Identification of Metabolites in Muscles of Lueyang Black-Bone Chickens: A Comparative Analysis of Caged and Cage-Free Rearing Modes Using Untargeted Metabolomic Techniques

Abstract

The Lueyang black-bone chicken is a specific native chicken strain in China. This study aimed to investigate the effects of different rearing systems on the meat quality of Lueyang black-bone chickens. Six hundred Lueyang black-bone hens were randomly divided into two groups at 7 weeks of age and raised in cage and cage-free systems for 20 weeks. The carcass yield, meat quality, and total metabolites were measured in both the leg and breast muscles. By comparison, the carcass yield of hens in the cage-free (CF) group (1.26 ± 0.09 kg) was significantly lower than that in the caged rearing (CR) group (1.52 ± 0.15 kg). However, the shear force of leg muscles in the CF group (27.98 ± 2.43 N) was significantly greater than that in the CR group (24.15 ± 1.93 N). In addition, six samples from each group were randomly selected and their metabolites were detected by the non-targeted metabolomics technique. Among these metabolites, 408 and 354 significantly differentially abundant metabolites were identified in breast and leg muscles, which were mainly involved in glycerophospholipid metabolism, unsaturated fatty acid biosynthesis, arginine and proline metabolism, and nucleotide metabolism. We found that the levels of 19 phospholipids, mainly phosphatidylcholines and lysophosphatidylcholines, were significantly greater in the CF group than in the CR group. Additionally, the contents of eight unsaturated fatty acids, linoleic acid, and linolenic acid were dramatically greater in the CF group than in the caged group. The accumulation of 4-hydroxy-proline, glutamate, and adenosine 3'-monophosphate (AMP) was enhanced in the CF group. Moreover, many more volatile organic compounds were identified in the muscles of the cage-free group, enhancing the flavor of the chicken meat. In conclusion, the cage-free rearing mode facilitates the accumulation of nutrients and flavor substances in the chicken meat and is a better rearing system for Lueyang black-bone chickens.

Keywords: black-bone chicken; meat quality; metabolomics; rearing system.

Figure 1

Overview of the experimental design. BM: breast muscle. LM: leg muscle. LC-MS: liquid chromatograph–mass spectrometer. GC-IMS: gas chromatography–ion migration spectrometry.

Figure 2

Multivariate analysis of metabolomics data from different chicken samples. (A,B) PCA score plots based on metabolites from negative and positive ion modes; (C,D) PLS-DA score plots based on metabolites from negative and positive ion modes. (E,F) Permutation tests were used to assess the accuracy of the PLS-DA models in negative and positive ion modes.

Figure 3

Analysis of metabolites among the four groups. (A) Heatmap of hierarchical clustering of differentiated metabolites among the four groups. (B) Venn analysis of shared metabolites in negative and positive ion modes.

Figure 4

Analysis of DMs in skeletal muscles. (A) Venn diagram of the total DM analysis. Heatmap of hierarchical clustering of DMs in the breast and leg muscles between cage-free and caged chickens. (B–E) The top 20 DMs in the leg and breast muscles.

Figure 5

Cluster analysis of DMs in the breast and leg muscles between the caged and cage-free groups. HMDB compound classification of DMs in breast muscles (A) and leg muscles (B). KEGG enrichment analysis of DMs in breast muscles (C) and leg muscles (D). Hierarchical clustering analysis of DMs involved in glycerophospholipid metabolism (E), biosynthesis of unsaturated fatty acids (F), arginine and proline metabolism (G), purine metabolism (H), and pyrimidine metabolism (I). * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 6

Analysis of volatile organic compounds in leg muscles (A) and breast muscles (B) between the caged and cage-free modes. Colors represent the concentration of substances; deep blue indicates background; light blue indicates low concentration; yellow indicates high concentration; red indicates higher concentration. The darker the color, the higher the concentration.

Figure 7

Correlations between the DMs and volatile organic compounds in the breast muscles (A) and leg muscles (B) of the caged and cage-free rearing modes.