Metabolomic markers of fertility in bull seminal plasma

Abstract

Metabolites play essential roles in biological systems, but detailed identities and significance of the seminal plasma metabolome related to bull fertility are still unknown. The objectives of this study were to determine the comprehensive metabolome of seminal plasma from Holstein bulls and to ascertain the potential of metabolites as biomarkers of bull fertility. The seminal plasma metabolome from 16 Holstein bulls with two fertility rates were determined by gas chromatography-mass spectrometry (GC-MS). Multivariate and univariate analyses of the data were performed, and the pathways associated with the seminal plasma metabolome were identified using bioinformatics approaches. Sixty-three metabolites were identified in the seminal plasma of all bulls. Fructose was the most abundant metabolite in the seminal fluid, followed for citric acid, lactic acid, urea and phosphoric acid. Androstenedione, 4-ketoglucose, D-xylofuranose, 2-oxoglutaric acid and erythronic acid represented the least predominant metabolites. Partial-Least Squares Discriminant Analysis (PLSDA) revealed a distinct separation between high and low fertility bulls. The metabolites with the greatest Variable Importance in Projection score (VIP > 2) were 2-oxoglutaric acid and fructose. Heat-map analysis, based on VIP score, and univariate analysis indicated that 2-oxoglutaric acid was less (P = 0.02); whereas fructose was greater (P = 0.02) in high fertility than in low fertility bulls. The current study is the first to describe the metabolome of bull seminal plasma using GC-MS and presented metabolites such as 2-oxoglutaric acid and fructose as potential biomarkers of bull fertility.

Fig 1. Representative GC-MS chromatogram of bull seminal plasma.

Peaks of lactic acid, glycerol, phosphoric acid, citric acid, fructose, heptadecanoic acid (internal standard) and cholesterol are indicated.

Fig 2. Number of metabolites per chemical class.

Metabolites identified were categorized according to their chemical classes, defined as amino acids, peptides and analogues; carbohydrates and carbohydrate conjugates; fatty acids and conjugates; steroids and steroid derivatives; nucleosides, nucleotides, and analogues; others organic compounds and inorganic compounds.

Fig 3. Abundance ratios of the most and least predominant metabolites present in bull seminal plasma.

(A) The five most abundant metabolites were fructose, citric acid, lactic acid, urea and phosphoric acid and (B) the five least metabolites were identified as androstenedione, 4-ketoglucose, D-xylofuranose, 2-oxoglutaric acid and erythronic acid. Abundance ratio of the metabolites was calculated by dividing abundance of target ions of metabolites by that of target ion of the internal standard. Error bars represent standard error of the mean.

Fig 4. PLS-DA score plot of seminal plasma from high (HF) and low fertility (LF) bulls.

The plots indicate that a separation could be observed between HF and LF bulls. Supervised PLS-DA was obtained with 2 components. The explained variances are shown in parentheses.

Fig 5. VIP scores of seminal plasma metabolites in high (HF) and low fertility (LF) bulls.

The selected metabolites were those with VIP score of greater than 1.5. Heat map with red or green boxes on the right indicates high and low abundance ratio, respectively, of the corresponding metabolite in HF and LF bulls. VIP score was based on the PLS-DA model.

Fig 6. Box plots of the abundance ratio of five metabolites in high and low fertility bulls.

(A) 2-oxoglutaric acid, (B) fructose, (C) ornithine, (D) L-leucine and (E) D-mannitol were significantly different (P ≤ 0.05) between high (HF) and low (LF) bulls.

Fig 7. Heatmap of Pearson’s correlations among metabolites identified in bull seminal plasma.

Fig 8. Metabolic networks associated with metabolites identified in bull seminal plasma.

(A) fructose, (B) citric acid, (C) lactic acid, (D) urea, (E) 2-oxoglutaric acid. Networks were generated using the Metscape plug-in for Cytoscape. Metabolites are shown in red hexagons. Compounds are represented as pink hexagons, reversible reactions as gray squares with purple text and non-reversible reactions as gray squares with orange text.