Bovine oocyte vitrification before or after meiotic arrest: effects on ultrastructure and developmental ability (original) (raw)
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Viability of bovine in vitro matured oocytes following ultra-rapid vitrification
Medical Journal of Cell Biology
The aim of the study was to examine viability of cattle oocytes after cryopreservation. Oocytes after in vitro maturation (IVM) were vitrified in minimum volume on the nickel electron microscopy grids by ultra-rapid cooling technique. After warming and subsequent in vitro fertilization the presumptive zygotes were cultured to reach the stage of the blastocyst (Bl). Several devitrified oocytes were processed for electron microscopy assay. Although, embryo cleavage and Bl percentages in the vitrified group were slightly lower than in the control group (P < 0.05), the Bl total cell number (TCN), apoptosis and dead cell percentages did not differ between both groups. However, significant difference was found between day 7 (D7) and day 8 (D8) Bl in the TCN in control (108.0 vs. 90.5) and vitrified group (103.75 vs 98.14). Electron microscopy of frozen oocytes revealed slight reversible injuries in mitochondria and the smooth endoplasmic reticulum (SER), nevertheless, the development o...
Journal of Assisted Reproduction and Genetics, 2009
Purpose The objectives were to test how the source of oocytes and semen impacted vitrification of large numbers of bovine oocytes and subsequent IVF and early embryo development to test procedures that may assist with assisted reproductive technologies in humans. Methods Bovine oocytes were vitrified from follicles of different diameters, small (≤4 mm) and medium (4 to 10 mm), using nylon mesh. Oocytes were exposed to the cryoprotectant composed of 40% (v/v) ethylene glycol, 18% (w/v) Ficoll-70, and 0.3 M sucrose in three stepwise dilutions. Thawing was conducted with a series of 0.5, 0.25 and 0.125 M sucrose dilutions in 20% fetal bovine serum. Results The cleavage (39.1% vs. 58.5%) and blastocyst rates (5.1% vs. 22.9%) were significantly lower for the vitrified oocytes. Follicle size had a significant impact on the development of embryos. Sires had significant effects on embryonic developmental rates. Conclusions We conclude that differences in development exist due to follicle source and sire used for IVF after vitrification.
Meiotic Resumption and Completion In Vitro of Immature Buffalo Oocytes after Vitrification
2017
Cryopreservation of buffalo oocytes can be done in using various methods of vitrification with different success rate. In this study, germinal vesicle (GV) stage buffalo oocytes were exposed to VS1 (7.5% EG + 7.5% DMSO in BM) for equilibration (10 min) before transferring to VS2 (15% EG + 15% DMSO + 0.6 M sucrose in BM) for 45 sec loaded in cryoloop and plunged directly into LN2 or directly plunging into LN2 in microdrops. After a few weeks of storage, the oocytes were warmed in step-wise dilution pattern for 3 min each in 0.6 M, 0.3 M and 0.15 M sucrose in BM, transferred to a washing solution (3x) before culturing in maturation droplets. In using the cryoloop method, the survival rate recorded was 86.5% (64/78) with a meiotic resumption rate of 85.9% (55/64). The maturation rate was 34.4% (22/64). In using the MDS method, the survival rate was 88.5% (54/75) with a meiotic resumption rate of 88.9% (48/54). The maturation rate was 42.6% (23/54). These findings indicate that both vit...
The cryopreservation of female gametes is still an open problem because of their structural sensitivity to the coolingand-freezing process and to the exposure to cryoprotectants. The present work was aimed to study the effect of vitrification on immature bovine oocytes freed of cumulus cell investment before freezing. To verify the feasibility and efficiency of denuded oocyte (DO) cryopreservation, the cytoplasmic alterations eventually induced either by cell removal or by the vitrification process were analyzed. In particular, the migration of cortical granules and Ca ++ localization were studied. In addition, the localization and distribution of microtubules and microfilaments in immature fresh and vitrified DOs were evaluated. Finally, to establish whether the removal of cumulus cells influenced developmental competence, DOs were thawed after vitrification, matured in vitro and fertilized; then presumptive zygotes were cultured to reach the blastocyst stage. The results indicate that mechanical removal of cumulus cells from immature bovine oocytes does not affect their maturation competence but reduces the blastocyst rate when compared with intact cumulus oocyte complexes (COCs). The findings indicate further that the vitrification process induces changes of cytoplasmic components. However, the composition of the manipulation medium used to remove cumulus cells plays a crucial role in reducing the injuries caused by cryopreservation in both cytoplasmic and nuclear compartments. In fact, the presence of serum exerts a sort of protection, significantly improving both oocyte maturation and blastocyst rates. In conclusion, we demonstrate that denuded immature oocytes can be vitrified after cumulus cells removal and successfully develop up, after thawing, to the blastocyst stage, following in vitro maturation and fertilization. 337 b 37.4±2.8 c 35.4±2.9 c n= number of oocytes; values are expressed as mean ± SE; a, b, c values within columns with different superscripts are significantly different (p<0,05)