Stimulatory effect of Rho-associated coiled-coil kinase (ROCK) inhibitor on revivability of in vitro-produced bovine blastocysts after vitrification

Inhibition of Rho-associated kinase (ROCK) activity promoted recovery and growth of frozen-thawed human embryonic stem cells. The primary objective was to determine if a ROCK inhibitor (Y-27632) in post-thaw culture medium unproved revivability of vitrified IVP bovine blastocysts. Expanding or expanded blastocysts (7 d after IVF) were vitrified (minimum volume cooling procedure, using a Cryotop) in 15\% ethylene glycol. 15\% DMSO and 0 5 M sucrose When post-warm blastocysts were cultured in inSOF medium, survival rate (re-expansion of blastocoel at 24 h of culture) was improved (P < 0 05) by the addition of 10 mu M Y-27632 (94 9 +/- 2 4\%, mean +/- SEM) compared to a control (78 0 +/- 6 0\%) Conversely. after 48 h of culture, there were no significant differences in hatching late (62.8 +/- 11 1 vs 59 6 +/- 9.4\%) and mean total cell number (135 2 +/- 13 1 vs. 146 7 +/- 13 3) In non-vitrified IVP bovine blastocysts, the hatching rate on Day 9 was improved by Y-27632 (91 7 +/- 3 8 vs 54 7 +/- 8 9\%. P < 0 05). with no difference in mean total cell number of blastocysts (230 0 +/- 23 0 vs 191 2 +/- 22 2, P = 0 23). In an additional experiment, Y-27632 was added to culture medium on either Day 0, Day 2. or Day 4 (and remained present until Day 8). resulting in no improvement in blastocyst yield compared to a control group (7.5 +/- 2 1, 31 4 +/- 2 3, 36 2 +/- 3.2. and 28.6 +/- 6.9\%. respectively) In conclusion, adding a ROCK inhibitor to post-thaw culture medium improved revivability of IVP bovine blastocysts after vitrification and warming (C) 2010 Elsevier Inc. All rights reserved