Table 1.
ESI and mass spectrometer parameters.
Fig 1.
Characterization of porcine milk extracellular vesicles (EV) in different stages of lactation from sows receiving diets with either a high (13:1, soybean oil [GS]) or a low (4.6:1, linseed oil [GL]) ratio of omega-6 to omega-3 (ω-6:ω-3) fatty acids.
A. The ANOVA mixed model comparison using data from nanoparticle tracking analysis (NTA) of mean size (nm) and EV concentration (particles/mL) in relation to the diets, time, and interaction of diet and time, with raincloud plots showing data distribution. Data are presented as means ± SEM. B. in relation to the diets, time, and interaction of diet and time, and raincloud plots with respective data distribution. Data are presented as means ± SEM. C. Pearson’s correlation test (R2 = 0.802, p-value < 0.05) comparing protein concentration (in mg/mL) and EV concentration (in particles/mL); D. Western blotting (WB) of EV marker TSG 101 in samples from both groups after ultracentrifugation coupled with size exclusion chromatography (SEC). Fractions 2 and 3 from SEC contained EV and were pooled together for further analysis. E. Representative electron micrograph of porcine milk exosomes in a close-up of a single vesicle and wide view. F. The bar scale is equal to 100 nm in all micrographs.
Fig 2.
Summary of 947 lipid elements identified in colostrum and milk extracellular vesicles (EV) by liquid chromatography–quadrupole time-of-flight mass spectrometry lipidomics analysis, color-coded by lipid category and stratified in lipid main classes.
Fig 3.
Principal component analysis (PCA) plot illustrating the lipidomic profiling of extracellular vesicles (EV) from porcine colostrum and milk, showing a distinct lipidomic profile between colostrum (day 0) and mature milk (days 7 and 14) EV.
There was no clear separation between feeding groups receiving diets with either a high (13:1, soybean oil [GS]) or a low (4.6:1, linseed oil [GL]) ratio of omega-6 to omega-3 (ω-6:ω-3) fatty acids. The color legend differentiates the time points: red for day 0, blue for day 7, and green for day 14. The shape legend distinguishes the dietary groups: circles for the GL group and squares for the GS group.
Fig 4.
Hierarchically clustered heatmap of the top 50 statistically significant lipids with the smallest P-values, grouped by day and time in milk extracellular vesicles (EV) from sows fed diets with either a high (13:1, soybean oil [GS]) or a low (4.6:1, linseed oil [GL]) ratio of omega-6 to omega-3 (ω-6:ω-3) fatty acids on days 0, 7, and 14.
The colors in the heat map reflect the different abundance of the lipids in milk EV and colostrum EV (mean smashed and divided by the range of each variable).
Fig 5.
(A) Volcano plot of log2 fold changes (x-axis) and their associated -log10 false discovery rate (FDR) adjusted p-values (y-axis) of all identified lipids in the comparison between colostrum extracellular vesicles (EV) (day 0) and milk EV at day 7. Significantly different lipids (t-test FDR adjusted p-value < 0.05, fold change > 2) are highlighted, indicating up (red) and down (blue) regulated differentially expressed lipids. (B) The circular bar plots represent the number of lipid elements in each subclass that were up- or down-regulated on EV from colostrum compared to milk at day 7.
Fig 6.
(A) Volcano plot of log2 fold changes (x-axis) and their associated -log10 false discovery rate (FDR) adjusted p-values (y-axis) of all identified lipids in the comparison between colostrum extracellular vesicles (EV) (day 0) and milk EV at day 14. Significantly different lipids (t-test FDR adjusted p-value < 0.05, fold change > 2) are highlighted, indicating up (red) and down (blue) regulated differentially expressed lipids. (B) The circular bar plots represent the number of lipid elements in each subclass that were up- or down-regulated on EV from colostrum compared to milk at day 14.
Fig 7.
Volcano plots showing the log2 fold changes (x-axis) and their associated -log10 false discovery rate (FDR) adjusted p-values (y-axis) for all identified lipids in the comparison of milk extracellular vesicles (EV) from sows fed with soybean oil (GS) and linseed oil (GL) at different lactation stages: A. Day 0, B. Day 7, and C. Day 14 postpartum. No significant lipid differences were observed at time points A and B, while one lipid was found to be downregulated in the comparison between GS and GL at day 14 (C).