In vitro embryo production has vastly improved over the past decade through the study of the in vivo environment and the metabolic requirements of embryos. In contrast, in vitro oocyte maturation ( IVM ) culture conditions have remained relatively unchanged and are suboptimal. The aim of this thesis was to create improved systems for bovine IVM by studying the metabolic profiles and requirements of intact cumulus oocyte complexes ( COCs ) during IVM and determining the ion and energy substrate composition of bovine follicular fluid ( FF ). Glucose, pyruvate and oxygen consumption of bovine COCs increased 2 - fold over the 24 h IVM period, with glucose being the preferred energy substrate. While initially the majority of glucose consumed by COCs is metabolised via glycolysis ( Llactate production ), a considerable proportion of glucose is used as a substrate for extracellular matrix ( ECM ) synthesis towards the end of IVM. Glucosamine ( an intermediate substrate of hyaluronic acid ) supplementation of IVM media lead to decreased glucose consumption and incorporation into ECM during FSH - stimulated expansion. Biochemical analyses of bovine FF demonstrated that the concentration of some ions and energy substrates varied with follicle size. Although follicular glucose concentrations increased with follicle size, levels were ~ 2 - fold lower than that found in Tissue Culture Medium ( TCM199 ), the most commonly employed medium for bovine IVM. Synthetic Follicular Fluid Medium ( SFFM ) was created, based on the FF data and also contained glucosamine. Two different glucose concentrations were examined, 2.3 mM glucose to represent physiological concentrations and 5.6 mM glucose, the same concentration as is in TCM199. Culturing COCs in different glucose concentrations manipulated the completion of nuclear maturation and this was dependant on concentration, gonadotrophin supplementation and the timing of media changes, demonstrating the importance of this substrate to meiotic competence. Although glucosamine had no effect on oocyte nuclear maturation, supplementation during IVM led to a dose - dependent decrease in blastocyst rates. The detrimental effects of glucosamine manifested during early cleavage and were associated with a 0.6 - fold decrease in protein synthesis levels within the oocyte compared to oocytes cultured in media with no glucosamine, suggesting a detrimental effect on developmental competence. Interestingly oocytes cultured in media containing glucosamine and EGF had significantly higher protein synthesis compared to the control group. The biochemical profiles of COCs during IVM and FF were determined and used to create new media that allowed manipulation of oocyte nuclear maturation but compromised cytoplasmic maturation. Further research is required to optimise SFFM and to investigate the detrimental effects of glucosamine on developmental competence. / Thesis (Ph.D.)--Medical School, 2004.
| Identifer | oai:union.ndltd.org:ADTP/263727 |
| Date | January 2004 |
| Creators | McDowall, Melanie Lisa |
| Source Sets | Australiasian Digital Theses Program |
| Language | en_US |
| Detected Language | English |
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