**2. Results**

Gas chromatography–mass spectrometry (GC-MS) analysis detected 67 metabolites in the sirloin samples of Wagyu and Holstein. Full results are shown in Supplementary Table S1. The principal component analysis (PCA) score plots showed that the metabolomic profile was divided into Wagyu and Holstein groups (Figure 1). The heatmap of metabolites also showed a di fference between Wagyu and Holstein groups (Figure 2). Metabolites contributing to cluster 7, which characterized the Holstein sample, were mainly composed of amino acids (proline and glycine), amino compounds (succinic acid, amino propanoic acid, creatinine, and pyruvic acid), and nucleic acid metabolites (inosine and ribose). In contrast, cluster 1, which characterized the Wagyu sample, was mainly composed of sugar components (maltose and xylitol) and fatty acids (stearic acid, palmitic acid and nonanoic acid). Table 1 shows the di fferences in the relative quantity of the main metabolite compounds in Wagyu and Holstein samples. The amount of glutamine, ribose-5-phosphate, uric acid, inosine monophosphate, 5-oxoproline, and glycine in Holstein samples was significantly higher than in Wagyu samples. In contrast, the amount of maltose and xylitol in Wagyu samples was significantly higher than that in Holstein samples.

**Figure 1.** Principal component analysis (PCA) of metabolome data from Japanese Black Wagyu (JB) and Holstein (HO) sirloin samples. •: JB (*n* = 4), •: HO (*n* = 4).

**Figure 2.** Heatmap of metabolites in Japanese Black Wagyu (JB) and Holstein (HO) sirloin samples. The upregulated metabolites are shown in red, and the downregulated metabolites are presented in green. Cluster 1, which characterized the Wagyu sample, was mainly composed of maltose, xylitol, stearic acid, palmitic acid, and nonanoic acid. Cluster 7, which characterized the Holstein sample, was mainly composed of proline, glycine, succinic acid, amino propanoic acid, creatinine, pyruvic acid, inosine, and ribose. B1-B4: Japanese Black Wagyu (*n* = 4); H1-H4: Holstein (*n* = 4).


**Table 1.** Main metabolite compounds in Japanese Black Wagyu and Holstein sirloin samples.

Values are expressed as means and S.D. ratio: fold intensity of metabolite compounds (JB/Ho). Annotation and relative quantification of metabolites was measured by each peak using the gas chromatography–mass spectrometry (GC-MS) solution (Shimadzu) and GC/MS Metabolite Database Ver. 2 (Shimadzu). Japanese Black Wagyu (JB, *n* = 4), Holstein (HO, *n* = 4) \* *p* < 0.05, \*\* *p* < 0.01.
