Demethylation dynamics of the paternal genome in pronuclear-stage bovine zygotes produced by in vitro fertilization and ooplasmic injection of freeze-thawed or freeze-dried spermatozoa

This study was designed to investigate the dynamics of the paternal genome demethylation in pronuclear-stage bovine zygotes produced either by in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) using freeze-thawed (FT) as well as freeze-dried (FD) bull sperm stored at +4 or -196 C for one year. Zygotes were fixed and immunostained using anti-5-methyl-cytosin at 8, 10, 14 and 18 h post IVF (hpi) and at 6 and 12 h post ICSI (hpic). In conventional IVF-derived zygotes, the overall average of the relative methylation (RM; male/female) decreased from 0.92 at 8 hpi to 0.69 at 10 hpi (P0.05) without any additional decrease at 14 and 18 hpi (0.67 and 0.64, respectively; P0.05). This was accompanied by higher proportions of zygotes showing RM0.6 (45.5, 37.5 and 38.2% at 10, 14 and 18 hpi, respectively; P0.05) compared with 3.7% at 8 hpi. The overall averages of the RM in the FT-ICSI derived zygotes (0.79 and 0.66 at 6 and 12 hpic, respectively) were similar to those in the corresponding IVF-derived zygotes (8 and 14 hpi), but a higher proportion of the 6 hpic zygotes (37.8%; P0.05) showed an RM0.6 compared with the 8 hpi zygotes (3.7%). The proportions of FD-ICSI derived zygotes at 12 hpic showing an RM0.6 (60.6 and 62.4% for +4 and -196 C storage, respectively) were higher than that of the FT-ICSI derived zygotes (39.4%; P0.05). Thus, the bovine paternal genome rapidly demethylated within 10 h after IVF and 6 h after ICSI, and the freeze-drying and/or the storage process had no adverse effect on demethylation of the paternal genome. The extent of demethylation in the pronuclear-stage bovine zygotes was moderate, with 0.4=RM0.6.