The yaks were obtained from the same herd in Tianzhu Tibetan Autonomous County, Gansu Province, China, and five 4.5-year-old Tianzhu white yaks (males; n = 5) with similar weight (217.50±17.08) and good health status were selected as experimental animals with the consent of the herders. Animals were clinically examined to ensure the absence of disease, injury, or abnormal growth. All yaks had ad libitum access to forage from the same pasture-based feeding system and free access to water. Prior to slaughter, animals were fasted for 24 h with free access to water. The yaks were transported to “Huarui Star Slaughterhouse” a commercial abattoir located in Tianzhu Tibetan Autonomous County, Gansu Province, China, with an approximate transportation duration of 30 min. Slaughter procedures were conducted by professional staff in accordance with standard commercial practices. Yaks were slaughtered in commercial abattoirs by professional slaughterhouse staff.

The LT, TB, and SM were collected, the LT, predominantly composed of slow-twitch fibers, TB and SM contain more fast twitch fibers, specialized for rapid) rinsed with PBS, and rapidly frozen in liquid nitrogen for the determination of flavor compounds, metabolites, amino acids, fatty acids, and gene expression. Additional LT, TB, and SM tissue (2×2×2 cm) was fixed in 4% paraformaldehyde.

The method for Oil red O staining follows the procedure described by Wang et al [15], with specific steps as follows: the tissue was embedded in OCT Reagent (Servicebio), rapidly frozen in liquid nitrogen for 15 s, and frozen in −70°C for 30 min, subsequently thawed in −40°C for 30 min, followed by −20°C for 30 min, and then sectioned into two 10 μm slices. Subsequently, the tissue pieces were fixed on anti-dehiscence slides. For Oil red O staining, sections were rinsed with distilled water for 2 min, immersed in 60% isopropanol for 2 s, stained with oil red O working solution for 15 min, briefly differentiated in 60% isopropanol, rinsed with tap water for 2 min, counterstained with hematoxylin for 30 s, and sealed with neutral resin.

10% homogenate preparation: weigh 1.0 g of muscle tissue, add 9 mL of pre-cooled saline at the ratio of mass (g) volume (mL) = 1:9, homogenize under the condition of ice-water bath, and centrifuge for 15 min at 3,500 r/min under the condition of 4°C, and then aspirate the supernatant for the assay. The enzyme activity indexes mainly included lactate dehydrogenase (LDH), lactate content (LD) and succinate dehydrogenase (SDH) activity, and ATP were detected using VarioskanLUX multifunctional enzyme labeling instrument (Thermo Fisher Scientific). The steps were performed in strict accordance with the instructions of the kit (Jiangsu Enzyme Labeling Biotechnology).

The method for fatty acids extraction was adapted from Wang et al [16], with the specific steps outlined below:1 g of the ground sample was weighed, and 0.7 mL of 10 mol·L⁻¹ KOH solution and with 5.3 mL of anhydrous methanol were added. The mixture was incubated in a water bath at 55°C for 1.5 h, followed by addition of 0.58 mL of H₂SO₄ solution and further incubated at 55°C for 1.5h. After the reaction, the mixture was cooled to room temperature with tap water, followed by the addition of 3 mL of hexane. The supernatant was collected and filtered through an organic phase membrane into a sample vial. Gas chromatography (GC-2010 Plus; Shimadzu) was performed using an HP-88 fused silica capillary column (100 m×0.25 mm×0.20 μm; Agilent Technologies) and a flame ionization detector (FID). Gas chromatography was conducted with nitrogen as the carrier gas, at a flow rate of 450 mL·min⁻¹. The injector temperature was set at 260°C; the detector temperature was set 250°C. The oven was initial held at 140°C for 5 min, then increased to 240°C at 4°C·min⁻¹ and held for 15 min, with a total run time of 45 min. Identification and quantification were performed using the external standard method with the application of a 37-component fatty acid methyl esters (FAMEs) mixture standard (CRM47885; Supelco; Sigma-Aldrich).

