Metabolomic analysis of testis tissues in swamp eels (Monopterus albus)

Male swamp eels were purchased from the local fish market (Wuhan, China) in July. These males were caught from the wild during the breeding season. Male swamp eels were euthanatized by immersion in the buffered MS-222 solution (E10521, Sigma-Aldrich). The testis was then isolated and cut into three parts for each swamp eel. One part was used for sperm preparation by squeezing with tweezers, and the other two parts were immediately frozen in liquid nitrogen for 5 minutes and then stored at -80 °C. All experiments involving animals were approved by the Ethics Committee of Institute of Hydrobiology, Chinese Academy of Sciences.After sperm motility analysis, the testis samples were divided into high-quality and low-quality groups. Full-spectrum and non-targeted metabolomic analysis was performed on the two groups of testes in the Metware Biotechnology (Wuhan, China) by using the Ultra Performance Liquid Chromatography (UPLC, ExionLC AD) and tandem mass spectrometry (MS/MS, QTRAP) system. The qualitative analysis of metabolites was performed according to the retention time (RT), ion-pair information, and secondary spectrum data based on the self-built target Metware Database (MWDB) of Metware Biotechnology (Wuhan, China). The quantitative analysis of metabolites was based on the multiple reaction monitoring mode (MRM) of triple quadrupole mass spectrometry. Unsupervised PCA (principal component analysis) was performed by statistics function prcomp within R (www.r-project.org). The data was unit variance scaled before unsupervised PCA. Orthogonal signal correction and Partial Least Squares-Discriminant Analysis (OPLS-DA) was performed by the R opls software package. The data was log transform (log2) and mean centering before OPLS-DA. In order to avoid overfitting, a permutation test (200 permutations) was performed. HCA (hierarchical cluster analysis) and PCC (pearson correlation coefficients) were carried out by R package ComplexHeatmap. The HCA results of samples and metabolites were presented as heatmaps with dendrograms, and normalized signal intensities of metabolites (unit variance scaling) were visualized as a color spectrum. The PCCs between samples were presented as only heatmaps. Significantly differential metabolites between groups were determined by VIP (variable importance in projection) >= 1 and absolute Log2FC (fold change) >= 1. VIP scores were extracted from the OPLS-DA result. The identified metabolites were annotated using KEGG Compound database and then mapped to KEGG Pathway database. Significantly enriched pathways are identified with a hypergeometric test’s p-value for a given list of metabolites.