Lipid profile analysis of bovine in vitro blastocysts deriving from insulin treated oocytes by desorption electrospray ionization – mass spectrometry (DESI-MS)

The aim of this study was to characterize the lipid profile of bovine blastocysts produced from oocytes exposed to different insulin concentrations during maturation by DESI-MS. Insulin is a key metabolic hormone and its concentration in blood and follicular fluid changes in situations of metabolic imbalance as obesity, diabetes or negative energy balance (NEB). The impact of insulin on the lipid profile of blastocysts can provide important insights on the metabolic changes induced by this hormone on early development. Blastocysts were produced from abattoir derived oocytes according to standardized IVP-protocols in our laboratory. Insulin treatment was performed during 22 h of maturation using 0 (INS0); 0.1 (INS0.1) or 10 (INS10) µg/ml bovine insulin. After maturation, all treatment groups were submitted to equal conditions during fertilization and culture. On day 8, blastocysts were separately frozen at -80oC in PBS with 0.1% PVA and individually transferred to glass sides in randomized order. A total of 63 blastocysts were used for DESI-MS lipid profile analysis. Lipids such as diacylglycerols (DAG), triacylglycerols (TAG) cholesteryl esters (CE), squalene and ubiquinone were detected in positive ion mode as silver adducts. Average full scan mass spectra of the three different treatment groups indicated few changes in the lipid profiles. Multivariate statistics by PCA (Principal Component Analysis) was used to comprehensively explore the chemical information of the full mass spectral dataset and visualize the grouping of samples resulting from chemical similarity. PCA showed some extent of discrimination between INS0 and INS10 whereas the discrimination between INS0 and INS0.1 was less evident. Data suggests down-regulated mitochondrial metabolism (indicated by ubiquinone abundance) in INS10 as well as few changes in TAG- and cholesterol metabolism comparing the treated groups (INS10 and INS0.1) with the control (INS0). Overall, the low extension of changes observed in the DESIMS lipid profiles indicates minimal impact of insulin exposure during oocyte maturation on lipid content during preimplantation embryo development. The results of the lipid profile analysis shows that the lipid profile was not significantly different in the day 8 blastocyst after exposure of insulin during maturation. Possible explanations could be that the insulin exposure during the IVM period is not sufficient to promote extensive end-point metabolism changes in the lipids detected during preimplantation development, or that the early embryo strongly compensates for the impact of a metabolic stressor as insulin during oocyte maturation by a subsequent change in gene expression, leading to compensating mechanisms to obtain balance in the chemical profile and permitting a viable phenotype.