Transcriptome and targeted metabolome analysis of lipid profiles, nutrients compositions and volatile compounds in longissimus dorsi of different pig breeds

Animal care

The present experiment was reviewed and approved by the ethics committee of Shanghai Jiao Tong University approved this study (202201188).

Sampling and intramuscular fat analysis

The fresh LD of castrated male LW (Body weight: 115.875 ±7.817 kg, n = 8), SW (102.400±2.011 kg, n = 8) and DLY (107.250±2.340 kg, n = 8) kg pigs were obtained from Jinan Laiwu Pig Industry Technology Research Institute Co., Ltd (Laiwu, China), Shanghai Swine Testing Center in Songjiang District (Shanghai) and Chongming district (Shanghai), respectively. The average slaughter ages were 177.500±0.945 d (SW), 300.375±1.194 d (LW), 173.600±1.580 d (DLY) respectively, which had reached the standardized market slaughter age and weight. All the pigs were reared under standard ventilation conditions and free access to diets and water. The dietary ingredients and nutritional levels were listed in Supplement 1, 2. Samples were immediately placed on ice or stored in liquid nitrogen and transferred to the laboratory for analysis. The IMF content was analyzed in muscle tissues using the Soxhlet method with petroleum ether as the solvent and quantified as the weight percentage of wet muscle tissue.

Morphological analysis

The fresh LD were also fixed in 4% paraformaldehyde and embedded in paraffin, sectioned and stained with hematoxylin and eosin (H&E) for morphological analysis. The muscle samples were also embedded in frozen liver section for oil red staining to visualized lipid droplets.

Triglyceride, total cholesterol and lactate dehydrogenase determination

The muscle tissues were homogenized in phosphate-buffered saline buffer and the supernatants were gathered after centrifuging 3,000×g for 15 min. Protein concentration in the supernatants was measured through the bicinchoninic acid (BCA) protein assay kit. The content of triglyceride (TG), total cholesterol (TC) and activity of lactate dehydrogenase (LDH) in these samples were determined according to the manufacturers’ instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

Redox parameters determination

The redox parameters were determined according to the manufacturers’ instructions. Protein concentration was measured through BCA protein assay kit. Superoxide dismutase (SOD), malondialdehyde (MDA), GSH/GSSG (Nanjing Jiancheng Bioengineering Institute) and NAD⁺/NADH (Shanghai Beyotime Biotechnology, Shanghai, China) were measured and the absorbance at 450, 530, 405 and 450 nm was recorded, respectively.

Transcriptome sequencing

The RNA of muscle tissues were extracted with the RNAiso Plus (#9108, TaKaRa, Kyoto, Japan). The results of RNA quality assessment were shown in Supplement 3. After mRNA purification and fragmentation, RNA was reverse transcribed into cDNA. Polymerase chain reaction (PCR) amplification was used for enrichment of library fragments, and 450 bp fragments were selected. Library quality was assessed on the Agilent 2100 Bioanalyzer. The library preparations were sequenced on an Illumina Hiseq platform and paired-end reads were generated. To ensure the RNA-seq data quality for subsequent analysis, the sequencing error rate distribution and A/T/G/C content distribution were checked, and low quality (average base mass value<20) and N>5 (undetermined base) were filtered out to obtain clean reads. Reads were mapped to the rat genome and were annotated. The differential expression analysis, enrichment analysis and clustering analysis of the samples were further performed.

RNA isolation, cDNA synthesis and quantitative polymerase chain reaction

The total RNA from muscle tissues was extracted following the instructions of the RNA extraction kit. The concentration of RNA was quantified using a spectrophotometer (ThermoFisher Scientific, Chadds Ford, PA, USA). Then 1 μg of RNA was reverse transcribed into cDNA using the PrimeScrip RT reagent Kit. The real-time quantitative PCR reaction was applied to quantify the gene expression with the LightCycler96 system (Roche, Basel, Swiss). The reaction was performed in a total volume of 20 μL, including 10 μL SYBR Green mix, 2 μL cDNA, 7.2 μL H2O, and 0.4 μL each of forward and reverse primers. Amplification conditions were initial 95°C for 30 s, 40 cycles of 95°C for 5 s and 60 °C for 20 s, and a melting curve data. The primers were designed and listed in Supplement 4. The quantification of the genes in each sample was performed in triplicate. β-actin was used as a housekeeping gene to normalize target gene transcript levels. The relative expression of target genes can be calculated by comparing the CT values of target genes and β-actin. The values of SW and DLY pigs were expressed as a fold of the LW pigs, using the formula 2-(^ΔΔCt), where ΔΔCt = (Ct _Target–Ct _β-actin) treatment–(Ct _Target–Ct _β-actin) model.

