## 3 Results and Discussion  
### Identification of Characteristic Bioactive Metabolites  
A total of 624 metabolites were identified in the breast muscle of 30 Silkie chickens and 30 Wuding chickens. Based on chemical classification, these metabolites mainly included organic acids and derivatives (40.77%) and lipids and lipid-like molecules (23.69%) (Figure 1A). The constructed PLS-DA model successfully distinguished between Silkie and Wuding chickens (Figure 1B), indicating significant differences in metabolite levels between the two breeds. Based on the criteria of VIP > 1 and \(p\) < 0.05, 38 metabolites were significantly upregulated and 88 metabolites were significantly downregulated in Silkie chickens (Figure 1C, Table S1). KEGG pathway enrichment analysis of these differential metabolites showed that arginine and proline metabolism and purine metabolism were significantly upregulated, while aminoacyl-tRNA biosynthesis, sphingolipid metabolism, and glycine, serine, and threonine metabolism were significantly downregulated in Silkie chickens (Figure 1D).  
Thirteen significantly upregulated metabolites with health-promoting effects were identified as characteristic bioactive metabolites of Silkie chickens (Figure 1E). Estradiol plays a role in regulating women's menstrual cycles [22] and effectively prevents menopausal syndrome [23; 24]. These effects are consistent with the traditional Chinese patent medicine \"Black Chicken Pills\". \(S\)-Adenosylmethionine, as a nutritional supplement, has been shown to maintain liver health [25], improve mood [26], and comfort joints [27]. Homo-l-arginine supplementation can attenuate atherosclerosis [28]. Nicotinic acid has pharmacological activities such as vasodilation and blood lipid-lowering effects [29]. Phytosphingosine can exert antidiabetic effects by reducing serum cholesterol levels, reducing free fatty acid levels, improving insulin sensitivity [30; 31], and alleviating inflammatory diseases [32]. Glyceric acid can activate mitochondrial metabolism and reduce inflammation in healthy volunteers aged 50-60 years [33; 34]. D-Phenyllactic acid can alleviate colitis by regulating microbiota composition, short-chain fatty acid production, and inflammatory responses [35]. Uridine monophosphate (UMP) can enhance cognitive functions [36]. Creatine is the most common dietary supplement with beneficial effects, enhancing exercise performance and protecting nervous and cardiac tissue [37]. Guanine has neurotrophic and neuroprotective effects [38]. 4-acetamidobutanoic acid is strongly correlated with antioxidant activity [39]. Biotin has health benefits [40] and improves skin, hair, and nails [41].  
Figure 1: Identification of characteristic bioactive metabolites. (**A**) Classification and percentage of metabolites in the breast muscle of 30 Silkie chickens and 30 Wuding chickens. (**B**) PLS-DA based on overall metabolites. (**C**) Cluster heat map of the differential metabolites. The abscissa represents samples, and the ordinate represents differential metabolites. (**D**) KEGG pathway enrichment analysis of differential metabolites. Each circle shows a corresponding pathway. Red circles represent significantly upregulated pathways, and blue circles represent significantly downregulated pathways. (**E**) Characteristic bioactive metabolites and their related metabolic pathways in Silkie chickens.  
The efficacy of these characteristic bioactive metabolites indicates that Silkie chickens are suitable for the elderly and women to nourish the body.