The Effect of the Duration of In Vitro Maturation (IVM) on Parthenogenetic Development of Ovine Oocytes

Abstract

The aim of this study was to compare the effect of time of parthenogenetic activation (22 hr versus 27 hr after In Vitro Maturation-IVM) on in vitro development of ovine oocytes using either single (Ionomycin 5 µM for 5 min or Ethanol 7% for 7 min) or combined (ionomycin and ethanol with 6-DMAP 2 mM for 3 hr) activation treatments. The abattoir-derived in vitro matured activated oocytes were cultured in modified synthetic oviductal fluid and assessed for the cleavage, blastocyst, and hatching rates. The single-activated oocytes had a reduction in cleavage, blastocyst and hatching rates compared to the combined-activated oocytes (except for the cleavage at 27 hr). In single-treated groups the rates of cleavage and blastocyst were increased as the maturation time was extended from 22 hr to 27 hr. The numbers of total cells and Inner Cell Mass (ICM), though insignificant, were greater in combined-treated groups compared to the single treatment. The number of ICM in Eth + 6-DMAP group activated at 27 hr was lower than 22 hr. Nonetheless, irrespective of the activation protocol, development to the blastocyst stage, the numbers of total cell, ICM, and cell allocation (ICM/total cells) were significantly lower in parthenogenetic than fertilized embryos. In conclusion, though the cleavage and blastocyst rates in single-treated groups were positively influenced by the extension of duration of IVM (27 hr), there was a trend of decreased numbers of total cells and ICM in slightly aged oocytes. Moreover, developmental potential of ovine parthenotes, especially in young oocytes, was improved by the addition of 6-DMAP to the activation regimen.

Keywords: Blastocyst; Ethanol; Ionomycin; Parthenogenesis; Sheep.

Figure 1

Epifluorescent microscopic images of bovine blastocysts derived from parthenogenetic activation and IVF. Trophectoderm and inner cell mass nuclei were labeled with propidium iodide (red) and Hoechst 33342 (blue), respectively. (A-B) Partenogenetic blastocysts derived from single treated groups (Ionomycin or ethanol). C-D) Partenogenetic blastocysts derived from combined treated groups (Ionomycin or ethanol with 6-DMAP). E-F) IVF-derived blastocysts