Medium-long chain fatty acids determination

A targeted metabolomics approach was used to study the compositions of medium-long chain fatty acids in LD according to our previous study [10]. Briefly, 10% H₂SO4-CH₃OH solution (600 μL) were added into 50 mg sample, suspended for 1 min and bathed in water at 62°C for 2 h. After cooling, anhydrous sodium sulfate and 600 μL n-hexane were added and suspended for 1 min. Then, the solution was centrifuged at 3,500 r/min for 5 min. The supernatants were dried vacuum, dissolved in 200 μL n-hexane and analyzed by gas chromatograph/mass spectrometer (GC-MS). The samples were separated by gas chromatography on a DB-5MS capillary column (30 m×0.25 mm ID×0.25 μm). A standard sample mixture was used to identify the fatty acids profiles. The concentrations were calculated based on the chromatogram peak areas.

Amino acids determination

A targeted metabolomics approach based on multiple reaction monitoring (MRM) was used to quantify the contents of AAs according to our previous study [10]. The sample pretreatment was the same to the UHPLC-QTOF-MS. The samples were separated by Agilent 1290 Infinity LC ultra-performance liquid chromatography. Mobile phase A were 25 mM ammonium formate+0.08% FA aqueous solution, and mobile phase B was 0.1% FA acetonitrile. The sample was placed in an automatic sampler at 4°C. The column temperature was 40°C and the flow rate was 250 μL/min. The injection volume was 2 μL. The gradient of liquid phase is as follows: mobile phase B decreases linearly from 90% to 70% during 0 to 12 min, decreases linearly from 70% to 50% during 12–18 min, decreases linearly from 50% to 40% during 18 to 25 min, increases linearly from 40% to 90% during 30 to 30.1 min and maintained at 90% during 30.1 to 37 min. A 5500 QTRAP mass spectrometer (AB SCIEX) was used for mass spectrometry analysis under positive ion mode. The conditions are as follows: source temperature 500°C, ion source gas1 (GAS1): 40, GAS2: 40, curtaingas: 30, ionsapary voltage floating: 5500 V. The ion pair was detected using the MRM mode. The contents of AAs were expressed as ng/mg in muscle tissue.

Volatile compounds determination

Analyses of volatile compounds were performed on an Agilent 7890A gas chromatograph coupled with an Agilent 5975C series mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). Briefly, one gram of samples were weighed in sample bottle. 3 mL of 1 mol/L sodium chloride solution were added, balanced at 60°C for 40 min and analyzed in GC-MS (Agilent Technologies). GC conditions were: inlet temperature 250°C, high purity helium, 1 mL/min carrier gas flow rate, 300°C transmission line temperature, DB-5MS column (30 m×0.25 mm×0.25°C). Heating procedure were: the initial temperature was 40°C and maintained for 1 min, then it was heated to 250°C at a speed of 5°C/min and maintained for 2 min. MS conditions were: 230°C ion source temperature, 70 eV ionization energy, 50 to 500 mz mass scanning range.

Statistical analysis

The data in three groups were analyzed with one-way analysis of variance (ANOVA) followed by post-hoc Duncan’s test using the statistical software SPSS 17.0 (SPSS Inc., Chicago, IL, USA), while AAs data were analyzed with independent sample t test. Data were presented as mean±standard error of the mean. A p-value of <0.05 was considered statistically significant.