Approximately 1 g of yak muscle sample was placed into a centrifuge tube, followed by the addition of 600 μL of methanol formate (Thermo Fisher Scientific). The mixture was centrifuged at 12,000 ×g for 5 min, after with the supernatant was diluted 50-fold with methanol formate and shaken for 30 s. Then, 100 μL of the diluted supernatant was spiked with 100 μL of the Trp-d3 internal standard (100 ng/mL) (Nanjing Reagent), filtered through a 0.22 μm membrane and analyzed.

The LC analysis was performed on EXion LC Liquid chromatography (AB SCIEX). Mass spectrometric detection of metabolites was performed on AB6500 Plus (AB SCIEX). Chromatographic separation was performed on an ExionLC system (AB SCIEX) coupled to a SCIEX QTRAP 6500+ mass spectrometer (AB SCIEX). ACQUITY UPLC BEH C18 Column (2.1×100 mm, 1.7 μm; Waters), the injection volume was 5 μL, the column temperature was set at 40°C, and the mobile phase were A:50 % methanol in water (containing 0.1% formic acid) (Thermo Fisher Scientific) and B:10% methanol in water (containing 0.1% formic acid) (Tokyo Chemical Industry). The ionization was performed using electrospray ionization (ESI) in positive mode. The ion source temperature was set at 500°C, the ion source voltage was 5,500 V, the collision gas was pressure at 6 psi, the curtain gas pressure at 30 psi, and both the atomizing and auxiliary gases at 50 psi. Scans were performed using multiple reaction monitoring (MRM).

A 500 mg meat sample was placed in a 15 mL centrifuge tube, frozen in liquid nitrogen for 5 min, and homogenized using a pulverizer for 3 min. Then, 0.6 g of the homogenized sample was transferred into a headspace vial containing 10 μL of internal standard solution of n-Hexyl-d13 (C/D/N Isotopes), incubated at 80°C for 10 min, and conditioned at 270°C for 10 min. The conditioned SPME fiber was inserted into the incubation chamber and exposed to the sample headspace for 40 min at 80°C. After adsorption, the fiber was transferred to the GC injection port and desorbed at 250°C for 5 min. For retention index calibration, 10 μL of n-alkanes standard solution (Sigma-Aldrich) was placed in a 20 mL headspace vial. Analyses were carried out using a LECO Pegasus 4D instrument (LECO) consisting of an Agilent 8890A GC (Agilent Technologies) system equipped with a split/splitless injector, and dual stage cryogenic modulator (LECO) coupled with TOFMS detector (LECO). The separation system consisted of a one-dimensional column:Separation was achieved using a DB-Heavy Wax column (Agilent Technologies) for the first dimension and an Rxi-5Sil MS (Restek) for the second dimension. High-purity helium was used as the carrier gas at a constant flow of 1.0 mL·min⁻¹. The temperature program of the oven was as followed: the oven temperature was held at 50°C for 2 min at first, then raised to 220°C at the rate of 4°C/min and held for 13 min. The secondary oven temperature was operated at 5°C higher than the first oven. The temperature of the modulator is always 15°C higher than that of the second column. The injector temperature was set to 250°C. Mass spectrometry detection was performed using a LECO Pegasus BT 4D system (LECO) with a transfer line temperature of 250°C, ion source temperature of 250°C, acquisition rate of 200 spectra·s⁻¹, electron ionization energy of 70 eV, and detector voltage of 2,031 V.

To compare flavor components with contents across different orders of magnitude, the raw data were normalized using either total peak area normalization or internal standard normalization. VOCs were identified based on the NIST2020 database through annotation with Chroma TOF software. The classification and analysis of detected flavor compounds, including the evaluation of their quantity and relative content, were conducted using the PubChem database and ClassyFire software. Additionally, the sensory characteristics of these compounds were examined and compared using the FlavorDB and Order database. Statistical significance was determined based on variable influence in VOCS (VIP) values>1 and p<0.05.

