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Title: A metabolic approach towards optimising equine in vitro maturation
Author: Lewis, Niamh Lucille
ISNI:       0000 0004 7964 2048
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2019
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In the horse, there is a paucity of species-specific IVM optimisation and energy metabolism pathways in COCs during IVM are undefined. Studies were undertaken to characterise glucose, pyruvate and lactate (GPL) and oxidative metabolism (OXPHOS) in equine COCs during IVM and further investigate these parameters under varying clinical conditions. Gene expression in single oocytes, cumulus cells and blastocysts was also explored. Novel technology for the horse; enzyme-linked metabolic assays, the Seahorse XFp Analyser, PolyA PCR and Primovision™ were employed to generate data. Data demonstrated that COCs had high glucose consumption, accounted for by lactate production (glycolytic index ~100%). Up to 24% COCs depleted all available glucose during IVM, which was not associated with a change in maturation or cleavage rate. COCs produced and secreted pyruvate into the medium. Glucose/lactate (GL) metabolism was independent of cumulus/meiotic status but correlated to COC DNA content. Pyruvate production was independent of DNA content. GPL metabolism displayed a temporal pattern; GL increased during 10-20 h and GL decreased and pyruvate production increased during 20-30 h. Oxygen consumption rate (OCR) was high, demonstrating OXPHOS plays a larger role in equine COC energy metabolism than in other species. Examination of mitochondrial function showed that ~50% of OCR was coupled to ATP production, ~35% was non-mitochondrial and 17% due to proton leak and did not vary over time. In contrast, spare capacity fell from 85% to 5% over the course of IVM. Addition of 0.15 mM pyruvate increased glucose consumption and at 5% oxygen, 0.15 mM pyruvate decreased pyruvate production. Pyruvate concentration (0-0.5 mM) had no effect on OCR or mitochondrial function. Addition of 0.5 mM pyruvate to 17 mM glucose medium increased expression of G6PD in cumulus cells and resulted in a switch to pyruvate consumption. Glucose concentration (5.6/17 mM) had no impact on developmental, morphokinetic, GPL or OCR measurements but the proportion of OCR coupled to ATP and non- mitochondrial processes were decreased and increased respectively. Taken together, these results demonstrate the plasticity of the equine COC in terms of substrate utilisation but that this plasticity incurs a change at the metabolic level that warrants further investigation.
Supervisor: McGowan, Cathy ; Brison, Daniel ; Hinrichs, Katrin ; Argo, Caroline ; Peffers, Mandy ; Grove White, Dai Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral