blastocyst formation (15.4% vs 10.9%, p < 0.05) using demecolcine auxiliary enucleation were significantly higher than those after blind enucleation. These results demonstrated that sika deer oocytes could be enucleated quickly and effectively using demecolcine auxiliary enucleation, which could enhance the enucleation rate, cell fusion rate and blastocyst rate of cloned embryos in vitro.

No abstract