Production performance and Meat quality
There is no significant difference among the H, L and COMB group in corresponding to FI, BW, BWG and FCR (P > 0.05) (Table 3). Compared with the L group, the H group showed significantly increased cooking loss of breast muscle (P < 0.05). The COMB group showed decreased cooking loss compared with the H group (P < 0.05). Besides, the drip loss in the COMB group was lower than that in the L group, as well (P < 0.05) (Figure 1).
The 45-min pH value in the H group was higher than that in the other 2 groups (P < 0.05) while there was no significant difference in 24-h pH values among the groups. Thus, the pH decline during 45 min to 24 h in the H group was significantly higher than that in the other 2 groups, indicating that the H group had rapid pH drop rate, which was attenuated in the COMB group under high stocking density (Figure 2).
The stocking density significantly altered the activity of LDH (P = 0.022). The activity of LDH in the H group was higher (P < 0.05) than that in the L group. The COMB group had significantly decreased (P < 0.05) activity of LDH when compared with the H group. However, stocking density had no significant effect on the activities of CK, T-AOC, MDH, anti-superoxide anion and the content of hydroxyproline (Table 4).
RNA sequencing data and differentially expressed genes (DEGs)
In the principal component analysis (PCA), there was a clear divergence among the H, L and COMB groups. In the Venn diagram, the number of identified genes in the H, L and COMB were 11777, 12554 and 11633, respectively (Figure 3). Compared with the H group, the number of DEGs in the L group and COMB group were 3752 and 773, respectively (Figure 4).
The gene sets were produced by DEGS. From Venn analysis of genes sets, we found that there were 1310 genes shared in common between the COMB group and the L group. Nevertheless, there were only 6 genes owed by both the COMB group and the H group. Similarly, from the iPath map of metabolic pathways, there were a total of 830 pathways annotated in common. In contrast, there was only 1 pathway owed by both the COMB group and the H group (Figure 5).
Up-regulated genes in the H group
Compared with those in the L group, a total of 1894 genes were up-regulated in the H group (Figure 4), which were mainly involved in muscle contraction, cell localization, ion transport, lipid metabolism, glycolysis, proteolysis, and immune stress (Figure 6).
Muscle contraction-related pathways were enriched in the H group. They involved vital genes including MYLK2, NOS1, TMOD4, and Six1 (Table 5). The H group was enriched for cell-localization-related genes such as KEAP1, CDKN1A, ERBB4, and TMOD4 (Table 5). Additionally, high-density up-regulated ion and amino acid transport-related genes included KCNJ12, KCNA7, SLC38A3 and SLC38A4, which are involved in ion transmembrane transport and transporter activity (Table 6). High-density enriched glycolysis-related pathways included fructose metabolism, fructose-2,6-diphosphate 2-phosphatase activity, and fructose 2,6-diphosphate metabolism (Table 7). The lipid metabolism-related genes such as MID1IP1, ACACB and Lpin1 were up-regulated in H group, which are involved in lipid synthesis and lipid oxidation (Table 7).
Stress response pathways including non-biologically stimulated cellular responses, extracellular stimuli response and nutritional level response were also enriched in the H group. Furthermore, high-density up-regulated proteolysis-related genes include TINAG, USP24, OTUD1, KEAP1, KLHL34, and SMCR8. Also, high-density enriched immune pathways include the regulation of host defence responses to viruses and prostaglandin receptor-like binding (Table 8).
In Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, genes involved in calcium signalling pathway (RYR), inflammatory mediator regulation of RTP channels (PLA2) and chemokine signalling pathway (SOS) (Fig. S1-S3) were enriched in the H group.
Down-regulated genes in the H group
Compared with those in the L group, a total of 1858 genes were down-regulated in the H group (Figure 4), which were involved in cell adhesion, cell matrix, and cell migration, etc (Figure 7).
The genes involved in muscle development include muscle fibre assembly and binding (LMOD2, MYOZ2 and ACTN1, etc.) and muscle fibre development (DSG2, LMOD2 and FSCN1, etc.), which were down-regulated in H group (Table 9). High-density also down-regulated genes related to cell-matrix pathways such as MMP9, FBLN1, THBS4, and VCAN. High-density also down-regulated collagen synthesis and collagen binding related genes including ADAMTS3, ADAMTS14, COL1A2, and LUM (Table 10). Besides, the adhesion-associated genes including DSG2, CSTA, THY1, TGFBI, NOV, CDH11 and FN1 were diminished. Additionally, antioxidant genes including MGST2, PTGS2, NCF1, SOD3, and CYBB were also down-regulated (Table 11).
In KEGG enrichment analysis, down-regulated genes in the H group were involved in ECM-receptor interaction (COL1A, THBS1, FN1, TN, ITGA5, ITGA8 and ITGB8), adherens junction (SHP-1, TGFβR, α-Actinin and Slug) and focal adhesion (Actinin and MLC) (Fig. S4-S6).
Up-regulated genes in the COMB group
Compared with those in the H group, up-regulated genes in the COMB group were involved in muscle development, hyaluronic acid synthesis, mitochondrial function, and redox pathway (Figure 8).
The muscle development-related pathways enriched in the COMB group included positive regulation of muscle tissue development and muscle cell decision processes, which involved key genes such as MYF6, LMCD1 and TRPC3. Besides, the COMB group was enriched for mitochondria-associated pathways such as electron transport chains, mitochondrial respiratory chain complex I and mitochondrial protein complex pathways, which involved genes including TOMM6, NDUFV1, NDUFS5, NDUFB2, NDUFA2, LMCD1, ZNF593 and COASY (Table 12). The hyaluronic acid-related genes up-regulated in the COMB group included HYAL1 and HYAL3. Besides, the redox-related genes including LDHD, CPOX, SUOX, NDUFV1, GRHPR, DOHH and NDUFA2 were up-regulated in the COMB group, which were involved in the pathways such as redox process, NAD binding, NADPH binding and NADH dehydrogenase complex (Table 13). In KEGG enrichment analysis, up-regulated genes in the COMB group were involved in oxidative phosphorylation (NDUFS5, NDUFV1, NDUFA2, NDUFA13, NDUFB2, NDUFB7 and NDUFC2) (Fig. S7).
Down-regulated genes in the COMB group
Compared with those in the H group, down-regulated genes in the COMB group were involved in the inflammatory response, acid metabolism, fatty acid metabolism, and glycolysis-related pathways (Figure 9).
The inflammatory response-related genes down-regulated in the COMB group included CCR5 and ALOX5 while the immune response-related genes included C1S, BLK, CCR5 and MARCH1 (Table 14). The acid metabolism-related pathways include organic acid synthesis process, oxoacid metabolism process and carboxylic acid synthesis process, which involved genes such as PSAT1, SCD, MAT1A, ALOX5, ST3GAL1 and ALDOB. The genes involved in fatty acid metabolism pathways include SCD and ALOX5. In addition, down-regulated genes in the COMB group were involved in glycolytic and carbohydrate metabolism, which included GALNT16, ST3GAL1, ALDOB and MAT1A (Table 15).
In KEGG enrichment analysis, genes involved in the regulation of lipolysis in adipocytes (PLIN), glycolysis/gluconeogenesis (ALDO) and arachidonic acid metabolism (ALOX5) were down-regulated in the COMB group (Fig. S8-S10).
Transcriptome differential gene verification
The transcriptome differential genes were verified by real-time PCR, and the gene expression pattern was consistent with the transcriptome results (Figure 10).