Metabolites were extracted according to the method of Want et al [17]. The procedure was as follows: 100 mg of yak meat from different anatomical regions was weighed and placed into a centrifuge tube containing 1 mL of 70% methanol (Sigma-Aldrich) with internal standard. Samples were homogenized e grinder at 50 Hz for 60 s, and repeated twice. The homogenate was then sonicated at room temperature for 30 min, followed by incubation on ice for 30 min. Samples were centrifuged at 12,000 ×g, and 4°C for 10 min. The supernatant was collected, concentration and dried. Dried exacts were reconstituted in 200 μL of 50% acetonitrile containing 2-chloro-L-phenylalanine (Aladdin) as internal standard, filtered.

The LC analysis was performed on a Vanquish UHPLC System (Thermo Fisher Scientific). Chromatography was carried out with an ACQUITY UPLC HSS T3 (2.1×100 mm, 1.8 μm) (Waters). The column maintained at 40°C. The flow rate and injection volume were set at 0.3 mL/min and 2 μL, respectively. For LC-ESI (+)-MS analysis, the mobile phases consisted of (B2) 0.1% formic acid in acetonitrile (v/v) and (A2) 0.1% formic acid in water (v/v). Separation was conducted under the following gradient: 0–1 min, 8% B2; 1–8 min, 8%–98% B2; 8–10 min, 98% B2; 10–10.1 min, 98%–8% B2; 10.1–12 min, 8% B2. For LC-ESI (−)-MS analysis, the analytes was carried out with (B3) acetonitrile and (A3) ammonium formate (5 mM). Separation was conducted under the following gradient: 0–1 min, 8% B3; 1–8 min, 8%–98% B3; 8–10 min, 98% B3; 10–10.1 min, 98%–8% B3; 10.1–12 min, 8% B3.

Mass spectrometric detection of metabolites was performed on Orbitrap Exploris120 (Thermo Fisher Scientific) with ESI ion source. Simultaneous MS1 and MS/MS (Full MS-ddMS2 mode, data-dependent MS/MS) acquisition was used. The parameters were as follows: dependent MS/MS acquisition was used. The parameters were as follows: sheath gas pressure, 40 arb; aux gas flow, 10 arb; spray voltage, 3.50 kV and −2.50 kV for ESI (+) and ESI (−), respectively; capillary temperature, 325°C; MS1 range, m/z 100–1,000; MS1 resolving power, 60,000 FWHM; number of data dependant scans per cycle, 4; MS/MS resolving power, 15,000. Dynamic exclusion was employed to eliminate redundant MS/MS information.

Relative metabolite abundances were normalized and orthogonal partial least squares-discriminant analysis (OPLS-DA) using R software. Differential metabolites were identified based on variable importance in projection (VIP>1) and p<0.05. Functional pathway enrichment analysis of the filtered differentially expressed metabolites was performed using the Metaboanalyst software.

Approximately 100 mg of muscle tissue were ground into 1 mL of AG RNAex Pro reagent (Accurate Biotechnology) using a tissue grinder, and subsequent were performed were carried out according to the manufacturer’ instructions (Accurate Biotechnology). The cDNA was synthesized from the extracted RNA according to the reverse transcription kit. Primer5.0 software was used to design the gene primers, and the primer information is shown in Table 1. Relative gene expression was calculated using the 2^−ΔΔCT method.

Reverse transcription quantitative polymerase chain reaction (RT-qPCR) data, Oil red O area, amino acids and fatty acids content were statistically analyzed using SPSS 26.0 one-way ANOVA and Tukey post-hoc test. The results have been presented as mean±standard deviation, with statistical significance determined at p<0.05.

As shown in Figure 1, intramuscular fat in the three muscles was mainly distributed as lipid droplets within the inter-fascicular spaces between muscle fiber bundles. Lipid signals were quantified by image analysis based on Oil Red O–stained sections. The results showed that the lipid droplet area fraction in LT was significantly higher than that in SM and TB (p<0.05). In addition, the difference between SM and TB was not significant (p>0.05). The higher intramuscular fat deposition in LT suggests a greater reserve of lipid-derived precursors.

