Amino Acids of Seminal Plasma Associated With Freezability of Bull Sperm

Abstract

Sperm cryopreservation is an important technique for fertility management, but post-thaw viability of sperm differs among breeding bulls. With metabolites being the end products of various metabolic pathways, the contributions of seminal plasma metabolites to sperm cryopreservation are still unknown. These gaps in the knowledge base are concerning because they prevent advances in the fundamental science of cryobiology and improvement of bull fertility. The objective of this study was to test the hypothesis that seminal plasma amino acids are associated with freezability of bull sperm. To accomplish this objective, amino acid concentrations in seminal plasma from seven bulls of good freezability (GF) and six bulls of poor freezability (PF) were quantified using gas chromatography-mass spectrometry (GC-MS). Multivariate and univariate analyses were performed to identify potential freezability biomarkers. Pathways and networks analyses of identified amino acids were performed using bioinformatic tools. By analyzing and interpreting the results we demonstrated that glutamic acid was the most abundant amino acid in bull seminal plasma with average concentration of 3,366 ± 547.3 nM, which accounts for about 53% of total amino acids. The other most predominant amino acids were alanine, glycine, and aspartic acid with the mean concentrations of 1,053 ± 187.9, 429.8 ± 57.94, and 427 ± 101.3 nM. Pearson's correlation analysis suggested that phenylalanine concentration was significantly associated with post-thaw viability (r = 0.57, P-value = 0.043). Significant correlations were also found among other amino acids. In addition, partial least squares-discriminant analysis (PLS-DA) bi-plot indicated a distinct separation between GF and PF groups. Phenylalanine had the highest VIP score and was more abundant in the GF groups than in the PF groups. Moreover, pathway and network analysis indicated that phenylalanine contributes to oxidoreductase and antioxidant reactions. Although univariate analysis did not yield significant differences in amino acid concentration between the two groups, these findings are significant that they indicate the potentially important roles of amino acids in seminal plasma, thereby building a foundation for the fundamental science of cryobiology and reproductive biotechnology.

Keywords: amino acids; bull sperm; freezability; metabolomics; seminal plasma.

FIGURE 1

Representative GC–MS chromatogram of bull seminal plasma. Total of 21 amino acids were identified using SIM (selected ion monitoring), Norvaline used as an internal standard with a concentration of 200 nM.

FIGURE 2

Concentrations of the most and the least abundant amino acids in bull seminal plasma. (A) The most abundant amino acids in bull seminal plasma was glutamic acid. Alanine, glycine, aspartic acid, and serine were the other predominant amino acids in bull seminal plasma. (B) The least predominant amino acids were tyrosine, methionine, alpha aminobutyric acid, allo-isoleucine, and asparagine.

FIGURE 3

Pearson’s correlations among amino acids identified in bull seminal plasma. (A) Correlation matrix of amino acid concentrations in seminal plasma [shaded lines: Pearson correlation coefficients (r); white boxes: P-value; highlighted boxes: P < 0.05). (B) Heatmap of Pearson’s correlations among amino acids identified in bull seminal plasma.

FIGURE 4

Partial least squares discriminant analysis (2D PLS-DA) of the seminal plasma amino acids from good freezability (GF) and poor freezability (PF) bulls. The plots indicate a separation between GF and PF bulls. PLS-DA was obtained with two components.

FIGURE 5

Variable importance in projection (VIP) plot displays the top 15 most important amino acid features identified by PLS-DA. Colored boxes on right indicate concentration of corresponding amino acid from GF and PF samples. VIP score is a weighted based on PLS-DA model.

FIGURE 6

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FIGURE 7

Pathway and network analyses of the amino acids with highest VIP scores (phenylalanine, threonine) and the most abundant amino acids (glutamic acid, alanine) were performed using MetScape. (A) Phenylalanine, (B) threonine, (C) glutamate, and (D) alanine. Amino acids are shown in red hexagons. Gray square: Reaction node with reaction ID; Pale red hexagon: Compound node; Green square: Enzyme node; Blue circle: Gene node.