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Stimulatory effect of Rho-associated coiled-coil kinase (ROCK) inhibitor on revivability of in vitro-produced bovine blastocysts after vitrification
- Hochi, S., Abdalla, H., Hara, H., Shimoda, M., Morita, H., Kuwayama, M., Hirabayashi, M.
- Theriogenology 2010 v.73 no.8 pp. 1139-1145
- cattle, assisted reproductive technologies, embryo (animal), in vitro fertilization, embryogenesis, blastocyst, cryopreservation, cryoprotectants, glycerol, dimethyl sulfoxide, freeze-thaw cycles, thawing, culture media, additives, kinases, inhibitors, physiological response, protective effect, viability, cell cleavage, yields
- Inhibition of Rho-associated coiled-coil 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 improved 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 0.5M sucrose. When post-warm blastocysts were cultured in mSOF medium, survival rate (re-expansion of blastocoel at 24h of culture) was improved (P <0.05) by the addition of 10μM Y-27632 (94.9±2.4%, mean±SEM) compared to a control (78.0±6.0%). Conversely, after 48h of culture, there were no significant differences in hatching rate (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.