**2. Results**

## *2.1. Phenotypic Characteristics*

Pigs were separated into low and high IMF groups at two locations of the National Livestock Breeding Center (NLBC), based on IMF (%) content in the loin eye muscle. The mean proportion of IMF content in pigs at the Miyazaki (MIY) station were 3.1% and 8.7%, respectively. The mean proportion of IMF content in pigs at the Ibaraki station (IBR) were 2.9% and 5.4%, respectively (Table 1). The differences were highly significant at both stations. Moisture (%) measured in the muscle tissues of low IMF pigs were significantly higher than in the high IMF pigs at both stations. At the MIY station, protein (%) and loin eye area (cm2) in low IMF pigs were significantly 8% and 11% higher than in high IMF pigs, respectively. At the IBR station, cooking loss (%) was significantly 15% higher than those of high IMF pigs.


**Table 1.** Comparison of growth performance and carcass characteristics between high and low intramuscular fat (IMF) groups at two stations.

\*1 Statistical significance is defined when *p* < 0.05. *N.S.* denotes no significance (*p* > 0.05). \*2 WHC: Water-holding capacity (%) by centrifugation at 10,000× *g*. \*3 WBSF: Warner-Bratzler Shear Force value (Kg) measured after cooling of the carcass.

#### *2.2. Screening of Di*ff*erential Metabolomics Profiles with CE-TOFMS*

The total number of metabolites detected in the MIY samples were 201 (144 cations and 57 anions), which were used as the semi-quantitative values (relative area based on the internal standard). Likewise, the IBR metabolites detected were 152 (104 cations and 48 anions). The mean detection rates of metabolites, which are defined as the proportion of metabolites identified among either 201 (MIY) or 152 (IBR), were 82.0% and 88.6%, respectively. The mean detection rate of metabolites at IBR was greater than at MIY, despite the fact that more metabolites were detected at MIY than IBR.

Principal component analysis (PCA) was carried out based on the semi-quantitative values (relative area) of all detected metabolites. At MIY, the high IMF group tended to be in the PC1 positive and PC2 negative regions, while the low IMF group showed higher variability (Figure S1 PCA (A)). The high IMF group at IBR was found in the PC3 and PC4 positive regions, but high and low IMF groups were neither separated by PC1 nor PC2 (Figure S1 PCA (B and C)).

Partial least squares (PLS) was then used to identify possible relationships between IMF content and metabolite profiles at MIY and IBR (Figure 1. Tables S1 and S2). Since the PLS was performed between two groups (low or high IMF), we only considered PLS1 separation. All high IMF pigs and low IMF pigs were clearly in the positive and negative PLS1 scores. Therefore, we considered that positive loading values were associated with high IMF and negative loading values were associated with low IMF (Table 2). Statistically significant metabolites with positive loading values at MIY were leucine (Leu), *O*-acetylhomoserine/*2*-aminoadipic acid, 1-methylnicotinamide, choline, and phosphorylcholine, while all 10 metabolites detected with negative loading values were significant. Statistically significant metabolites with positive loading values at IBR were urea and gluconic acid, while those of the negative loading values were threonine (Thr) and diethanolamine. At both stations a BCAA, valine (Val) was identified from the positive loading metabolites. At MIY, Leu and isoleucine (Ile) were also identified from the positive loading metabolites. However, as we observed in the PCA analysis, BCAAs (Leu, Ile, and Val), tryptophan (Trp) and amino acid metabolites (*O*-acetylhomoserine/2-aminoadipic acid, *N,N*-dimethylglycine) were also detected in the list of positive PLS1 loading metabolites at MIY. Negative PLS1 loading metabolites at MIY included amino acids (Gly and beta-alanine (β-Ala)), amino acid metabolites (*N*-acetylornithine, *N*-acetyllisyne, 5-oxoproline, *N5*-ethylglutamine), peptides (anserine-divalent, carnosine) and organic acids (creatine, cis-aconitic acid). At IBR, urea, *3*-hydroxybuyric acid, nicotinamide, mucic acid and homocitrulline were commonly observed in the top positive loading metabolites on PCA3 and PCA4, While negative PLS1 loading metabolites consisted of several amino acids (Thr, Asn, Arg, Lys, methionine (Met), and Tyr) and amino acid metabolites (5-hydroxylysine and hydroxyproline).

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**Figure 1.** Partial least squares (PLS) analysis by the metabolites detected from pig plasma samples showing the di fference in the intramuscular fat contents at the two stations. Blue and red bars indicate low and high IMF pigs, respectively. Top and bottom panes show the results of PLS in MIY and IBR stations, respectively.


**Table 2.** Top 10 metabolites with positive and negative high loading values in partial least square at two stations.

\*1 *R* indicates correlation coefficient between PLS score and each metabolite levels. Since *O*-acetylhomoserine/ 2-aminoadipic acid were detected within the identical single peaks having the consistent *m*/*z* (molecular mass/electric charge of ions) andMT/RT (migration time/retention time) by the CE-TOFMS system employed in this study, the values in the table indicated the combination of two compounds: The *m*/*z* and MT/RT for *O*-acetylhomoserine/2-aminoadipic acid is 162.076 and 12.10, respectively.