Figure 2 demonstrates significant differences in enzymatic activity among the three muscle types. LDH activity was markedly higher in the SM group compared to TB and LT (p<0.05). In contrast, SDH activity was significantly higher in the LT group relative to the other two muscle types (p<0.05).

As shown in Table 2, a total of 29 fatty acids were identified in the three parts of yak, included 13 saturated fatty acids, 8 monounsaturated fatty acids, and 8 polyunsaturated fatty acids. In yak muscle, 18:1n9c and C18:0 was identified as the predominant fatty acids. A total of 12 fatty acids exhibited significant differences among different parts of the muscle, with C18:0, C18:1n9c, C18:2n6c, and C18:2n6t were the highest in TB (p<0.05). Conversely, C14:0, C16:0, C21:0, C23:0, and C18:3n6 were significantly higher in SM compared to the other two groups (p<0.05), while C14:1 and C20:1 were higher in the LT group than in the SM and TB (p<0.05).

Compared to yak TB, the concentrations of Orn, Gln, Glu, Arg, flavor amino acid (FAA), bitter amino acid (BAA), and non-essential amino acids (NEAA) in LT and SM were lower (p<0.05). In contrast, Hcy, Tyr, Ser, and His were higher in LT than in TB (p<0.05), while the content of NEAA was higher in TB than in LT (p>0.05), and the total free amino acid content was significantly higher in TB than in LT (p<0.05) (Table 3).

As shown in Figure 3, the acetyl-CoA carboxylase-α (ACACA), carnitine palmitoyltransferase-1b (CPT1B), Fatty acid swasynthase (FASN), and Lipoprotein lipase (LPL) gene expression was significantly higher in the TB and SM groups than in the LT group (p<0.05). The relative expression of myosin heavy chain 7 (MYH7), diacylglycerol acyltransferase 1 (DGAT1), Elongation of Very Long Chain Fatty Acids 4 (ELOVL4), Hormone-sensitive lipase (HSL), and Sterol regulatory element-binding factor 1 (SREBP1) in the TB group was significantly greater than the in LT and SM groups (p<0.05). The relative expression of Acyl-CoA synthetase long-chain family member 4 (ACSL4), Fatty acid-binding protein 4 (FABP4) in the TB group significantly greater than the LT and SM groups (p<0.05).

A total of 273 flavor compounds were identified after quantifying flavor compounds through a standardized normalization process. These included benzene-type compounds (34), alcohols (35), ethers (4), aldehydes (34), carboxylic acids and derivatives (4), hydrocarbons (34), ketones (23), organic compounds (12), organic heterocyclic compounds (29), lipids and lipid-like molecules (11), esters (33), and others (20) as shown in Figure 4A. The relative content of alcohols in LT was higher than in the other two groups, while the relative content of aldehydes and ketones was lower than in the other two groups. The number of hydrocarbons in SM was higher than in the other two groups, however, the number of heterocyclic compounds, ketones, aldehydes, alcohols, and carboxylic acids in TB was higher than in the other two groups (Figure 4B).

Through differential analysis, we found that the flavor compounds in the three muscle types were primarily alcohols (Figure 5). Among these, TB contained 5-Hepten-2-ol, 6-methyl-, 2-Penten-1-ol, (E)-, 1-Octen-3-ol, 1-Hexanol, 5-methyl-2-(1-methylethyl), 2-Heptanol, 4-methyl-, 1-Hexanol, 2-ethyl-, 2-Pentanol, 2-Nonanol, and 1-Hexanol were significantly higher in TB than in SM and LT (p<0.05), while the content of 2-Heptanol was significantly higher in LT than in SM and TB (p<0.05). The content of panic acid, ethyl ester, ethyl acetate, undecanoic acid, ethyl esters, and L-valine, ethyl esters in the TB group, was significantly higher than that in the SM and LT groups (p<0.05). For ketone compounds, we found five different flavor compounds, with the content of isophore in SM significantly higher than that in TB and LT (p<0.05). The content of 2-octanone, 2-undecanone, 2-pentadecanone, and 2-tridecanone in TB was significantly higher compared with LT and SM (p<0.05).

