*4.1. Animals*

This study used castrated boars from the Duroc breed, which has been genetically bred for high IMF content as reported in the previous study [23]. All pigs used in this study were fed identical diets based on the Japan Nutrition Standards (NILGS 2016) and were raised at two locations of the NLBC (IBR and MIY) in Japan.

At each location, five high IMF content pigs and five low IMF content pigs were chosen from among the herd, for a total of 20 animals. Other than IMF content, growth performance and carcass characteristics of pigs were compared (Table 1). Aside from the fact that low IMF pigs took longer to gain slaughter weight, there were no significant phenotypic variables between any of the four IMF groups.

Blood samples were collected in a vacuum blood collection tube with EDTA/2Na from the jugular vein of each pig after fasting overnight. After blood samples were collected, the pigs were slaughtered at the abattoir located at each station. The blood collection tube was kept on ice and centrifuged with 3000× *g* at 4 ◦C for 10 min. Plasma was removed from the blood collection tube and placed into a new sample tube, then snap frozen, and stored in −80 ◦C freezer until use. All procedures involving animals were performed in accordance with the National Livestock Breeding Center's guidelines for care and use of laboratory animals.

#### *4.2. Semi-Quantitative Metabolomics (Basic Scan) by Capillary Electrophoresis-Time of Flight Mass Spectrometry (CE-TOFMS)*

Preprocessing was initiated by adding 50 μL pig plasma to 450 μL methanol containing 50 μM Internal Standard Solution 1 (H3304-1002, Human Metabolome Technologies (HMT), Tsuruoka, Yamagata, Japan) followed by mixing. To the mixture, 500 μL chloroform and 200 μL ultrapure water were added and then mixed. After centrifugation at 2300× *g*, in 4 ◦C for 5 min, 400 μL supernatant of the mixture was removed to ultrafiltration using UltraFree MC PLHCC (HMT) and centrifuged at 9100× *g* at 4 ◦C for 120 min. The filtrate was once frozen-dried and dissolved in 50 μL ultrapure water just before applying to Agilent CE-TOFMS system (Agilent Technologies, Santa Clara, CA, USA). Metabolome analysis was performed by Basic Scan package of HMT using the CE-TOFMS based on the methods described previously [29,30]. Briefly, CE-TOFMS analysis was carried out using an Agilent CE capillary electrophoresis system equipped with an Agilent 6210 time-of-flight mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). CE-TOFMS was operated using Agilent G2201AA ChemStation software version B.03.01 (Agilent Technologies, Santa Clara, CA, USA), compounds were detected using the cation or anion modes. Briefly, the compounds were electrophoresed through fused silica capillaries (50 μM × 80 cm, Agilent Technologies, Santa Clara, CA, USA) with 30 kV under 50 mbar and 10 s pressure injections. The electrophoresed compounds were ionized with either ESI Positive or Negative MS ionization. The scan range for the compounds was between 50 and 1000 *<sup>m</sup>*/*<sup>z</sup>*. Detected peaks from the CE-TOFMS system were processed by MasterHands ver2.17.1.11 [31]. Signal peaks corresponding to isotopomers, adduct ions, and other product ions of known metabolites were excluded, and remaining peaks were annotated according to the HMT metabolite database library, which includes 900 metabolites, based on their *m*/*z* values with the MTs determined by TOFMS. Areas of the annotated peaks were then normalized based on internal standard levels and sample amounts to obtain relative levels of each metabolite.

#### *4.3. Absolute Quantification of 110 Target Metabolites for CE-TOFMS Analysis*

Based on the same preprocessing and the analysis system, primary 110 metabolites involved in the pathways of glycolytic/gluconeogenesis, TCA cycle, pentose phosphate, lipid metabolites, and nucleic acid metabolites were absolutely quantified based on one-point calibrations using their respective standard compounds.

#### *4.4. Measurement of Free Amino Acids, Peptides, and Carcass Traits in Muscle Tissues*

The procedure used to analyze the free amino acids and peptides was carried out using the exact same method described in [32]. Briefly, 0.10 g samples of loin eye muscle tissues were drawn from minced raw meat (about 15 g in total). Samples were homogenized with 4.24 mL of ultrapure water, 4 mL of N-hexane and 0.16 mL of internal standard solution mixed with norvaline (5 nmol/μL) in ultrapure water, and then centrifuged at 1750× *g* for 5 min. The underlayer was mixed with 4 mL of N-hexane and centrifuged at 1750× *g* for 5 min. The resultant underlayer was then mixed with 3.6 mL of acetonitrile and centrifuged at 1750× *g* for 10 min. The resulting supernatant was filtered through a 0.45-μm microfilter (Millex-LH; Merck Millipore, Billerica, MA, USA), and the filtrate was then mixed with 45% acetonitrile solution and analyzed for free amino acids and peptides using an Agilent 1260 infinity high performance liquid chromatograph equipped with an Agilent 1260 Binary Pump, 1260 HiP Degasser, 1260 HiP ALS autosampler, 1290 thermostat, 1260 Thermostatted column compartment control module, 1260 diode array detector and a Poroshell 120 EC-C18 column (3.0 × 100 mm, 2.7 μm; Agilent). The eluents used were: (i) 20 mmol/L disodium hydrogen phosphate (pH 7.6); and (ii) acetonitrile/methanol/water (5:5:1, *v*/*v*/*v*). Amino acids and peptides were identified through the comparison of their retention times with those of established standards. The concentrations of each were calculated using internal and external standard solutions and expressed as μmol per g of meat and μmol per 1% of moisture in meat, respectively. The internal standard solutions were used to account for matter lost during analysis, and the external standard solutions (1, 10, 50, and 100 pmol/μL) were used to plot a calibration curve for each amino acid and peptide. Carcass traits were analyzed according to the methods described previously [32,33]. Moisture content was determined in duplicates by drying 2 g samples of meat drawn from the minced raw meat (approximately 30 g in total) for 24 h at 105 ◦C. IMF content was determined by Soxhlet extraction of the dried samples with diethyl ether for 16 h. Crude protein content was determined using 1 g samples of meat drawn from the minced raw meat (approximately 30 g in total) by the Kjeldahl method using a nitrogen distillation titration device (2400 Kjeltec auto sampler system, FOSS, Hillerod, Denmark). Cooking loss was determined by the weight di fference of meat samples (approximately 50 g) before and after heating at 70 ◦C for 1 h in a water bath. After cooking loss measurement, the meat sample was then used for WBSF measurement with crosshead speed of 200 mm/min using Instron (model 5542; Instron, University Avenue Norwood, MA, USA). WHC was determined using meat samples (approximately 0.5 g) drawn from the minced raw meat which were centrifuged by 10,000× *g* for 30 min. Determination of tenderness, pliability, toughness, and brittleness were performed using meat samples prepared for the same as the WBSF using Tensipresser (model TTP-50BX II, Takemoto Electric Inc., Tokyo, Japan).
