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Global gene expression analysis of in vitro produced cryopreserved bovine embryos using vitrification and slow freezing techniques



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The overall objective of this thesis was to analyze developmental and gene expression changes in cryopreserved in vitro produced (IVP) bovine embryos using two techniques; vitrification and slow freezing. Specifically, the first objective was to study and compare the blastocyst development of IVP bovine day 6 morulae [day 0 = in vitro fertilization (IVF)] after cryopreservation using vitrification and slow freezing (end point = development to expanded blastocyst stage on day 7-8). The blastocyst development rate for the vitrification group (52±4.6%) was higher (p<0.001) than that of the slow freezing group (35±4.2%). Blastocyst development rate was the highest in unfrozen control group (78 ± 3.6%). Re-expansion of vitrified morulae upon warming and correlation with subsequent blastocyst conversion rate was studied. No significant correlation was found between the vitrified morula re-expansion upon warming and blastocyst rate (Pearson’s correlation = -0.048; p>0.05). Data obtained from above study of cryopreserved embryos was also utilized for comparing the effect of season (cleavage rate and morula rate) and season x cryopreservation interaction on blastocyst conversion rate. Fall season had lower cleavage (67±1.6%; day 2) and morula (22±1.4%; day 6) rates than other seasons (74±1.1% and 30±1.2%, respectively; p<0.05). Spring, summer and winter seasons did not differ (p>0.05). Blastocyst conversion rate differed (p<0.05) between summer (63±4.5%) and spring (85±3.3%) seasons in unfrozen groups, between summer (20±4.1%) and all other seasons (43±8.3%) in slow freezing groups and did not differ among the seasons in the vitrification group (p=0.19). Another aim of the study was to determine the effect of different cryoprotectants (glycerol and ethylene glycol) on the blastocyst conversion rates from slow frozen morulae. The blastocyst conversion rate had a tendency (p=0.065) to be higher in the ethylene glycol group (31±5.3%) than the glycerol group (18±4.3%). A final set of experiments was designed to compare the global gene expression analysis of vitrified and slow frozen IVP embryos to control IVP embryos. Day 6 IVP bovine morulae were randomly distributed across three groups: unfrozen control, vitrified and slow frozen. These embryos were allowed to grow to expanded blastocyst stage in culture for 24-48 h without treatment (control) or after warming (vitrification and slow freezing groups). Four successful replicates were conducted for each group on separate dates (6-7 embryos per group per replicate). Total RNA was extracted, RNA quality was tested using bio-analyzer and samples were hybridized on microarray slides. Images acquired from microarray slides were analyzed using ArrayPro™, ELMA, FlexArray and Ingenuity pathway analysis softwares. The vitrified group had 64 differentially regulated (up and down regulated) genes as compared with control group, while a total of 162 genes were differentially expressed in slow frozen group as compared with control group. Upon in silico analysis differential gene expression showed 76 genes up and downregulated between slow freezing and vitrification groups. Interestingly, vitrification embryos showed highest gene expression changes in lipid metabolism, cellular movement, cell-cell signaling, molecular transport and vitamin and mineral metabolism. Detailed pathway analysis showed the affected mechanisms of lipid peroxidation and steroid biosynthesis pointing towards the impairment of further embryo development. In addition, pathways involved in blastocyst hatching and apoptosis were affected. In slow frozen embryos, only three genes were affected. The pathways suggest involvement in cell structure and cell adhesion/MAPK pathways. The study suggests that even if the survival of vitrified embryos is significantly higher than the slow frozen embryos, the viability, innate developmental capacity and future uterine implantation capability of vitrified embryos may be lower than that of slow frozen survivor embryos.



Bovine embryo, Vitrification, Slow freezing, Lipid metabolism, Microarray, Real time PCR, Cryopreservation, Morula, IVF, implantation



Master of Science (M.Sc.)


Veterinary Biomedical Sciences


Veterinary Biomedical Sciences



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