This approach allows for the calculation of the relative odor activity value (ROAV), which assesses the individual impact of volatile compounds on the overall odor. A ROAV value of ≥1 indicates that a compound is a significant contributor to the overall scent. From the Table 4, it can be seen that SM contains 8 key odor compounds, 7 key odor compounds in LT and 6 key odor compounds in TB. The highest flavor compounds in all samples were 2-Nonenal, (E)-, and 2,3-Butanedione.

To obtain a comprehensive understanding of the sensory characteristics variation in yak meat from different parts, we conducted a sensory evaluation using the Flavordb and order database. Radar plots of flavor profiles were created, and as depicted in the figure, the sensory evaluation results indicated that yak meat exhibited higher sweetness and green aroma, contributing to a fresh and pleasant odor. Among these, the TB group displayed the strongest sweet and fruity flavors, followed by the LT group, while the SM group had relatively weaker odors. The radar chart illustrates that the TB part of yak provides a general profile of pleasant flavor characteristics (Figure 6A).

We created network maps of flavor compounds and their sensory properties using Cytoscape. We chose three sets of different volatile flavor compounds, with the 10 most common flavors for constructing the network map. 3-heptanal, (E)-, 2-undecanone, ethyl undecanoate, and 2-tridecanone are associated with a fatty flavor. 2-Heptanol, 3-Heptenal, (E)-, 2-Undecanone, 2-Tridecanone, 1-Hexanol, 2-Ethyl- play a crucial role in yak flavor (Figure 6B).

Broadly targeted metabolomics analysis revealed 270 metabolites in different parts of all yaks belonging to 12 classes. This suggests that the models generated from OPLS-DA were well-fitted and had a high level of predictability, making them suitable for further analysis of the data. Subsequently, the groups were carefully examined and classified for distinguishing metabolites using the OPLS-DA results (Figure 7A). There are three differentially enriched metabolites common to all three comparison groups (Figure 7B). A total of 11 differential metabolites were identified in SM vs LT (8 upregulated, 3 downregulated). In SM vs TB, 22 differential metabolites were identified (10 upregulated, 12 downregulated), and 37 metabolites were identified in TB vs LT (20 up-regulated, 17 down-regulated) (Figure 7C). In general, comparisons between the three parts identified 28 differential metabolites (Figure 7D).

Subsequently, a trend analysis was performed on 270 metabolites using the fuzzy c-means algorithm, which revealed four main clusters. Additionally, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was conducted for each trend cluster (Figure 7E). Metabolites highly expressed in TB muscle were primarily enriched in pathways related to amino acid, fatty acid, and purine metabolism, all of which are associated with the synthesis of flavor precursor compounds. These metabolites, which are highly abundant in TB muscle, may be one of the primary reasons for the high content of flavor compounds in TB.

Functional enrichment analysis of different metabolites was conducted to identify enriched metabolic pathways in the KEGG pathway. The metabolic pathways of differential metabolites included metabolic pathways, fatty acid biosynthesis, and amino acid metabolism biosynthesis. Enriched metabolic pathways between SM and LT were found to be tyrosine metabolism, phenylalanine, tryptophan metabolism, thyroid hormone synthesis, and Glutathione metabolism (Figure 8A). Additionally, tyrosine, alanine, phosphatase lipid D, and fatty acid biosynthesis were significantly enriched in SM vs TB (Figure 8B), while fatty acid biosynthesis and tyrosine. Synthesis-related pathways were significantly enriched in TB and LT (Figure 8C). The biological processes of these differential metabolites-enriched KEGG pathways indicated their involvement in amino acid, carbohydrate, and lipid metabolism. In addition, considering that flavor compounds are mainly produced with lipid metabolism and amino acid reactions, thus differential metabolites related to fatty acid and amino acid metabolism were identified. Functional enrichment analyses of these precursors that may be useful for flavor revealed that these differential compounds were mainly enriched in the pathways of fatty acid and unsaturated fatty acid and glycerophospholipid metabolism, and therefore we hypothesized that changes in fatty acid content were the main reason for the flavor differences (Figure 8D).

