Quality analysis of buffalo blastocysts derived from oocytes vitrified before or after enucleation and reconstructed with somatic cell nuclei

Abstract

We investigated the potential of vitrified-warmed buffalo oocytes to develop to blastocysts after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT). In vitro-matured oocytes before and after enucleation (M-II oocytes and enucleated oocytes, respectively) were put in 7.5% DMSO and 7.5% ethylene glycol (EG) for 4, 7 and 10 min, and then vitrified (Cryotop device) after 1-min equilibration in 15% DMSO, 15% EG and 0.5 M sucrose. Following 4-, 7- and 10-min exposure, proportions of the post-warm oocytes with a normal vitelline membrane were similar (66–71% in M-II oocytes and 69–71% in enucleated oocytes). However, 18–20% of the normal M-II oocytes had no detectable first polar body in their perivitelline space (no potential for subsequent enucleation). When the post-warm M-II oocytes were treated for PA by 7% ethanol, 10 μg/mL cycloheximide and 1.25 μg/mL cytochalasin-D, parthenogenetic development into Day-7 blastocysts occurred in 10–13% of cultured oocytes, lower ( P < 0.05) than fresh (control) oocytes (24%). In the absence of the cooling and warming, blastocyst rates in the 4-min exposure group (22%), but not in the 7-min and 10-min exposure groups (14–15%), were similar to that in the fresh group (23%). The total cell number (group average 117–132 cells) and the ICM ratio (22–24%) of the PA blastocysts derived from vitrified M-II oocytes were comparable with fresh oocytes (127 cells and 25%). After SCNT (with fibroblast cells and vitrified-warmed oocytes), blastocyst rates were similar for the three exposure periods for M-II oocytes (8–10%) and enucleated oocytes (7–9%), but were lower ( P < 0.05) than in the fresh group (15%). The total cell number of the SCNT blastocysts derived from vitrified M-II and enucleated oocytes (80–90 and 82–101 cells) was smaller ( P < 0.05) than from fresh oocytes (135 cells); the ICM ratio of blastocysts derived from the M-II and enucleated oocytes after vitrification in 7- or 10-min exposure groups (20–22%) was not different ( P > 0.05) from fresh control oocytes (24%) or those in 4-min exposure group (M-II 23%, enucleated 24%). Thus, SCNT of swamp buffalo oocytes following vitrification before or after enucleation resulted in blastocysts with a slightly decreased cell number.