Comparative Metabolomics Analysis Reveals the Unique Nutritional Characteristics of Breed and Feed on Muscles in Chinese Taihe Black-Bone Silky Fowl

Abstract

The Chinese Taihe Black-bone silky fowl (TBsf) is the homology of medicine and food and has high nutritional and medical value all over the world. However, the nutritional compositions and potential metabolite biomarkers of Taihe silky fowl in muscles are still poorly understood. In this study, we investigated the differences in nutritional components between TBsf and another similar breed (Black Feathered chicken and laid green-shelled eggs, BF-gsc). Meanwhile, we also explored the divergences in muscle characteristics of Taihe silky fowl fed with two different diets; that is, normal chicken feed (TBsf-ncf) and Broussonetia papyrifera-fermented feed (TBsf-bpf). Firstly, the growth performance and biochemical index of Taihe silky fowl was significantly different compared with black-feathered chicken. Secondly, we identified the metabolic alterations in Taihe silky fowl by performing an un-targeted UHPLC-Q-TOF-MS/MS analysis. Our results suggested that all the metabonomic characteristics had obvious separation between TBsf-ncf, TBsf-bpf and BF-gsc groups, both in the positive and negative ion mode by PCA analysis. Next, OPLS-DA multivariate analysis revealed that 57 metabolites (in positive mode) and 49 metabolites (in negative mode) were identified as differential metabolites between the TBsf-ncf and BF-gsc groups. These differential metabolites were mainly enriched to ABC transporters, biosynthesis of amino acids and aminoacyl-tRNA biosynthesis. Besides, 47 metabolites (in positive) and 13 metabolites (in negative) were differentially regulated between the TBsf-ncf and TBsf-bpf groups, which were majorly involved in histidine metabolism and linoleic metabolism. Furthermore, the integrated network analysis suggested that DL-arginine, DL-isoleucine, linoleoylcarnitine, stearoylcarnitine (positive) and ricionleic acid, D-proline, and uric acid (negative) were the significant metabolic biomarkers in Taihe silky fowl. Moreover, the metabolites of primaquine, ticlpoidine, riboflavin, acetylcarnitine (positive) and salicylic acid, acetaminophen sulfate, and glutamic acid (negative) were markedly changed in the Taihe silky fowl fed with BP-fermented feed. In summary, a global survey of the nutritional components and metabolite differences was performed in muscle tissues of Taihe silky fowl between various breeds and feeds. Meanwhile, our study provided valuable information for nutritional components and metabolic biomarkers in Chinese Taihe silky fowl, which greatly promoted the economic value of the black-boned chicken industry and laid a solid theoretical foundation for the development of chicken products with greater added value in future.

Keywords: Taihe silky fowl; biosynthesis of amino acids; breed and feed; metabolic components; un-targeted metabolome.

Figure 1

The growth performance and biochemical component of Taihe silky fowl was significantly different from Black-feathered chicken. (A) The morphological characteristics of each chicken in TBsf-ncf, TBsf-bpf and BF-gsc, respectively. The egg was shown in the lower left corner and meat color was shown in the lower right corner. (B) The body weight of three kinds of chicken at 20 weeks after birth was measured in each group (n = 10). (C) The egg mass of three kinds of chicken was calculated in each group (n = 20). (D) The contents of triglyceride (TG) in three kinds of chicken were detected in blood samples (n = 12, four biological replicates * three techniques replicates). For all experiments, the value was represented as mean ± S.D. * p < 0.05, ** p < 0.01. Abbreviations: Taihe black-bone silky fowl fed with normal chicken feed (TBsf-ncf); Taihe black-bone silky fowl fed with Broussonetia papyrifera-fermented feed (TBsf-bpf); Black-feathered chicken and laid green-shelled eggs (BF-gsc).

Figure 2

Quality assessment of UHPLC-Q-TOF-MS/MS metabolomic data in Taihe silky fowl. (A) The PCA scores of the chicken muscle samples in each group with the ESI positive ion mode. (B) The PCA scores of the chicken muscle samples in each group with the ESI negative ion mode. T[1] represents principal component 1 and T[2] represents principal component 2, which the aggregation degree of QC samples reflects the repeatability of the experimental data. Abbreviations: green dots indicate the BF-gsc samples, blue dots indicate the TBsf-ncf samples, purple dots indicate the TBsf-bpf samples and yellow dots indicate the QC samples.

Figure 3

OPLS-DA plots derived from UHPLC-Q-TOF-MS/MS spectra in Taihe silky fowl. (A,B) The OPLS-DA score map between TBsf-ncf (green dots) and BF-gsc group (blue dots) both in the positive and negative ion mode, respectively. (C,D) The OPLS-DA score map between TBsf-bpf (green dots) and BF-gsc group (blue dots) both in the positive and negative ion mode, respectively. (E,F) The OPLS-DA score map between TBsf-ncf (green dots) and TBsf-bpf group (blue dots) both in the positive and negative ion mode, respectively.

Figure 4

The differential metabolites were identified in Taihe silky fowl by metabolomic analysis. (A) The number of metabolites extracted from positive and negative ion peaks in HILIC column were presented. (B) The proportion of identified metabolites in each chemical classification. The specific chemical categories of each metabolite can be found in the legend, and the proportion of each superclass was presented in the corresponding pie chart. (C) Venn diagram showing the shared and unique differential metabolites between Taihe silky fowl and black-feathered chicken in positive ion mode. (D) Venn diagram showing the shared and unique differential metabolites between Taihe silky fowl and black-feathered chicken in negative ion mode. The overlapping regions represent metabolites that are concomitantly regulated in two or three samples.

Figure 5

The KEGG enrichment analysis revealed the pathways of protein synthesis and transport were mainly activated in Taihe silky fowl. (A) The KEGG pathway enrichment analysis of differential metabolites in Taihe silky fowl compared with the black-feathered chicken. (B–E) Hierarchical clustering analysis of differential metabolites in ABC transporters, biosynthesis of amino acids, aminoacyl-tRNA biosynthesis, and glycine, serine, and threonine metabolism, respectively.

Figure 6

The integrated networks showed the correlation in various differential metabolites of Taihe silky fowl. (A) Network data integration and visualization of differential metabolites of Taihe silky fowl in the positive ion mode. (B) Network data integration and visualization of differential metabolites of Taihe silky fowl in the negative ion mode. The circle represents the significantly enriched differential metabolites. The size of the circle is related to the degree of connectivity: the larger the degree, the larger the circle.

Figure 7

The functional analysis of differential metabolites in Taihe silky fowl fed by BP-fermented feed. (A) The KEGG pathway enrichment analysis of differential metabolites in Taihe silky fowl fed with Broussonetia papyrifera-fermented feed compared with normal chicken feed. (B) The hierarchical clustering analysis of differential metabolites between TBsf-ncf and TBsf-bpf groups. (C) The integrated networks of enriched differential metabolites in the positive ion mode between TBsf-ncf and TBsf-bpf groups. (D) The integrated networks of enriched differential metabolites in the negative ion mode between TBsf-ncf and TBsf-bpf groups.