Muscle fiber types influence metabolic rate, connective tissue abundance, intramuscular fat deposition, and postmortem proteolysis, thereby affecting meat color, tenderness, texture, flavor, juiciness [18]. Meat quality is primarily reflected through indicators such as muscle tenderness, cooked yield, water loss rate, and intramuscular fat content. Among these, intramuscular fat content plays a decisive role in determining meat tenderness, juiciness, and flavor. Previous studies have demonstrated a positive correlation between intramuscular fat content and meat tenderness [19]. In this study, Oil-red O staining showed that lipid droplets were primarily located in the muscle fibers, and the Oil-red area of LT was significantly greater than that of TB and SM, suggesting higher intramuscular fat content in LT and potentially improved sensory attributes, including tenderness and palatability. The differences in Oil-red O staining among the three muscles may reflect variation in muscle fiber composition and functional demands; compared with the LT, TB and SM are more specialized for locomotion and weight-bearing, which may limit intramuscular fat deposition. Bai et al. reported that the infraspinatus muscle displayed a more intense color and higher intramuscular fat content that thefi longissimus thoracis and semitendinosus muscles [20]. These findings are consistent with previous studies that muscle fiber type is a key determinant of intramuscular fat distribution, which in turn influences meat tenderness and flavor [21].

Muscle fiber type composition is a key determinant of muscle metabolic characteristics and plays a critical role in shaping meat quality, particularly flavor development. Lactate dehydrogenase is involved in the glycolytic pathway and catalyzes the production of lactic acid from pyruvate, a hallmark enzyme that measures the body’s capacity for anaerobic fermentation and aerobic metabolism [22,23]. Oxidative muscles typically contain more mitochondria and phospholipids, which can increase the availability of specific PUFAs and lipid-derived flavor precursors. Therefore, the combined differences in intramuscular fat deposition and metabolic enzyme activities likely contribute to muscle-specific precursor availability and oxidative stability. Succinate dehydrogenase activity is positively correlated with the number of oxidized muscle fibers and negatively correlated with the number of enzymatic muscle fibers in muscle [24]. Succinate dehydrogenase is not only involved in meat energy metabolism, but also plays a central role in flavor generation. It affects the formation of color, aroma and “umami” by regulating succinic acid metabolism, electron transport and myoglobin status. In the meat industry, the precise regulation of SDH activity is a potential path to improve flavor quality. For example, Gao et al in the higher class added different concentrations of succinate to the longissimus thoracis of beef, and found that higher concentrations of succinate could enhance the myoglobin and flavor of meatloaf [25]. In this study, the content of SDH in the LT group was higher than that in TB and SM, which may be due to structural differences within the muscles. This result suggested that differences in muscle fiber type drive distinct energy metabolic patterns, which in turn influence the activity of key metabolic enzymes like SDH and LDH. Therefore, fiber-type composition indirectly but significantly affects the flavor profile and overall eating quality of yak meat.

