204 Lipid profiling of bovine oocytes matured in vivo and in vitro

It is well established that events occurring during in vivo oocyte maturation (IVO) contribute to increased developmental potential compared with IVM. One factor influencing developmental competence is hypothesised to be altered lipid metabolism during IVM. Attempts have been made to improve synchrony between meiotic and cytoplasmic maturation in vitro through addition of C type natriuretic peptide (CNP) in prematuration culture followed by culture without CNP to the metaphase II stage. In this study, metabolic profiling was performed on germinal vesicle (GV), IVM, CNP-IVM, and IVO oocytes to identify differences in cytoplasmic maturation, potentially affecting developmental competence. Follicle aspiration was performed on cross-bred beef cattle for collection of either oocytes for IVM or after IVO. Recovered GV oocytes were either stored for later analysis or allocated to IVM or CNP-IVM. Media for both IVM systems were prepared in TCM-199 with 10% FBS, 1% penicillin-streptomycin, 2 mM glutamine, 0.2 mM sodium pyruvate, 5 μg mL⁻¹ follicle-stimulating hormone, and 50 ng mL⁻¹ epidermal growth factor. Cumulus–oocyte complexes were either cultured for 22 h in conventional IVM or for 6 h in prematuration culture with 200 nM CNP prepared in maturation media followed by 22 h of IVM. Oocytes from each maturation treatment were denuded and polar body visualisation was confirmed before analysis. In vivo-matured oocytes were evaluated immediately, without any culture after recovery. Raman spectroscopy was used for single cell metabolic profiling and a principal component (PC) analysis (PCA) was applied to visualise variation between treatments. Raman spectra provide a molecular fingerprint based on wavelength shift from energy transfer between incident light to the molecules encountered. A total of 17 spectra were obtained per oocyte (n = 3 per treatment) with the laser focused on the cross section of cytoplasmic lipid droplets chosen at random. The PCA revealed differences (PC1 = 82.5%, PC2 = 14.9%) in the molecular composition between each maturation system, where IVM resulted in the most similar spectra to GV oocytes while CNP-IVM altered the molecular profile of mature oocytes. All groups were significantly different than those resulting from IVO. Oocytes from IVO and CNP-IVM groups also displayed less variation within each system compared with those from GV and IVM. Mapping was also performed, where spectra were obtained in 1 μM intervals over each sample. Spatial heat maps compared with a reference library identified fewer fatty acids in CNP-IVM and IVO oocytes than IVM and GV. We hypothesise that greater levels in IVM oocytes are an indication that fatty acids are not used appropriately during final maturation. Molecular differences in lipid droplet composition suggest that the CNP pathway regulating resumption of meiosis also affects cytoplasmic metabolism. The CNP-IVM system resulted in less variation within the treatment group; however, further intervention is required to achieve cytoplasmic maturation comparable to IVO oocytes. The use of Raman spectroscopy allows for single cell metabolic profiling in oocytes, which will be valuable for further optimization of maturation systems.