The ability to successfully cryopreserve mammalian oocytes has numerous practical,
economical and ethical benefits, which may positively impact animal breeding programs
and assisted conception in humans. However, oocyte survival and development following
vitrification remains poor. The aim of the present study was (1) to evaluate the effect
of the presence of cumulus cells on the outcome of vitrification of immature (GV)
or mature (MII) bovine oocytes, (2) to compare empirical and theoretical vitrification
protocols, and (3) to assess the effect of adding ice blockers to vitrification media
on survival and development competence of bovine oocytes following vitrification using
the Cryotop method. In Experiment 1, cumulus-enclosed and partially-denuded GV and
MII oocytes were vitrified in 15% EG+15% Me(2)SO+0.5M sucrose in two steps. In Experiment
2, GV oocytes were vitrified either as above or using theoretical modeling based on
permeability and osmotic tolerance characteristics in 30% EG+11.4% trehalose in three
steps or 40% EG+11.4% trehalose in four steps. In Experiment 3, GV oocytes were vitrified
in media supplemented or not with 1 of 2 ice blockers (21st Century Medicine, Fontana,
CA) 1% X-1000, 1% Z-1000 or both in three steps. In Experiment 1, the survival, cleavage
and blastocyst rate of cumulus-enclosed oocytes was significantly higher than those
of partially-denuded oocytes when vitrified at the GV stage (93.8% vs. 81.3%, 65.8%
vs. 47.3%, 11.3% vs. 4.0%, respectively, P<0.05). However, no significant effect of
cumulus cover was detected between the two groups when vitrified at MII (93.0% vs.
91.8%, 35.2% vs. 36.8%, 5.0% vs. 4.4%, respectively). Furthermore, cumulus-enclosed
oocytes vitrified at the GV stage exhibited significantly higher developmental competence
than those vitrified at the MII stage (P<0.05). In Experiment 2, there were no significant
differences in the survival, cleavage and blastocyst rate among three protocols (86.0%
vs. 92.8% vs. 91.2%, 44.8% vs. 54.4% vs. 45.6%, 5.0% vs. 5.4% vs. 4.0%, respectively).
However, cleavage and blastocyst rate were significantly lower (P<0.05) than non-vitrified
control oocytes. In Experiment 3, the presence of ice blockers did not alter the cleavage
rate or blastocyst development (P>0.05). In conclusion, cumulus-enclosed GV bovine
oocytes survived vitrification and subsequently developed at higher rates than MII
oocytes using Cryotop method and conventional IVF procedure. Theoretical analysis
of permeability characteristics and tolerance limits could not explain the low developmental
competence of vitrified oocytes.
(c) 2010 Elsevier Inc. All rights reserved.