#### *2.3. Integrated Analysis of Metabolomics Data Using Absolute Quantification*

With the CE-TOFMS metabolomics analysis system, the concentration in micro molar (mM) of 110 metabolites was calculated using a standard curve method. We then focused on the differences in these selectively quantified metabolites between the low and high IMF groups at the two stations. Consequently, the total number of metabolites detected in any of the eight samples from MIY and IBR was 59 mM. The mean number of cations detected at MIY was 38.7, and the mean number of anions at MIY was 34.6. At IBR, cations were 13.7, while anions were 11.9. The mean detection ratios of quantified metabolites at MIY were 88.8% and 78.9% at IBR. This indicates that there were a greater number of quantified metabolites identified at MIY than IBR. For the 59 quantified metabolites, we analyzed relationships between IMF content and the metabolites.

We used PLS to further determine which metabolites are definitively associated with IMF content. The PLS score plot showed that the low and high IMF groups at both stations were more separated by PLS2 than PLS1 or PLS3 (Figure 2), while the two stations themselves were clearly separated by PLS1 (Figure 2). We then extracted the top five metabolites with both positively and negatively high loading of PLS2 (Table 3, Table S3). Positively loading PLS2 metabolites included the BCAAs Leu, Ile, and Val, and creatine, which were found to be significant metabolites with relatively moderate correlation coefficient (R). Comparison of these BCAAs and creatine concentrations at MIY indicated

that the high IMF group displayed a significantly higher amount or tended to show greater amounts of the metabolites than those in the low IMF group. Basically, these same metabolites, BCAAs and creatine in the high IMF group at IBR also showed a greater amount than those of low IMF, although the difference was not as large as at MIY.

**Figure 2.** Integrated analyses of partial least squares by the metabolites detected from pig plasma samples showing the difference in the intramuscular fat contents at the two stations. Blue and red dots indicate low and high IMF pigs from MIY station, respectively. Cyan and magenta dots indicate low and high IMF pigs from IBR station, respectively. Top and bottom panes show the results of PLS analyses with PLS1 and 2 and with PLS2 and 3, respectively.

All metabolites from the negatively loading PLS2 metabolites were determined significant with slightly higher correlation coefficients as compared to those in the positively loading metabolites. Similar to the positive loading metabolites, the differences in the negatively loading PLS2 metabolites concentrations between the high and low IMF groups at MIY were clearer and more significant than those at IBR. Consequently, all metabolites identified by PLS2 factor loadings were commonly found in the list of metabolites which were identified with the one-factor PLS analysis at MIY and IBR (Table 2).



The unit of metabolites concentration is represented in μM. \*1 *R* indicates correlation coefficient between PLS score and each metabolite levels. \*2 Values are denoted with means ± S.E.M. Statistical significance is defined as *tnd.* (*p* < 0.10), \* (*p* < 0.05), \*\* (*p* < 0.01), and \*\*\* (*p* < 0.001). *N.S.* indicates no significance.

#### *2.4. Comparison of Amino Acids and Related Metabolites*

Since several amino acids and their related metabolites were identified as the differential metabolites between IMF groups, we next focused our analysis on those metabolites derived from BCAAs such as 3-methyl-2-oxovaleric acid (2K3MVA)/4-methyl-2-oxovaleric acid (2-oxoleucine), 2-oxoisovaleric acid (2-KIV) (Figure 3). The combined amount of Ile and Leu in the high IMF group at MIY was significantly higher than in the low IMF group. The amount of Ile and Leu at IBR, and Val at both stations showed a similar tendency, although the difference was not significant. Likewise, the amount of 2K3MVA/2-Oxoleucine and 2-KIV in the high IMF groups at both stations were greater than in the low IMF groups. There was no significant difference in the total free amino acid contents between the low and high IMF groups. The mean concentrations and standard deviations were 3428 ± 240 (μM) and 3258 ± 182 (μM), respectively.

**Figure 3.** Comparisons of branched-chain amino acids and their degraded metabolites detected from pig plasma samples showing the difference in the intramuscular fat contents at the two stations. Blue and red bars indicate low and high IMF pigs, respectively. Total shows the sum of MIY and IBR. *Y*-axis indicate the relative area of each metabolites normalized by the internal standard. 3-methyl-2-oxovaleric acid (2K3MVA) and 4-methyl-2-oxovaleric acid (2-oxoleucine), which are the degraded metabolites of Ile and Leu, respectively, could not be separately detected by the capillary electrophoresis-time of flight mass spectrometer (CE-TOFMS). Statistical significance is defined as \* (p < 0.05).

#### *2.5. Amino Acid Content Analysis in the Muscle Tissue of Pig Carcass*

Per our CE-TOFMS metabolomics results, we concluded that BCAAs were associated with high IMF content in loin eye muscle. We examined the free amino acid contents of 20 amino acids with taurine, carnosine and anserine (Asn) in the muscle tissues of pig carcasses in order to better understand the mechanism by which high IMF content had either resulted from or was caused by high BCAAs in plasma (Table 4).

At MIY, high IMF pigs had significantly higher concentrations of Asn in loin eye muscle tissue than low IMF pigs, while Gly, carnosine and Asn in high IMF pigs were significantly lower than low IMF. The results of Gly and Carnosine matched the results shown in Tables 2 and 3. At IBR, low IMF pigs had greater concentrations of Asp, His, Met, Trp, phenylalanine (Phe), Ile, and Leu in loin eye muscle tissues than those of high IMF pigs. The BCAA contents of loin eye muscles in low IMF pigs were significantly higher than those of high IMF pigs. This result is quite opposite from the results obtained from the plasma metabolomics. As seen in the plasma metabolomics, the total free amino acid content of loin eye muscle tissues displayed no di fference between the low and high IMF groups.
