Effects of Taxol on immature bovine oocyte vitrification using Open Pulled Straw (OPS)


Shayegh M *, Barati F , Mohammadi Gh ,

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Objective: In spite of the good sperm and embryo response to cryopreservation programs oocyte response (post-thaw maturation and fertility) to cryopreservation procedures is very poor. Special cellular status such as very sensitive cytoskeletal system early release of cortical granules and pre-fertilization zona hardening and changes in position and structures of ZP glycoproteins are the consequences of freezing procedures on the oocytes. However matured oocytes can tolerate the freezing procedures better than immature oocytes. Cytoskeleton stabilizer compounds like Cytochalasin D and B improved the vitrification outcome mature and immature oocytes. Taxol is a cytoskeleton stabilizer and used for human immature bovine and murine mature oocyte vitrification successfully. The aim of this study is to investigate the effects of taxol on immature bovine oocyte vitrification. Materials and Methods: Material and Methods Indigenous bovine ovaries transported to the Lab in the physiologic saline contained penicillin streptomycin in 39oC. Cumulus Oocyte Complexes (COC) aspirated form follicles (2-8 mm) and their qualification assessed in the medium TCM supplemented with 10%FCS. Grade A oocytes subjected to five groups of experiments: Control (n=108): oocytes with the routine IVM procedure CRP (n=51): oocytes which exposed to the vitrification solutions without vitrification and subjected to IVM procedure Tax (n=51): oocytes which exposed to Taxol for 15 min then transferred to IVM medium OPS (n=50): oocytes which were vitrified thawed and subjected to IVM procedure and OPS-Taxol (n=50): oocytes which exposed to the Taxol after 15 min vitrified thawed and transferred to the maturation medium. IVM medium was TCM 199 supplemented with 10mg FSH 10mg LH 10% Bovine follicular fluid and 5% FCS. During maturation oocytes incubated in the atmosphere with 5% CO2 95% humidity for 24 hours. After the period of maturation COCs denuded and stained with the conventional procedure of aceto-orcein satin for evaluating nuclear maturation. Before vitrification COCs exposed to three medium: HM: TCM 199+10% FCS V1: HM+10% EG+ 10% DMSO and V2: HM+20% EG+ 20% DMSO+0.05m sucrose for 5 min within each medium then loaded in the Open Pulled Straw (OPS) and plunged into the liquid nitrogen tank. At least after 48 hours of vitrification COCs thawed in the thawing media composed of T1: HM+0.25m sucrose and T2: HM+0.15m sucrose. COCs held within each media for 5 min and transferred to the IVM medium finally. Taxol (Abetaxel® ) with the maturation medium with dose of 1 nmol/ml. Percentage of matured oocytes were analysed with General Linear Model of SAS and means separated with the Tukey multiple comparison test. Data expressed as Least square means with SEM. Results and Discussion: The percentage of matured oocyte was not different (p>0.05) between CRP (71.9 ± 5.4) and Tax (73.1 ± 6.8) groups compare to Control (80.5 ± 6.8). Vitrification (9.1 ± 6.2) significantly affected the maturation rate compare to control (p05) in Taxol treated vitrified oocytes compare to the oocytes which were not treated with Taxol. Conclusion: These results show the impact of vitrification on oocyte maturation of immature bovine oocytes with no significant improvement with pre-freeze Taxol treatment.