Downsizing cumulus cell layers to improve cryotolerance of germinal vesicle-stage bovine oocytes.

This study was undertaken to investigate whether complete removal or downsizing of the cumulus cell layers in germinal vesicle (GV)-stage bovine cumulus-oocyte complexes (COCs) can improve blastocyst development rate following Cryotop vitrification. Downsized COCs (196 µm in mean diameter) and denuded oocytes (141 µm in mean diameter) were prepared by vortex-mixing of full-sized COCs (330 µm in mean diameter) retrieved from abattoir-derived ovaries. Nuclear maturation rates, assessed by the first polar body extrusion, after vitrification and the subsequent 22-h IVM were comparable (61.9-62.9%). Approximately one-third (30.5-31.2%) of the matured oocytes derived from the downsized COCs could develop into high quality blastocysts after 6-h IVF and 8-d IVC, while 13.4 and 23.7% of the matured oocytes derived from denuded oocytes and full-size COCs reached to the blastocysts, respectively. Cytoplasmic lipid droplets of matured oocytes in vitrification group were more clustered with decreased number and increased size of the droplets, when compared to those in fresh control group. However, individual oocyte culture in well-of-the well system suggested that change of lipid droplet distribution in the matured oocytes had no adverse effect on their subsequent developmental competence up to the blastocyst stage. In conclusion, Cryotop vitrification of downsized GV-stage bovine COCs allowed blastocyst yields as high as >30%.

Direct comparison of Cryotop® vitrification and Bicell® freezing on recovery of functional rat pancreatic islets.

Two protocols, Bicell® freeze-thawing and Cryotop® vitrification-warming, were compared for suitability in cryopreserving rat pancreatic islets (101-150 µm in mean diameter). Immediate survival rates of post-thaw and post-warm islets (50 and 57%, respectively), assessed by FDA/PI double staining, were lower than that of fresh control islets (90%). Most of the PI-positive dead cells were detected in peripheral area of post-warm islets, and were removed after subsequent 24 h culture (survival rate; 85% vs 59% in post-thaw islets). Quantitative PCR analysis showed that Bicell® freeze-thawing compromised expression of genes relating to ß-cell function (Pdx1 and Glut2), but not to one of apoptotic pathways (Bax/Bcl2 ratio). Expression of these genes was maintained in islets before and after the Cryotop® vitrification-warming. Values of stimulus index (SI) for 20 mM/3 mM glucose-stimulated insulin secretion were 6.7, 1.9 and 3.9 in fresh control, post-thaw and post-warm islets, respectively. The SI values after 24 h culture were 4.1, 1.9 and 3.1, respectively. Larger islets (>150 µm in diameter) had comparable survival rates, but lower SI values after Cryotop® vitrification-warming when compared to smaller counterparts. These results suggest that rat pancreatic islets can be cryopreserved by Cryotop® vitrification-warming rather than Bicell® freeze-thawing, without considerable loss of in vitro ß-cell function.

HIGH INCIDENCE OF POLYSPERMIC FERTILIZATION IN BOVINE OOCYTES MATURED IN VITRO AFTER CRYOTOP VITRIFICATION.

BACKGROUND: Vitrification with the Cryotop device is the most promising technique for oocyte cryopreservation, but the high post-warming morphological survival of bovine oocytes does not guarantee high developmental competence after in vitro fertilization (IVF). OBJECTIVE: This study was designed to examine achievement of normal fertilization in bovine oocytes vitrified-warmed with the Cryotop device. MATERIALS AND METHODS: Oocytes were matured in vitro and vitrified-warmed after complete removal of the cumulus layers. Distribution of cortical granules (CGs) was assessed by Lens culinaris agglutinin (LCA) lectin staining. Ten hours after IVF, presumptive zygotes were analyzed for pronuclear formation. Day-8 blastocysts were harvested and stained with Hoechst-33342 for total cell counting. RESULTS: Both yield and mean cell number of the blastocysts were impaired by Cryotop vitrification. Incidence of polyspermic fertilization was three-times higher in vitrified oocytes compared to fresh oocytes. No difference in CG distribution was found between vitrified and fresh oocytes. CONCLUSION: Polyspermic fertilization induced in vitrified-warmed bovine oocytes may be one of the possible causes responsible for their low developmental potential.

High revivability of vitrified-warmed bovine mature oocytes after recovery culture with α-tocopherol.

The objective of this study was to investigate whether developmental competence of vitrified-warmed bovine oocytes can be improved by antioxidant treatment during recovery culture. In experiment 1, one of the two antioxidants (either l-ascorbic acid or α-tocopherol) was added as a supplement to the recovery culture medium to which postwarming oocytes were exposed for 2 h before IVF. The exposure to α-tocopherol had a positive effect on rescuing the oocytes as assessed by the blastocyst yield 8 days after the IVF (35.1-36.3% vs 19.2-25.8% in untreated postwarming oocytes). Quality of expanding blastocysts harvested on Day 8 was comparable between α-tocopherol-treated vitrification group and fresh control group in terms of total cell number and chromosomal ploidy. In experiment 2, level of reactive oxygen species, mitochondrial activity, and distribution of cortical granules in α-tocopherol-treated postwarming oocytes were assessed. No obvious differences from the control data were found in these parameters. However, the treatment with α-tocopherol increased the percentage of zygotes exhibiting normal single aster formation (90.3% vs 48.0% in untreated postwarming oocytes; 10 h post-IVF). It was concluded that α-tocopherol treatment of vitrified-warmed bovine mature oocytes during recovery culture can improve their revivability, as shown by the high blastocyst yield and the higher mean total cell number in the blastocysts.