Improved parthenogenetic development of vitrified-warmed bovine oocytes activated with 9% ethanol plus 6-DMAP.
The objective was to compare various activation protocols on developmental potential of vitrified bovine oocytes. Bovine oocytes matured in vitro for 23 h were vitrified with EDFSF30 in open pulled straws. After warming, they were cultured in vitro for 1h, followed by parthenogenetic activation. Vitrified-warmed oocytes had a morphologically normal rate similar to that of controls (nonvitrified oocytes cultured in vitro for 24h; 98.6% vs. 100%, P>0.05). When vitrified-warmed oocytes were first activated with 7% ethanol for 5 min and then incubated in 6-dimethylaminopurin (6-DMAP) for 4h, cleavage and blastocyst rates were 41.2% and 23.2%, respectively, which were lower than those of controls (77.5% and 42.0%, P < 0.05). Subsequently, we varied the ethanol concentration to increase the effectiveness of parthenogenetic activation. When either 5%, 6%, 7%, 8%, 9%, 10%, or 11% ethanol alone (for 5 min) or in combination with 6-DMAP (4h) was used to activate vitrified-warmed oocytes, cleavage rates ranged from 22.3% to 61.1% and blastocyst rates ranged from 1.1% to 30.6%. These rates were optimized when oocytes were treated with 9% ethanol plus 6-DMAP; this was verified in experiments evaluating other activation protocols with 9% ethanol, calcium ionophore A23187, or ionomycin alone, or in combination with DMAP or cycloheximide (CHX). In conclusion, the oocyte activation protocol affected developmental capacity of vitrified bovine oocytes; 9% ethanol (5 min) followed by 6-DMAP (4h) promoted optimal parthenogenetic activation.
Hou
Y-p
YP
State Key Laboratories for Agrobiotechnology, College of Biological Sciences, China Agricultural University, People's Republic of China.
Liu
Y
Y
Dai
Y-p
YP
Li
R
R
Shi
W-Q
WQ
Wang
H-p
HP
Wang
L-l
LL
Li
N
N
Zhu
S-e
SE
D000225:Adenine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D002417:Cattle / Q000502:physiology; D002478:Cells, Cultured; D015925:Cryopreservation / Q000379:methods; D004338:Drug Combinations; D046149:Embryo Culture Techniques / Q000379:methods; D047108:Embryonic Development / Q000187:drug effects; D000431:Ethanol / Q000494:pharmacology*; D005260:Female; D005615:Freezing; D009865:Oocytes / Q000187:drug effects* / Q000502:physiology; D009994:Osmolar Concentration; D010312:Parthenogenesis / Q000187:drug effects* / Q000502:physiology
D004338:Drug Combinations; D000431:Ethanol; C001020:N(6),N(6)-dimethyladenine; D000225:Adenine