**3. Discussion**

This study sought to identify the metabolites associated with IMF content in pork loin eye muscle using CE-TOFMS metabolomics data detected in pig plasma before slaughter. We selected five castrated boars with low IMF content, and five castrated boars with high IMF content in the loin eye muscle from two NLBC stations in Japan (IBR and MIY), for a total of 20 pigs. The difference in IMF content in the high IMF pigs was 2.8 times greater than the low IMF pigs at MIY. At IBR, the difference in IMF content in high IMF pigs was 1.9 times greater than the low IMF pigs. In contrast the moisture content (%), crude protein content (%), and loin eye area (cm2) in both high IMF groups were either significantly less or tended to be less than those of the low IMF groups. Most of the growth-related traits and the eating-quality traits displayed no differences between the groups. The metabolites differentially identified between the IMF content levels were therefore considered to be primarily associated with IMF content, and secondly with moisture content. Possible partial relationships may have occurred between the candidate metabolites for IMF content, crude protein content, loin eye area or cooking loss (Table 1).

Plasma samples from each pig were profiled using semi-quantification results derived from CE-TOFMS metabolomics. As indicated in the PCA analysis results, large variations were observed, particularly in the low IMF groups at both stations, although the high IMF groups had relatively close PCAs plots. No nutritional controls or genetic factors were used to generate these di fferences. Since no obvious separation was observed in the IMF content di fferences from either station, it can also be concluded that none of these factors were involved in samples obtained during this study and it is natural to see such vague separations (Figure S1).

Next, we conducted the PLS analysis. Our results clearly depicted variations in the candidate metabolites in the first PLS1 screening; more specifically, that high IMF groups had positive PLS1 values, while low IMF groups had negative PLS1 values (Figure 1). The metabolites with high positive loading values of PLS1 consistently included BCAAs and amino acid metabolites. This led us to conclude that BCAAs are potential biomarkers for IMF content in pork loin eye muscles. Metabolites with negative loading values of PLS1 included several kinds of amino acids and amino acid metabolites, which suggests that an increased amount of amino acids and amino acid metabolites in plasma may represent lower IMF content (i.e., leaner meat) (Table 2).

We used the quantification results from the CE-TOFMS to validate these findings. We applied the integrated PLS analysis using quantification data from the four groups (Figure 2). Although the PLS1 accounted for 60.4% of the total IMF variability, it appeared to obviously discriminate station di fferences, which was not the original purpose of this study. The PLS2 showed the comprehensible separation between the IMF content groups. Even though the number of metabolites quantified in the integrated analysis was 59 out of 201 at MIY and 152 at IBR during the screening analysis, BCAAs were commonly extracted from the list of metabolites with high loading PLS2 values (Table 3).

According to the correlation coe fficients estimated in Tables 2 and 3, metabolites with higher R values showed clear di fferences between the high and low IMF groups. Additionally, metabolite concentration di fferences between the two IMF groups at MIY were more obvious than those at IBR. Nevertheless, the concentration of metabolites in the high IMF groups was consistently greater at both stations, with the opposite holding true for metabolites with negative loading PLS2 values.

Since BCAAs were indicated to correspond to IMF content, we examined the potential BCAA-related metabolites closer (namely 2K3MVA, 2-oxoleucine, and 2-KIV) (Figure 3). Since 2K3MVA and 2-oxoleucine could not be separately identified by the CE-TOFMS system, additional amounts of 2K3MVA and 2-oxoleucine and their original BCAAs (Ile and Leu) were applied (Figure 3). The relative amount of metabolites degraded from BCAAs (2K3MVA, 2-oxoleucine, and 2-KIV) indicated a tendency similar to the original BCAAs. Even if the di fferences were not statistically significant, all of these metabolites follow the patterns of "low in low IMF", and "high in high IMF". One explanation for this may be that the baseline amount of BCAAs in high IMF pig plasma is greater than in low IMF pigs, which means that the amount of metabolized BCAAs might follow the same pattern. If this is the case, then it is quite important to analyze the amino acid content in tissues that metabolize amino acids.