Intramuscular fat, lipid contents and redox parameters in longissimus dorsi of pigs

H&E staining showed that LD muscle fiber of LW pigs is closely arranged (Figure 1A). Muscle fiber diameter is small and muscle fiber density is high, while muscle fiber diameter and density in SW and DLY were large and low. Oil red staining results showed that the number of lipid droplets in LD of LW pigs were more than those in SW and DLY pigs (Figure 1A). IMF content (about 12%) was significantly higher in LD of LW pigs than that in SW and DLY pigs (p<0.05) (Figure 1B). No difference of IMF content was observed between SW and DLY pigs (p>0.05). The contents of TG and TC in LD of LW pigs were highest, followed by SW and DLY pigs. There were no significant difference of TG and TC between SW and DLY pigs (p>0.05) (Figures 1C, 1D).

Figure 1

The morphology, and contents of IMF, TG and TC, and redox parameters in the LD of LW, DLY and SW pigs. (A) HE staining and oil red staining. (B) IMF, (C) TG, (D) TC, (E) SOD, (F) MDA, (G) LDH. ^a,b Different letters represent significant differences (p<0.05). IMF, intramuscular fat; TG, triglyceride; TC, total cholesterol; SOD, superoxide dismutase; MDA, malonaldehyde; LDH, lactic dehydrogenase; LW, Laiwu pigs; DLY, Duroc×(Landrace Yorkshire); SW, Shanghai white.

The activity of SOD, ratios of GSH/GSSG and NAD⁺/NADH in LD of LW pigs were significantly higher (p<0.05) than those of SW and DLY pigs (Figures 1E–1G). The content of MDA in LD of LW and SW pigs was significantly lower than that of DLY pigs (Figure 1H). The activity of LDH in DLY pigs was significantly higher than that of LW pigs (Figure 1I). These results suggested that muscle tissues of LW pigs had better lipid deposition and higher antioxidant ability than SW and DLY pigs. No significant difference of lipid deposition and higher antioxidant ability were observed between SW and DLY pigs.

Medium-long chain fatty acids contents

The contents of medium-long chain fatty acids including saturated fatty acids (SFA), monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA) in the LD of pigs were determined by targeted metabolomics (Table 1). Our results showed that total SFA, MUFA and PUFA were significantly higher (p<0.05) in SW pigs compared with LW and DLY pigs, while total SFA, MUFA and PUFA were not significantly different between LW and DLY pigs (p>0.05). SFA including pentadecanoic acid, palmitic acid, margaric acid, stearic acid, arachidic acid, heneicosanoic acid and tricosanoic acid, MUFA including myristelaidic acid, Oleic acid, cis-11-eicosenoic acid and erucic acid, and elaidic acid, PUFA including linoelaidic acid, γ-linolenic acid, eicosapentaenoate, arachidonic acid, cis-11,14,17-eicosatrienoic acid, docosapentaenoate (C22:5n-6), adrenic acid and 13C,16C-docosadienoic acid were higher in SW pigs compared with LW and DLY pigs (p<0.05). MUFA such as heptadecanoic acid was significantly lower in SW pigs when compared with LW and DLY pigs. The SFA including lauric acid, MUFA including trans-10-heptadecenoic acid (C17:1T) were significantly higher in SW pig compared with DLY pigs (p<0.05). MUFA such as trans-11-eicosenoic acid was significantly higher in SW pig compared with LW pigs (p<0.05).

Table 1

The contents of medium-long chain fatty acids profiles (ng/g tissue) in LD of three pig breeds

Amino acids and derivatives contents

The 8 essential amino acids (EAAs) including Lys, Met, Thr, Trp, Phe, Leu, Ile and Val were identified in the LD of two breeding pigs (Table 2). There were no difference of EAAs (p>0.05) in the LD between LW and SW pigs. The non-essential amino acids (NEAAs) including Asp, Tyr, Ser, Gly, Glu, Pro, Arg, Gln, Asn and His were identified in the LD of pigs except for Ala. The content of Asp was significantly lower (p<0.05) in SW pig compared with LW pigs. AAs derivative γ-aminobutyric acid (GABA) was significantly higher (p<0.05) in SW pigs compared with LW pigs, while ornithine (Orn) content was not different between these two pigs (p>0.05).

