The effect of Glucose Concentration (5.6 mM vs. 17 mM) During IVM on Developmental and Metabolic Markers of Equine Cumulus-oocyte Complex Function

Lewis, N.; Hinrichs, K.; Brison, D.; Sturmey, R.; McG. Argo, C.

Abstract

Both M199 and DMEM/F-12 support in vitro maturation (IVM) of equine oocytes and production of viable embryos after ICSI. However, these media differ markedly in glucose concentration: 5.6 mM (M199) vs. 17 mM (DMEM/F-12). Given the known impact of embryonic nutritional programming on fetal and adult development, we evaluated the effect of glucose concentration during IVM on equine cumulus-oocyte complex (COC) function. Abattoir-derived COCs were held overnight, then cultured for 30 h either in individual 10-μl droplets within a petri dish (Study 1) or directly in a Seahorse XFp Bioanalyser plate (Study 2) in M199 with 10% FBS and 5 mU/ml FSH, with 5.6 or 17 mM glucose. After IVM, spent medium was analysed for glucose, pyruvate and lactate using enzyme-linked ultrafluorometry (n = 176 COCs, Study 1). Mature oocytes underwent ICSI, and time of fragmentation, first cleavage, and blastocyst formation were monitored using a Primovision™ Time–Lapse System. Basal oxygen consumption rate (OCR) of 54 COCs was recorded after IVM (Study 2), then respiratory inhibitors were sequentially added: 1) the ATP synthase inhibitor, oligomycin [1 μM]; 2) the protonophore uncoupler FCCP [5 μM]; and 3) inhibitors of the electron transport chain, antimycin and rotenone [2.5 μM]. This allowed calculation of the proportion of OCR coupled to ATP production, spare respiratory capacity, non-mitochondrial respiration and proton leak. Data were analysed via Student’s t-test or one-way ANOVA. There were no significant differences in maturation, cleavage, or blastocyst development between 5.6 mM and 17 mM glucose (63 and 51%; 76% and 76%, and 8.2% and 2.9%, respectively), nor in the evaluated morphokinetic timings. Strikingly, 18% of COCs in the 5.6 mM glucose group depleted all available glucose; however this was not associated with altered embryo development. Medium glucose concentration did not significantly affect glucose consumption, lactate or pyruvate production, lactate:glucose ratio, or basal OCR. The OCR coupled to ATP production was lower and the non-mitochondrial OCR higher in the 17 mM (42% and 48%, respectively) than in the 5.6 mM treatment (62% and 26%, respectively; p=0.02). In summary, glucose concentration did not affect glucose metabolism, oocyte maturation or embryo development, but high glucose decreased coupled oxidative metabolism and increased non-mitochondrial metabolism in COCs, which points to elevated production of reactive oxygen species. The effects of these changes in mitochondrial function on the resultant embryos and offspring require further investigation, so that an optimum glucose concentration for equine IVM can be determined.

Citation

Lewis, N., Hinrichs, K., Brison, D., Sturmey, R., & McG. Argo, C. (2018). The effect of Glucose Concentration (5.6 mM vs. 17 mM) During IVM on Developmental and Metabolic Markers of Equine Cumulus-oocyte Complex Function. Journal of Equine Veterinary Science, 66, 181. https://doi.org/10.1016/j.jevs.2018.05.073

• Journal Article Type: Article
• Acceptance Date: Jul 1, 2018
• Online Publication Date: Jun 22, 2018
• Publication Date: 2018-07
• Deposit Date: Jun 13, 2019
• Journal: Journal of Equine Veterinary Science
• Print ISSN: 0737-0806
• Publisher: Elsevier
• Peer Reviewed: Peer Reviewed
• Volume: 66
• Pages: 181
• DOI: https://doi.org/10.1016/j.jevs.2018.05.073
• Public URL: https://hull-repository.worktribe.com/output/1989705
• Publisher URL: https://www.sciencedirect.com/science/article/pii/S0737080618302776
• Additional Information: This article is maintained by: Elsevier; Article Title: The effect of Glucose Concentration (5.6 mM vs. 17 mM) During IVM on Developmental and Metabolic Markers of Equine Cumulus-oocyte Complex Function; Journal Title: Journal of Equine Veterinary Science; CrossRef DOI link to publisher maintained version: https://doi.org/10.1016/j.jevs.2018.05.073; Content Type: simple-article; Copyright: