# Effects of Roughage on the Lipid and Volatile-Organic-Compound Profiles of Donkey Milk
## 4 Discussion  
Donkey milk has a total protein content of 1.5-1.8%, a lactose content of 5.8-7.4%, and a fat content of 0.2-1.8% [37; 38]. Lipids are one of the main nutrients in donkey milk and an important indicator of milk quality in commerce [39; 40]. Mice fed donkey milk, especially the n-3 family, were found to show increased hepatic mitochondrial uncoupling, decreased energy efficiency, maintained energy balance, decreased body fat deposition, and improved antioxidant and anti-inflammatory defenses [41]. Therefore, donkey milk lipids represent a valid focus of research. This study employed UPLC-ESI-MS/MS, which is considered a novel cutting-edge technology that provides a platform for evaluating the lipids in donkey milk primarily on a minor scale with excellent precision and in a short period of time [42]. In this study, 1842 lipids were detected in the donkey milk. This is significantly higher than that for donkey milk (335 lipids), cow milk (557 lipids), goat milk (756 lipids), and human milk (617 lipids) identified by conventional LC-MS [13; 43; 44; 45]. Specifically, MG, PG, BisMePA, wax ester (WE), ZyE, Co, and AcHex were newly identified in the present study compared with previous studies [13; 37; 46; 47]. Clearly, the lipids detected in this study are more abundant than those previously studied, which could be  
Figure 5: Difference analysis of VOC compositions between G1, G2, and G3. OPLS-DA plot (**A**), corresponding OPLS-DA validation plots (**B**), heatmap of hierarchical clustering analysis (red indicates increased, blue indicates decreased) (**C**). due to the technological superiority of the technique used. TGs (a total of 1016) were discovered to be the dominant lipids in donkey milk, which is consistent with the results of previous studies [13; 48]. Interestingly, we detected PA and SiT in the dietary roughage, but they were not detected in the donkey milk. This may be due to the degradation of PA to DG [49] by phospholipid hydrolase. Previous studies have shown that different fermentation systems present different FA compositions, and the production rate of milk lipids increases with FA concentration [50; 51]. Dietary fiber has an impact on how the lipids are metabolized, which results in further differences in the lipid compositions of milk [52; 53]. A previous study indicated that the contents of specific FAs in TG molecules is positively correlated with the content of the corresponding dietary FAs [54]. The levels of PC, PE, PG, Cer, hexosylceramide, SM, and BisMePA in the G1 and G2 groups were significantly lower than those in the G3 group, suggesting that these lipids are affected by roughage. Interestingly, TGs, especially TG (18:4-8:0-18:1) and TG (8:0-10:0-18:3), were observed at higher levels in the G1 group than in the G2 and G3 groups. It is possible that the roughage in the G1 group contained more PUFAs, enhancing the diversity and content of TG species [55]. In the present study, the G2 and G3 groups contained higher levels of C16:0 in their diets, which resulted in higher levels of C16:0 in the milk lipids. This is consistent with the finding of a previous study, i.e., that changes in the equivalent FA content of milk fat can come from changes in the lipid composition of the crude feed [56]. In addition, monogalactosylmonoacylglycerollogcerol (MGMG), monogalactosyldialacylglycerollogcerol (DGDG) were detected in the donkey milk, and these lipids are present in wheat flour [57]. The main lipid class contents of the different groups have linear relationships with their dietary lipid profiles, and this phenomenon has been observed upon the modification of lipid intake in human, sheep, and cow diets [51; 53; 58; 59]. TGs were found to be most interactive with other lipid species, which indicates that TG may play an essential role in milk production in donkey fed on different roughages.