Quantitative and qualitative lipidomic analysis of in vitro produced (IVP) bovine embryos with different developmental kinetics

Lipid accumulation in in vitro produced (IVP) embryos has been related to lower cryopreservation efficiency and developmental potential, being indicative of inadequate in vitro culture conditions when compared with in vivo system. The hypothesis of this work is that embryos with different developmental kinetics metabolize and pile up lipids differentially, which may be reflected in the embryo viability. Therefore, our objective was to gather quantitative, by SUDAN BLACK B staining, and qualitative, by matrix assisted laser desorption Ionization – mass spectrometry (MALDI-MS), data about the lipid composition of embryos of fast (4 cells 40hpi) and slow (2 cells 40hpi) development. The IVP bovine embryos were produced and cultured individually, classified as fast and slow according to the number cells of the first cleavages (40hpi) and finally analyzed by staining and mass spectrometry at cleavage (40hpi), morulae (96hpi) and blastocyst (168hpi) stages. For staining, we calculated the number of pixels obtained from each image and converted into arbitrary units by a script created in the development environment Matlab using the Image Processing toolbox. For staining analysis, conventional group cultured used as control. The MS data were acquired directly on single embryos, without extraction, in the positive ion mode using an Autoflex III MALDI-TOF/TOF mass spectrometer (Bruker Daltonics) using 2,5-dihydroxybenzoic acid (DHB) matrix to favor the ionization of the lipids. For MALDI-MS analysis, in vivo blastocysts were included as control group. Both the qualitative and quantitative data were submitted to ANOVA with Tukey post test (SUDAN BLACK) and MetaboAnalyst 2.0 (MALDI-MS) for statistical analysis. No difference in the amount of lipids between cleavage (Fast: 93884±4331; Slow: 68911±7180; Fast Control: 74622±21180; Slow Control: 70763±20046) and morulae (Fast: 65803±13774; Slow: 43511±4097; Control: 4610±12298), groups has been observed by staining, although slow blastocyst presented lower amount of lipids when compared to the fast and control groups (Fast: 122626±30378; Slow: 38617±3379; Control: 95658±15138). These data were corroborated by MALDI-MS, where the highest variability of phospolipid species (of m/z 725.5, 730.5, 732.5, 752.5, 758.5, 780.6, 782.6) was observed in fast blastocysts, while phospholipid ions of m/z 704.5, 746.5, 756.5, 788.5, 810.5 look characteristic of slow blastocysts. Based on these data, we can conclude that embryo kinetic has an important role on lipid metabolism of IVP embryos.