Table 2

AAs contents and derivatives in LD between SW and LW pigs (ng/mg sample)

Volatile compounds analysis

The differential volatile compounds of LW and SW samples were further studied. Orthogonal partial least squares discriminant analysis (OPLS-DA) results showed that these samples were divided into 2 clusters (Figure 2A). OPLS-DA with 7-fold cross-validation showed R2Y = 0.954 and Q2 = 0.728. There was no overfitting in permutation test (Figure 2B). Totally, 745 metabolites were ultimately identified, including hydrocarbons (15.962%), wax monoesters (5.77%), fatty alcohols (5.23%), oxygenated hydrocarbons (2.82%), pyridine alkaloids (1.21%), acyclic monoterpenoids (1.07%), shikimic acids and derivatives (0.94%), phenylalanine-derived alkaloids (0.81%), halogenated hydrocarbons (0.67%), and others (58.12%) (Figure 2C). A variable importance in projection score>1 and p<0.05 were used as criteria for differential metabolite screening. Totally, 27 differential metabolites were identified. Compared with SW pigs, 26 differential metabolites were decreased, and one metabolite (cholesterol) was increased in LW pigs (Figure 2D). The down-regulated compounds including ketones (2-Octanone, 2-methyl-3-Nonanone, 3,4-dimethyl-2-Hexanone, 5-ethyl-4-methyl-3-Heptanone, 1-[2,3-dihydro-2,3-dihydroxy-2-(1-methylethenyl)-5-benzofuranyl]-Ethanone, 2′,4′-Dihydroxy-3′-methylpropiophenone), alkenes (2,4-Dimethyl-1-heptene,7-methyl-(E)-4-Decene, (E)-9-Octadecene, 1-Tridecene), alkanes (4-methyl-Heptane, 2,4-dimethyl-Heptane, 2,3-dimethyl-Heptane, 2,3,5-trimethyl-Hexane, 1-ethyl-2,4-dimethyl-Cyclohexane, 4,5-dimethyl-Nonane, 2,6-dimethyl-Nonane, 4-methyl-Octane, 2,3,6,7-tetramethyl-Octane, 2,6,11,15-tetramethyl-Hexadecane, 5,7-dimethyl-Undecane), aldehydes (Pentadecanal-, and E-15-Heptadecenal), alcohol (2-Isopropyl-5-methyl-1-heptanol), and others (tetrahydro-6-methyl-2H-Pyran-2-one and 2,6-Dimethyl-6-trifluoroacetoxyoctane) (Figure 2E).

Figure 2

The differential volatile compounds of LD between LW and SW pigs determined by GC-MS. (A) OPLS-DA score plot. (B) OPLS-DA permutation test. (C) The classification of identified metabolites. (D) Volcano plot. (E) Hierarchical clustering of differential volatile compounds between LW and SW. LD, longissimus dorsi; LW, Laiwu pigs; SW, Shanghai white; GC-MS, gas chromatograph/mass spectrometer; OPLS-DA, orthogonal partial least squares discriminant analysis.

Transcriptome analysis of differential expressed genes in LD

The differential expressed genes (DEGs) in LD of LW, DLY and SW pigs were further analyzed by transcriptome. The samples of three groups were basically divided into three clusters by principal component analysis (PCA) (Figure 3A). p<0.05 and |log2FC|>1 were used as the criterion for screening. The DEGs cluster analysis and volcano plot among these pig breeds were shown in Figures 3B–3E. Totally, 2120 DEGs were down-regulated, and 939 genes were up-regulated in LD of LW pigs compared with DLY pigs. 1524 genes were down-regulated, and 931 genes were up-regulated in LD of SW pigs compared with DLY pigs. 801 genes were up-regulated, and 1074 genes were down-regulated in LW pigs compared with SW pigs. Compared with SW pigs, go enrichment analysis of LD from LW pigs showed that several genes focused on biological process including low-density lipoprotein particle, contractile fiber, high-density lipoprotein particle and so on (Figure 3F). Several genes focused on molecular function including lipid transporter activity, hormone-sensitive lipase activity, steroid hormone receptor activity and so on. A few genes focused on cellular component including ventricular cardiac muscle tissue development, high-density lipoprotein particle assembly, lipid localization and so on. Compared with DLY pigs, go enrichment analysis of LD from LW pigs showed that several genes focused on biological process including contractile fiber, myofibril, supramolecular fiber and so on (Figure 3G). A few genes focused on molecular function including binding, cytoskeletal protein binding and so on. A few genes focused on cellular component including positive regulation of RNA metabolic process, positive regulation of nucleic acid-templated transcription and so on. Compared with DLY pigs, go enrichment analysis of LD from SW pigs showed that a number of genes focused on biological process including nucleosome, DNA packaging complex and so on (Figure 3H). Several genes focused on molecular function including DNA binding, DNA binding transcription factor activity and so on. A number of genes focused on cellular component including striated muscle contraction, carbohydrate metabolic process, muscle contraction and so on. The results of Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis did not show related pathways among LW, DLY, and SW pigs.