Free amino acids are crucial flavor precursors, as their types and concentrations directly influence the sensory quality of meat [26]. Notably, Lys, Arg, Asp, and Glu are recognized to flavor compound formation by interactions with soluble reducing sugars in meat [27]. Glu exhibited the highest concentrations and is recognized as a key flavor precursor, capable of synergize with inosinic acid to enhance a range of flavors, including acidic and alkaline flavors [28]. In this study, Glu levels were higher in TB than in LT, suggesting that yak TB meat exhibited a more pronounced flavor profile. Interestingly, total free amino acid content in TB was significantly higher than in LT, which further supports the notion that higher amino acid content in TB may facilitate the generation of more volatile flavor compounds. This could be particularly relevant in the context of the Maillard reaction, where amino acids interact with reducing sugars to form volatile compounds that contribute to the aroma and flavor of the meat. Although this study suggests a correlation between elevated amino acid levels and enhanced flavor profiles in TB, further studies are needed to confirm the specific pathways involved in flavor compound synthesis, particularly those linked to the Maillard reaction.

Fatty acids, amino acids, and proteins are important sources of nutrients in beef cattle, and studies have shown that the composition and content of fatty acids and amino acids have a significant effect on the flavor, juiciness, and texture of meat [29,30]. The impact of fatty acids on hardness is attributed to their varying melting points, which decrease as unsaturation increases [31]. Higher levels of UFAs in lamb lead to quicker fat oxidation and a shorter shelf life [32]. Oleic acid, due to its single double bond and relatively reactive molecular structure, oleic acid readily undergoes oxidative degradation and participates in complex chemical reactions such as the Maillard reaction and Strecker degradation. These processes lead to the formation of key volatile compounds including aldehydes, ketones, esters, and alcohols. These volatiles contribute to the characteristic aroma and flavor profile of cooked meat by providing notes ranging from fatty and buttery to green, fruity, and floral tones [33]. In this study, we found that the oleic acid content in the three groups there were significant differences, which may have contributed to the differences in the flavor of the muscles of different parts of the yak. Similarly, it was also found that the higher oxidative capacity of unsaturated fatty acids, produces a rich flavor profile during meat processing, and we found that the distinctive flavor of yak may be related to the richness of unsaturated fatty acids in the muscle [34]. In the present study, we found that the C16:0 content in SM was significantly higher than the other two groups, which may be beneficial for the synthesis of SM aldehydes. C16:0 is important for flavor formation and can be oxidized to form heptanal, nonanal, 2-heptanone, and D-limonene. Heptanal has a fishy and sweet almond flavor. Nonyl aldehyde gives fats an aromatic, herbaceous, and fishy flavor. 2-heptanone and D-limonene have a fruity flavor [33].

In this study, we analyzed the expression of genes related to fat deposition in different muscle fiber type of yak. ACACA and FASN, as key enzymes for fatty acid synthesis, promoted the synthesis of long-chain fatty acids, while LPL mediates the uptake of circulating lipids for storage in adipose tissues [35]. ELOVL4 is responsible for lengthening fatty acid chain lengths, promoting the formation of long-chain and ultra-long-chain fatty acids, which are readily converted into flavor substances [36]. SREBP1 is a critical transcription factor involved in fat synthesis [37]. Their high expression may promote the bidirectional regulation of lipid metabolism in the TB group, which allows for the co-existence of fat storage and mobilization, thus contributing to the formation of specific fat flavor profiles. There are significant differences in the expression characteristics of genes related to fat metabolism in different parts of yak, and these differences not only affect the fat deposition pattern, but also may regulate the fatty acid composition, accumulation of flavor precursors, and protein degradation pathways, which ultimately shape the flavor characteristics of meat from different parts of yak.

Among all compound classes, aldehydes are identified as the predominant contributors to yak meat aroma due to their low odor thresholds and strong sensory impact [38]. Aldehydes are primarily formed through oxidation of unsaturated fatty acids or the decomposition of lipid-derived radicals [31], with their relative abundance increases with intramuscular fat content, which serves as a substrate for lipid oxidation [39]. At lower concentrations, aldehydes contribute to characteristic fatty aroma, whereas higher concentrations may generate rancid or off-flavors [40]. Gkarane et al reported that the aldehydes are formed through the autoxidation of C18:1 n9 and C18:2 n6, imparting orange, onion, and green odor [41]. In this study, the levels of 2-methylpentanal, 2-methylpropanal, 3-methylbutanal, and 2-methylbutanal were significantly higher in TB than in LT. We hypothesized that the elevated 2-nonenal content in TB is attributable to its higher unsaturated fatty acids content compared to LT, which in turn leads to increasing aldehydes levels by lipid oxidation.

