UHPLC-MS/MS Untargeted Metabolomics Profiling of Cecal Content in Three Chicken Breeds

ABSTRACT:

This study applied an untargeted metabolomics approach to analyze cecal content samples from three chicken breeds (Wenchang chicken, White-ear Yellow chicken, and Recessive White chicken) using UHPLC-MS/MS (Vanquish UHPLC coupled with Q Exactive HFX Orbitrap). A total of 90 samples (30 per breed) plus 8 pooled QC samples were analyzed. Metabolites were extracted with methanol/acetonitrile (1:1, v/v), and data were acquired in positive and negative ionization modes. Raw data were converted to mzXML format by MSConvert (ProteoWizard v3.0.8789) and processed using the R XCMS package for peak detection, retention time correction, and alignment. Metabolites were annotated against HMDB, MassBank, LipidMaps, mzCloud, KEGG, and an in-house database. Data normalization was performed using QC-RLSC (LOESS signal correction), and features with QC RSD > 30% were filtered out. Multivariate statistics (PCA and OPLS-DA) were conducted using the R package ropls to distinguish metabolic profiles. Differential metabolites were screened using a two-tiered strategy: for 2 dpi, P < 0.05 and VIP > 1.0 were used; for 4 dpi and 7 dpi, VIP > 1.0, |log2FC| > 1, and FDR-adjusted P < 0.05 were applied. Pathway enrichment analysis was performed using MetaboAnalyst; no pathway reached P < 0.05, and pathways with P < 0.2 were considered as trends.

Breed name mapping: In the commercial dataset folders, breeds are abbreviated as WCJ (Wenchang chicken), BEJ (White-ear Yellow chicken), and YXB (Recessive White chicken). These correspond to WCC, WEYC, and RWFC, respectively, as used in this study and the manuscript.

Note: The associated manuscript analyzes and reports conclusions based exclusively on the Wenchang chicken breed; data from the other two breeds (YXB and BEJ) are included for project-level transparency. They have not been used in the analyses presented in this manuscript.

Data source clarification: The manuscript analyses are based on the 2023 commercial metabolomics report, which was generated from the complete original raw data at that time. The raw data recovered in 2026 are deposited in MetaboLights (MTBLS15811) and in the Zenodo record for archival and transparency purposes. A small number of recovered files have reduced scan counts due to the original corruption; however, all files can be opened, and this does not affect the manuscript conclusions, which rely on the 2023 commercial report.

INSTRUMENT(S): Liquid Chromatography MS - negative - reverse-phase, Liquid Chromatography MS - positive - reverse-phase

PROVIDER: MTBLS15811 | MetaboLights | 2026-10-08

REPOSITORIES: MetaboLights