The overall objective of this thesis was to investigate the vitrification of bovine cumulus oocyte complexes (COCs) as a tool for conservation of female genetics. Specifically, the first objective was to compare in vitro maturation rates (i.e. end point Metaphase II (MII) rate) of bovine COCs following vitrification using two different equilibration times (0 vs 10 min) in vitrification solution 1 (VS1) and two different cryodevices (cryotop vs 0.25 mL straw). The MII rate was higher in the non-vitrified control group than in vitrified groups (61 vs 16%, P<0.001). In the vitrified groups, equilibration time in VS1 [TCM-199 + 7.5% ethylene glycol (EG) + 7.5% dimethyl sulfoxide (DMSO) + 20% calf serum (CS)] did not affect MII rate (P=0.964); however, MII rate was higher in COCs vitrified on cryotops than in straws (23 vs 9%, P=0.007).<p>
The second objective of this thesis was to compare cleavage and subsequent developmental competence of bovine COCs vitrified using different vitrification solutions (fresh vs frozen) and different equilibration times in VS1 (0 vs 5 min). Immature bovine COCs were vitrified on cryotops using vitrification solutions either prepared fresh or frozen/thawed, and two equilibration times in VS1 (0 vs 5 min). Cleavage and blastocyst production rates were higher (P<0.001) in the non-vitrified control group than vitrified groups (cleavage rate 93 vs 42% and blastocyst rate 31 vs 0.4%). The cleavage rate of COCs vitrified in frozen/thawed solutions with 5 min equilibration was higher (P=0.05) than in other treatment groups. However, the blastocyst rate did not differ (P=0.993) among vitrified groups.<p>
We hypothesized that the nuclear stage of bovine COCs at the time of vitrification will affect post-warming in vitro maturation (IVM), cleavage and embryo development. Firstly, a preliminary study was conducted to validate our in vitro maturation system. The COCs were at germinal vesicle (GV, 89%), germinal vesicle breakdown (GVBD, 47%), metaphase I (MI, 90%), and metaphase II (MII, 84%) stages at 0, 6, 12 and 22 h of IVM respectively. In a subsequent study, bovine COCs were vitrified after 0, 6, 12 and 22 h of IVM by loading on cryotops and plunging in liquid nitrogen. Following 1 min in warming solution (TCM-199 + 17% sucrose + 20% CS), COCs were placed in IVM medium to complete 22 h of IVM and nuclear stages were evaluated using lamin A/C-DAPI staining. The nuclear maturation (MII)
rates of COCs were 23, 23, 35 and 89% (P<0.001) in the 0, 6, 12 and 22 h IVM groups, respectively. In the final nuclear stage experiment, cleavage and embryo development was determined after vitrification of COCs after 0, 12 and 22 h of IVM. The cleavage and blastocyst rates were higher (P<0.001) in the non-vitrified (control) group than vitrified groups (73 vs 15% and 22 vs 0.3%, respectively). The cleavage rates (14% in 0 h, 17% in 12 h and 14% in 22 h groups; P=0.825) and blastocyst rates (0% in 0 and 22 h and 1% in 12 h groups) did not differ. Results from this study indicated that a greater proportion of COCs progressed to MII if vitrification occurred at MI rather than at GV and GVBD stages. However, the nuclear status of vitrified COCs appeared to have no effect on fertilization or subsequent embryo development.<p>
A final set of experiments were designed to determine if the exposure of bovine COCs to cryoprotectant solutions and different warming time intervals affects their ability to become fertilized, cleave and develop into blastocysts. In both experiments, the cleavage and blastocyst rates in the vitrified group were lower (P<0.001) than in the non-vitrified control groups VS1 group, and in the VS1 + vitrification solution 2 (VS2; TCM-199 + 15% EG + 15% DMSO + 20% CS) group. However, the cleavage and blastocyst rates did not differ (P>0.05) in control, VS1 and VS1+VS2 groups. In the second experiment, there was no difference in cleavage rates between 1 and 5 min intervals in the warming solution.
In conclusion, cryotop can be used as a preferred cryodevice for vitrification of bovine COCs. Vitrification solutions (VS1 and VS2) and the warming solution can be prepared and stored in the freezer (-20 °C) for convenience. The pre-equilibration of bovine COCs in VS1 had no effect on the nuclear maturation of vitrified/warmed COCs, but 5 min pre-equilibration in VS1 improved cleavage rates. The nuclear stage of COCs at the time of vitrification and cryoprotectant exposure appeared to have no effect on subsequent cleavage. Furthermore, there was no difference in cleavage and blastocyst rates following vitrification and 1 or 5 min warming time intervals. When compared to non-vitrified controls, the vitrification of immature bovine COCs resulted in reduced nuclear maturation, cleavage and embryo development rates. Further studies are needed to elucidate the causes of the poor embryo development in vitrified-warmed bovine COCs at genomic, proteomics and metabolomics levels.
| Identifer | oai:union.ndltd.org:USASK/oai:usask.ca:etd-12082010-190458 |
| Date | 10 January 2011 |
| Creators | Prentice, Jennifer Rae |
| Contributors | Singh, Jaswant, Mapletoft, Reuben, Anzar, Muhammad, Baerwald, Angela, Muir, Gillian |
| Publisher | University of Saskatchewan |
| Source Sets | University of Saskatchewan Library |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://library.usask.ca/theses/available/etd-12082010-190458/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Saskatchewan or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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