Vitrification of immature and in vitro matured bovine cumulus-oocyte complexes: effects on oocyte structure and embryo development

This study aimed to verify the effects of cryoprotectant and Open Pulled Straw (OPS) vitrification on ultrastructural changes in bovine oocytes. In experiment 1, the cryoprotectant exposure was analyzed by distributing immature and matured cumulus-oocyte complexes (COCs) in groups: control immature, immature and exposed to one vitrification solution (IVS1), immatured and exposed to two vitrification solutions (IVS1-2), matured and exposed to one vitrification solution (MVS1), matured and exposed to two vitrification solutions (MVS1-2), control matured, IVS1 post in vitro maturation (IVS1 post IVM) and IVS1-2 post in vitro maturation (IVS1-2 post IVM). In experiment 2, immature and matured COCs were distributed in: control and vitrified by OPS using VS1-2. Sample COCs from both experiments were evaluated for mitochondrial distribution, nuclear lamins (immature COCs) and meiotic spindle (matured COCs). In both experiments, COCs were in vitro matured and fertilized. In experiment I, nuclear lamins in immature COCs exhibited higher proportion of structures with irregular shape when treated with VS1 and VS1-2. Immature and matured COCs treated with VS1-2, and matured COCs treated with VS1 had lower (P < 0.05) blastocyst development (29%, 20% and 8%, respectively) than control and immature COCs treated with VS1 (51% and 41%, respectively). In experiment 2, immature oocytes exhibited more (P < 0.05) structures with irregular lamin (72%) than control (9%). Vitrification of matured COCs did not induce abnormalities of meiotic spindle, compared to control matured (25% and 37%, respectively) (P > 0.05). The cleavage rate was higher (P < 0.05) in matured vitrified than immature vitrified group (19% vs. 8%). In conclusion, immature COCs were more tolerant to cryoprotectant exposure. However, matured oocytes showed better embryo development (cleavage) after vitrification

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 (AFP0) or supplemented with 500 (AFP500) or 1000 ng/mL (AFP1000) 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 AFP0, AFP500 and AFP1000, respectively. Similar cleavage rate was obtained among the vitrified groups (AFP0 = 17.9%, AFP500 = 16.9% and AFP1000 = 17.8%), but lower (P < 0.05) compared with control group (68.7%). At Day 5 of culture, embryo production rate of AFP500 (30.8%) and AFP1000 (25.0%) were similar to control group (49.4%). However, at Day 8 of culture, AFP0, AFP500 and AFP1000 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