Figure 3

Transcriptome analysis of DEGs in LD of LW, DLY and SW pigs. (A) PCA. (B) DEGs cluster analysis. (C–E) Volcano plot of LW vs SW pigs, LW vs DLY pigs and SW vs DLY pigs. p<0.05 and |log2FC|>1 were used as the criterion for screening. (F–H) GO pathways enrichment of LW vs SW pigs, LW vs DLY pigs and SW vs DLY pigs. PCA, principal component analysis; LW, Laiwu pigs; SW, Shanghai white; DLY, Duroc×(Landrace Yorkshire); DEGs, differential expressed genes; GO, Gene Ontology.

Quantitative polymerase chain reaction verification and correlation analysis

The gene expression of lipid reserve, synthesis, transport and hydrolysis were further verified. Results showed that gene expression of Plin1 was higher, but Plin3 was lower (p<0.05) in LW pigs than in LW and DLY pigs (Figure 4A). The gene expression of CD36 and FABP3 were significantly higher (p<0.05) in DLY pigs compare with SW pigs and there were no difference between LW and SW pigs (p>0.05). Mgll expression was significantly lower (p<0.05) in LW pigs compared with DLY pigs, and Mgll expression was not different (p>0.05) between LW and SW pigs. HSL expression was higher in LW pigs compared with SW pigs (p<0.05) and not different between DLY and SW pigs (p<0.05). ABCA4 and Nedd4 expression were significantly higher in LW pigs than in DLY pigs (p<0.05) (Figure 4B). PPARA expression was lower in LW and DLY pigs than in SW pigs (p<0.05). There were no difference of ABCG1, DGAT2 and PPARG expression among these three pigs (p>0.05).

Figure 4

Gene expression was verified by qPCR and spearman correlation between nutrients compositions volatile compounds, redox parameters and gene expression in LD. (A) qPCR results. β-actin was used as a housekeeping gene to normalize target gene transcript levels. Different letters represent significant differences (p<0.05). (B) Spearman correlation results, * p<0.05, ** p<0.01. LW, Laiwu pigs; DLY, Duroc×(Landrace Yorkshire); SW, Shanghai white; TG, triglyceride; TC, total cholesterol; IMF, intramuscular fat; SOD, superoxide dismutase; MDA, malondialdehyde; GABA, γ-aminobutyric acid; SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; TFA, trans fatty acids; PUFA, polyunsaturated fatty acids; qPCR, quantitative polymerase chain reaction; LD, longissimus dorsi.

Furthermore, the spearman correlation between the biochemical index (including lipid contents, redox parameters, fatty acids, AAs and volatile compounds) and gene expression in LD was analyzed (Figure 4B). The expression of Plin1 was positively (p<0.05) related to contents of TC. The expression of HSL and Nedd4 were positively related to contents of TG, IMF, GSG/GSSG, and activity of SOD, while negatively related to 2H-Pyran-2-one, tetrahydro-6-methyl-, 2-Octanone, SFA, trans fatty acids (TFA) and PUFA (p<0.05). The expression of Plin3 and Mgll were negatively (p<0.05) related to contents of TG, IMF, GSG/GSSG, cholesterol and Asp, while positively (p<0.05) related to MDA, LDH, 2-Octanone, and Nonane, 4,5-dimethyl-(p<0.05). Also, the expression of Plin3 was positively related to SFA, MUFA, TFA, PUFA and many volatile compounds such as Hexane, 2,3,5-trimethyl-, 2-Octanone and so on (p<0.05). The expression of PPARA was negatively (p<0.05) related to contents of TC and IMF, and activity of SOD, while positively related to many volatile compounds such as heptane, 4-methyl-, 2H-Pyran-2-one, tetrahydro-6-methyl- and so on, which were similar to ABCG1 expression (p<0.05).