## 3 Results and Discussion
### Identification of Characteristic Bioactive Lipids and Fatty Acids  
A total of 653 lipid molecules were identified in the breast muscle of 30 Silkie chickens and 30 Wuding chickens. The PLS-DA model could clearly distinguish between breast muscle samples from the two breeds (Figure 2A). These 653 lipid molecules were categorized into four major classes, which were further subdivided into 28 subclasses. The distribution of the lipid major classes and subclasses is presented in Figure 2B. Phospholipids accounted for the highest proportion (68%), followed by sphingolipids (21%). Based on the criteria of VIP > 1 and \(p\) < 0.05, differential lipid subclasses between the two breeds were identified. In the breast muscle of Silkie chickens, phosphatidylethanolamine (PE), phosphatidylinositol/glycerol (PG), cardiolipin (CL) and sulfatide (ST) were significantly upregulated, and sphingomyelin was significantly downregulated (Table S2). Among them, 27 PE molecules, five PG molecules, 24 CL molecules, and five ST molecules were significantly upregulated, and 11 sphingomyelin molecules were significantly downregulated in Silkie chickens (Figure 2C, Table S3). Figure 2: Identification of characteristic bioactive lipids and fatty acids. (**A**) PLS-DA based on overall lipid molecules. (**B**) Distribution of major lipid classes and subclasses. (**C**) The number of differential lipid molecules in each lipid subclass. Orange and green represent lipid molecules that were significantly upregulated and downregulated in Silkie chickens, respectively. (**D**) Heat map of differential fatty acids in Silkie and Wuding chickens. Orange, green, and gray squares indicate fatty acids that were significantly upregulated, significantly downregulated, and not differentially expressed in Silkie chickens, respectively. Transparent squares indicate the absence of this fatty acid. To further identify the differential lipid molecules that contributed significantly to the upregulation of CL, PE, PG, and ST, we calculated the relative contribution of each lipid molecule to differences in lipid subclasses. As illustrated in Figure S1A, CL(18:2_18:1_18:1_18:2)-H and CL(18:2_18:1_18:2_18:2)-H contributed to 31% and 25% of the variance in the CL subclass, respectively. As illustrated in Figure S1B, PE(18:0_20:4)-H, PE(16:0_20:4)-H, and PE(36:4)+H accounted for 32%, 13%, and 10% of the variance in the PE subclass, respectively. As illustrated in Figure S1C, PG(16:0_18:1)-H and PG(18:0_18:1)-H contributed 69% and 23% of the variance in the PG subclass, respectively. As illustrated in Figure S1D, ST(m38:0)-H accounted for 77% of the variance in the ST subclass. These eight lipid molecules play an important role in the formation of four characteristic bioactive lipids in Silkie chickens. PE is a lecithin with antioxidant and antiaging effects [42]. CL is a mitochondria-exclusive phospholipid. A reduction in CL content is related to aging and several neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, and traumatic brain injury [43]. ST is a multifunctional molecule, and ST deficiency is an important contributor to and driver of neuroinflammation and mild cognitive impairment in Alzheimer's disease [44]. The fatty acid composition is shown in Figure 2D. A total of 116 fatty acids were detected. Based on the criteria of VIP > 1 and \(p\) < 0.05, 19 fatty acids were significantly upregulated and 17 fatty acids were significantly downregulated in the breast muscle of Silkie chickens (Table S4). Among monounsaturated fatty acids, palmitoleic acid was significantly upregulated in Silkie chickens. Among polyunsaturated fatty acids, DPA and DHA were generally significantly upregulated in Silkie chickens. Palmitoleic acid can significantly ameliorate or prevent insulin resistance and diabetes [45]. DPA and DHA exert myriad health benefits on cardiovascular disease, diabetes, cancer, depression, and cognitive decline [46]. DHA has unique actions in promoting normal brain function [47].