Metabolic Comparison of Mature and Immature Bovine Cumulus–Oocyte Complexes with Standardization of Bioenergetic Assessment

This study compared the bioenergetic profiles of immature and in vitro-matured bovine cumulus-oocyte complexes (COCs) using Seahorse extracellular flux technology, with the aim of establishing standardized conditions for real-time metabolic assessment during in vitro maturation (IVM). Groups of five COCs were analysed prior to maturation and after 22 h of IVM using the Seahorse XFp Analyzer to measure oxygen consumption rate (OCR, pmoL/min) and extracellular acidification rate (ECAR, mpH/min), providing dynamic readouts of oxidative phosphorylation and glycolysis that extend beyond conventional endpoint assays. To optimize assay performance, three media were first evaluated: TCM199, DMEM/F12, and HEPES-buffered synthetic oviductal fluid (HSOF). HSOF yielded the most reliable readings for immature COCs, whereas TCM199 provided superior conditions for mature COCs. Adhesion strategies were then tested by comparing uncoated wells with wells coated with fibronectin, concanavalin A, or Matrigel^®. Sequential injections of oligomycin and rotenone plus antimycin A enabled partitioning of mitochondrial and glycolytic contributions to ATP production. COC maturation was associated with a clear metabolic shift from glycolysis toward oxidative metabolism. Immature COCs displayed a predominantly glycolytic phenotype, while mature COCs showed increased active mitochondrial ATP production. Adhesion conditions markedly affected the detected metabolic profile: concanavalin A and fibronectin supported effective attachment and were associated with robust energy metabolism, whereas Matrigel^® and poor adhesion were linked to quiescent profiles with low OCR and ECAR signals. Together, these data define practical assay parameters for extracellular flux analysis of COCs and highlight the increasing reliance on mitochondrial function as a hallmark of oocyte maturation, supporting improved metabolic phenotyping for IVM optimization.