Evaluation of lipid content in the equine oocyte and the changes that occur during In Vitro maturation

Limited knowledge exists on equine cumulus-oocyte complexes (COCs) metabolism during in vitro maturation (IVM) and on the culture conditions necessary to optimise oocyte developmental competence. Previous studies have suggested that while glycolysis plays a role in energy provision, ATP is produced primarily through oxidative phosphorylation (OXPHOS) of non-glycolytic substrates in equine COCs. Endogenous lipids, especially fatty acids stored within lipid droplets as triglycerides (TGs), are increasingly recognised as prime candidates for providing energy-rich substrates through fatty acid oxidation (FAO), yielding high levels of ATP. This thesis aimed to quantify lipid content in equine oocytes for the first time using novel methods and explore the changes occurring during in vitro maturation that may be attributed to consumption for ATP production. Simultaneously analysing bovine and porcine oocytes provided comparative insights about technical validation against existing studies, allowing for a cross-species comparison. Data generated using fluorometric analysis of TG content did not detect significant differences in TG levels between immature and mature oocytes. The TG content of immature equine oocytes was 40.6 ± 4.9 ng/ml and 50.2 ± 4.3 ng/ml in MII oocytes. Porcine oocytes had higher levels of TG than equine (61.3 ± 4.3 ng/ml), and bovine oocytes had lower levels (34.7 ± 3.9 ng/ml). A significant difference was noted between porcine and bovine oocytes, and values aligned with those reported in previous studies. Data generated using high-content confocal screening of Nile Red-stained oocytes also did not detect any difference in lipid content between immature and mature equine oocytes; however, it did reveal significant interspecies variations in lipid droplet size, number, and intensity. Porcine oocytes had the largest liquid droplets (LDs), and equine oocytes had the smallest. Additionally, porcine oocytes contained significantly more LDs than the other species, while bovine oocytes exhibited significantly greater lipid intensity. The observed morphological differences between species emphasise the need for further investigation into changes occurring during maturation. Such studies could provide valuable insights into species-specific metabolic strategies for energy acquisition. Although spatial changes in lipid distribution were noted morphologically, additional refinement in pipeline analysis may be needed to achieve objective differentiation.
Taken together, these data show that equine oocytes are indeed rich in lipids. However, further studies are needed to confirm the contribution of these lipids to ATP production in equine COCs during IVM and how differences in culture conditions may modify their use.