Vitrification of Bovine Oocytes
Anchamparuthy, Vahida Muhammed Ismail
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Cryopreservation of oocytes is a challenge. Studies were conducted to vitrify mouse zygotes and cumulus-intact bovine oocytes from follicles of different diameters, small (â ¤ 4 mm) and medium (4 to 10 mm), using nylon mesh. The specific goals were to assess changes in apoptotic gene expression (Fas-FasL, Bax, Bcl-2, and survivin) in conjunction with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and caspase assays. Mouse zygotes were exposed to increasing concentrations of ethylene glycol (EG), Ficoll-70 and sucrose in phosphate buffered saline (PBS) for vitrification on nylon mesh and plunged into liquid nitrogen. Warming resulted in 81.7% morphological survival. The rate of blastocyst formation was 59.9% for vitrified zygotes but, this was significantly lower than that of non vitrified embryos (66.2%). There was no difference in the hatching rates between groups. Both Fas and FasL mRNA were detected at the 4-cell and morula stages, suggesting Fas signaling was operational in early embryos. The level of expression of Bax mRNA tended to increase, while expression of survivin mRNA was not different for 2- and 4-cell embryos. Fragmented embryos showed an increase in Bax mRNA levels, while survivin mRNA level was reduced. In the second experiment, vitrification of bovine oocytes was carried out. Pre-cooled cryovials resulted in 98.9% morphological survival. The oocytes from small and medium size follicles had a significant impact on cleavage (53.7 Â± 1.6% vs. 43.8 Â± 1.6%, respectively) and blastocyst rates were 16.9 Â± 1.0% and 11 Â± 1.2%, respectively. Follicle size for oocytes had no impact on the expression of apoptotic genes. The Fas-FasL and Bax-Bcl-2 mRNA showed increased expression after vitrification, but tended to decrease after 9 h of maturation. Yet, results from TUNEL and caspase assays did not support the evidence of the downstream apoptotic signaling pathway in embryos. The semen utilized for in vitro fertilization in both vitrified and control oocytes responded differently in the 4 tested bulls than their field fertility record. The altered transcriptional activities of apoptotic genes, Fas-FasL and Bax-Bcl-2, and survivin were indicative of possible apoptotic activity in vitrified embryos and oocytes subjected to in vitro fertilization.
- Doctoral Dissertations