Resumen de Bovine oocyte cryopreservation: optimization of vitrification protocols through different carrier systems and a taxol pretreatment
Morale. R Z008 Bovine oocyte cryopresen/ation: Optimization of vitrification protocols through different carrier systems and a Taxol pretreatment The aim of this thesis was to improve the bovine oocyte vitrification protocols using Taxo: a cytoskeletal stabilizer, ana three different types of vitrification supports: open pulled straws (OPS), flexipette denuding pipette iFDP) and cryotops. Spindle abnormalities, cortical granules distribution, ultrastructural features and embryo development of calf and cow oocytes were evaluated following vitrification.
Vitrification is a simple, efficient and cost-effective way to improve the cryopreservation of oocytes In the first study, the efficacy of pre-treating mature bovine oocytes with Taxol before vitrification by the OPS method was determined. Vitrification of calf or cow oocytes after Taxo! pretreatment significantly reduced the proportions of spindle abnormalities compared to non vitrified oocytes. Likewise, Taxol treatment had a beneficial effect on the cortical granule migration, on the cleavage rate and, oniy in cow oocytes on the blastocyst rates Calf oocytes were unable to develop to the blastocyst stage after vitrification, irrespective of Taxoi treatment.
Potential subcellular consequences of treatment with the microtubule stabilizer Taxol with or without subsequent vitrification of cow and calf oocytes by the OPS method were documented in the second study of this work. The main injuries of Taxol treatment and vitrification manifested themselves in the metaphase plate and spindle ultrastructure. In control oocytes (no Taxol treatment and no vitrification), the metaphase appeared as condensed chromosomes arranged in a well-organized metaphase plate and the spindle showed well organized microtubules in both cow and calf oocytes. However, in cow oocytes vitrified without Taxol treatment, the metaphase plate was disorganized into scattered chromosomes or the chromosomes were condensed into a single block of chromatin. In addition, microtubules were not organized in a typical spindle. In contrast, cow oocytes submitted to Taxol treatment as weii as both cow and calf oocytes submitted to Taxol treatment, but not vitrification, snowed well-organized metaphase plates and normal spindle morphology Ail vitrified calf oocytes displayed a single block of chromatin and lack of microtubules independent of Taxol treatment. These results support the findings of the first work with solid ultrastructural data demonstrating a beneficial affect of Taxoi on the metaphase plate and its sssociated spindle apparatus.
The following study, presented a straight forward functional comparison of two vitrification methods which is supported by cell biological data demonstrating effects en the metaphase plate and the associated spindle apparatus. The main aim was to determine the effectiveness of two different vitrification supports for oocyte cryopreservation: the OPS and the flexipet denuding pipette. These two methods were evaluated with respect to their effect on cytoskeletal components and embryo developmental capacity of the oocytes. Those oocytes virrified by the FDP system evidenced a lower cleavage rate than such vitrified by the OPS system, which again displayed a lower cleavage rate than the non vitrified oocytes. Blastocysts were only obtained using OPS method and at a lower rate than in the control oocytes. Oocytes vitrified in FDP displayed higher rates of spindle and chromosome abnormalities than those vitrified in OPS No differences between cow and calf oocytes were observed in these experiments. Finally, the effectiveness of another and recent vitrification carrier system, the cryotop, a specially constructed fine polypropylene strip attacned to a plastic handle, was compa-'ed with the OPS method. Cleavage rates, although iower than non vitrified control oocytes, were significantly higher for both cow and calf oocytes vitrified by the Cryotop system as compared with the OPS system. Only control oocytes ana oocytes vitrified by the Cryotop method de.veiopea to the blastocyst Mage: the latter at significantly reduced rates, in calf oocytes, vitrification using OPS straws resulted in a significant reduction of the percentages of normal spindle configuration when compared to control oocytes and oocytes vitrified using cryotops. OPS or cryotop vitrified cow oocytss, showed significantly lower percentages of normal spindle morphology from non vitrified oocytes. In conclusion, the use of the cryotop system and Taxol pretreatment improve the potential for oocytes to deveiop to blastocyst stage during cryopresen/afon without compromising their survival. The data on the developmental potential of oocytes vitrified by different methods with c without microtubule stabilizing agent can directly ts translated into practical use. and the cell biological studies on metaphase piate and spindle morphology gives solid ceil biological support.