Post slaughter amino acid analysis was conducted on the loin eye muscle tissues (Table 4). Results of this analysis were inconsistent with the metabolomic plasma analysis, that is, the amino acid tissue analysis showed that high IMF pigs at MIY had significantly lower amounts of Gly, Car, and Ans than low IMF pigs. This suggests that the amount of Gly, Car, and Ans (divalent) detected in the plasma of high IMF pigs might be correlated with the intake of those metabolites into muscle tissues. The amino acid analysis at IBR indicated that the amount of Asp and Met in the low IMF group were significantly greater than those in the high IMF group, which matched our plasma screening results. It is interesting that the amount of BCAA in the muscle tissues of the low IMF group were significantly greater than in the high IMF group. Additionally, the BCAA di fferences at MIY were not as obvious as in the plasma samples (N.S.), although the same trend was observed in both the plasma and the muscle tissues. These results sugges<sup>t</sup> that the amount of BCAA in plasma is positively associated with high IMF content in the loin eye muscle.


**Table 4.** Analysis of amino acid contents in the loin eye muscle tissues from two stations.

The unit of amino acid concentration is represented in μmol/g. \*1 This list includes analysis result of amino acids together with Taurine, Carnosine and Anserine. \*2 Statistical significance is defined as *tnd.* (*p* < 0.10), \* (*p* < 0.05), \*\* (*p* < 0.01). *N.S.* indicates no significance. \*3 FAA denotes total amount of free amino acids.

Recently, it was demonstrated that surplus dietary Ile, one of BCAAs, increased the IMF content in skeletal muscle via the upregulation of lipogenic genes by stimulating lipogenesis in skeletal muscle tissue of finishing pigs [19]. In the previous study, Luo et al. clearly showed the e ffect of extra Ile in the

diet on lipogenesis in the muscle tissue, serum cholesterol levels, and fatty acid composition without affecting growth performance. However, it is not possible to further discuss whether Ile (BCAA) intake into the muscle cells affected lipid synthesis due to the lack of Ile level in muscle tissue [19]. In contrast, it has been well known that BCAA are effective amino acids to regulate protein synthesis because of a greater increase in protein synthesis than degradation [20–22].

Therefore, one possible explanation for this is that the availability of BCAA and/or the intake of BCAA in the muscle tissue of low IMF pigs are more effective than in high IMF pigs, which results in the pig building more muscle mass. It is possible, therefore, that the availability of BCAA in the muscle tissue of high IMF pigs is relatively low, which would mean that some kind of molecular mechanism(s) could be triggered that could enhance the preadipocytes near the muscle cells.

The Duroc pigs used for this study were derived from the one previously established to improve IMF content at NLBC. The heritability of IMF in the Duroc genetic line established at NLBC has been previously determined to be 0.52 [23]. Although there were certain differences in the metabolite profiles between MIY and IBR by the PLS1 (Figure 2), these observed differences may be due to environmental factors only, since the genetic Duroc line and the feeding regimes were nearly identical at each location.

Previously, Katsumata et al. [14] reported that a low lysine diet fed to finishing pigs increased IMF content, although the authors recognized that the influence of restrictive amino acid nutrition on IMF content needs to be researched further. Restricted amino acid diets may increase stress sensitivity in pigs, since it has been indicated to do so in other animals [14,24,25]. In the case of beef marbling improvements, vitamin A restriction in the diet of beef steers increased IMF content due to hyperplasia of the adipocytes, although subcutaneous fat depth was not affected [26,27]. This method of feeding beef cattle diets with low vitamin A is well-known and used worldwide. In addition to the vitamin A restriction, it is suggested that a genotype of alcohol dehydrogenase 1C (ADH1C) has an interactive effect with vitamin A on IMF content, because of the differential transcriptional regulation that potentially occurs in this genotype [28]. It is plausible that nutritional control combined with certain genetic backgrounds may increase or decrease the IMF content in meat animals. However, there is still an inevitable phenotypic variation in IMF content, as is the nature of quantitative traits. Therefore, a non-invasive method, such as plasma metabolomics by CE-TOFMS, could be of benefit to determine the physiological status of meat producing animals, since it can measure a broader range of compounds than other metabolomics systems.

In conclusion, in this study we found that pigs with high IMF content in the loin eye muscle also had an increased amount of BCAA in their blood plasma. We also found that pigs with low IMF content in the loin eye muscle had an increased amount of Gly, Ans, and Car in their blood plasma. We suggest, therefore, that BCAA, Gly, Ans, and Car are potential biomarkers that could be used to estimate IMF in the loin eye muscle before slaughter. Further study of these biomarkers is required, since their amounts were not always consistently demonstrated in this study.

#### **4. Materials and Methods**
