The use of antifreeze protein type III for vitrification of in vitro matured bovine oocytes

The aim of this study was to evaluate the use of antifreeze protein type III (AFP III) into vitrification medium on meiotic spindle morphology of in vitro matured bovine oocytes as well as the fertilization and blastocyst rates. Mature cumulus-oocyte complexes (COC) were distributed in four groups: control (untreated), vitrified without supplementation (AFP(0)) or supplemented with 500 (AFP(500)) or 1000 ng/mL (AFP(1000)) into vitrification solutions. Samples from each group were used to analyze the organization of meiotic spindle by confocal microscopy and the remaining COC were submitted to in vitro fertilization and culture for eight days. Control group exhibited only 15% of abnormal spindle. However, the spindle morphology was affected in all vitrified groups regardless to AFP concentration: 75.8%, 76.1% and 69.2% (P > 0.05) for AFP(0), AFP(500) and AFP(1000), respectively. Similar cleavage rate was obtained among the vitrified groups (AFP(0) = 17.9%, AFP(500) = 16.9% and AFP(1000) = 17.8%), but lower (P < 0.05) compared with control group (68.7%). At Day 5 of culture, embryo production rate of AFP(500) (30.8%) and AFP(1000) (25.0%) were similar to control group (49.4%). However, at Day 8 of culture, AFP0, AFP(500) and AFP(1000) groups exhibited lower (P < 0.05) blastocyst rates (10.0%, 3.8% and 9.4%, respectively) when compared to control (41.1%). In conclusion, AFP III did not preserve meiotic spindle organization against the cryoinjuries. However, the use of AFP III improved embryo development at Day 5 of culture, although this effect was not maintained up to the blastocyst formation. (C) 2016 Elsevier Inc. All rights reserved.