A total of 6 key flavor compounds were identified across different regions of yak meat. Among these, 2,3-Butanedione exhibited the highest ROAV value, indicating that it contributes most significantly to yak meat aroma. This finding is consistent with the report by Yu et al who identified 2-nonenal, (E)-, 2-octenal, heptanal, 1-octen-3-one, 2,3-butanedione, and furan, 2-pentyl- as the six key aroma compounds in Gannan yak across different age groups [42]. The convergence of these results underscores the importance of these 6 compounds in defining the characteristic aroma of yak meat. Furthermore, Wan et al. recognized 2,3-butanedione as a key aroma compound in both raw and cooked beef [43], and a comparative regional study on air-dired beef also highlighted 2,3-butanedione as a key flavor compound [44]. Overall, compared to LT and SM, TB exhibits enhanced flavor characteristics across most aroma attributes. Although the ROAV of 2,3-Butanedione in TB is lower than that in LT and SM, the higher abundance of multiple key flavor compounds in TB collectively shapes its aroma profile through complex cumulative, antagonistic, and synergistic interactions.

Flavor-associated metabolites differ among muscles, including O-Acetylcarnitine, taurine, -L-carnitine, arginine, and glycylcholic acid. In addition to contributing to nutritional value, taurine binds to bile acids [45], regulates of glucose and lipid homeostasis, membrane stabilization, antioxidant, and metabolic regulatory functions [46]. Bile acids play a critical role in signaling, maintaining cholesterol balance, and regulating glucose and lipid metabolism [47]. Primary bile acids are formed through conjugation with taurine, and these taurine-conjugated primary bile acids are subsequently secreted into bile and enter the intestines, enhancing the absorption of nutrients, lipids, and fat-soluble vitamins [48]. Therefore, taurine and cholic acid metabolites, by participating in the regulation of lipid, glucose and energy metabolism, may play potential bioactive roles in the synthesis and regulation of flavor compounds [49]. Additionally, carnitine plays a potential role in lipid and energy metabolism by facilitating and transport of fatty acid into mitochondria for β-oxidation [50]. Although taurine and carnitine are not directly involved in flavor compounds biosynthesis, they may indirectly influence meat flavor by modulating of lipid metabolism.

LysoPA (16:0/0:0) (lysophosphatidic acid with a 16:0 acyl chain) is a bioactive lysophospholipid that serves as an intermediate in phospholipid remodeling by the Lands’ cycle and as a signaling molecule modulates cellular responses to oxidative stress [51]. It is typically generated through phospholipase A1 or A2 (PLA1/PLA2) mediated cleavage of phosphatidic acid or other phospholipids [51]. Elevation LysoPA (16:0/0:0) levels may reflect enhanced membrane lipid turnover or stress-induced phospholipid catabolism, processes often accompanied by increased reactive oxygen species [52]. These oxidative conditions favor the peroxidation of polyunsaturated or monounsaturated fatty acids, leading to the formation of volatile aldehydes such as (E)-2-Nonenal and (E)-2-Octenal via β-scission of hydroperoxides formed through lipoxygenase (LOX) or non-enzymatic lipid oxidation pathways [53]. As shown in Figure 9, genes and metabolites associated with fatty acid synthesis are highly expressed in TB. We speculate that the increase in flavor compound content in TB muscle may result from the high expression of genes. This elevated gene expression may promote fatty acids synthesis and accumulation, thereby providing additional substrates or precursors for flavor compounds formation, ultimately enhancing flavor compound content. However, these findings require further validation. Future studies may validate these results by monitoring changes in lipids and flavor compounds through the overexpression and interference of genes involved in fat synthesis in yak adipocytes.