1H Nuclear Magnetic Resonance of Pig Seminal Plasma Reveals Intra-Ejaculate Variation in Metabolites

Abstract

In pigs, ejaculate is expelled in fractions, mainly the sperm-rich fraction (SRF) and the post-SRF (PSRF), which differ in both sperm content and origin. In addition, intra-ejaculate variability between fractions in terms of sperm reproductive characteristics has been previously reported, the highest sperm quality being observed in the first 10 mL of the SRF (SRF-P1). As seminal plasma (SP) composition has been purported to influence sperm physiology, the aim of this study was to profile pig SP metabolite composition and to find putative differences between the ejaculate portions (SRF-P1, the rest of SRF [SRF-P2], PSRF) and entire ejaculate (EE). To this end, ejaculates (n = 8, one per boar) were collected in fractions and SP was analyzed using ¹H Nuclear Magnetic Resonance spectroscopy. We identified 19 metabolites present in all ejaculate portions and the EE, and reported correlations between the metabolites. Additionally, and for the first time in mammals, we found intra-ejaculate variability in the SP metabolites, observing different relative abundances in choline, glycerophosphocholine and glycine. Regarding their influence in sperm physiology, we hypothesize that these metabolites may explain the specific reproductive characteristics of each ejaculate portion. Finally, the reported SP metabolites could serve as a first steppingstone in the study of quality, functionality, and fertility biomarkers.

Keywords: intra-ejaculate variability; metabolites; nuclear magnetic resonance; pig; seminal plasma.

Figure 1

¹H Nuclear Magnetic Resonance (¹H NMR) Carr-Purcell-Meiboom-Gill (CPMG) spectrum of pig seminal plasma in phosphate buffer recorded at 400 MHz and 298 K. Sodium trimethylsilylpropanesulfonate (DSS) was used as internal standard. Uridine (Uri); Histidine (His); Phenylalanine (Phe); Tyrosine (Tyr); Citrate (Cit); Cytidine (Cyt); Adenosine Triphosphate (ATP); Lactate (Lac); Glycine (Gly); Choline (Cho); Glycerophosphocholine (GPC); Creatinine (Cr); Glutamic acid (Glu); Glutamine (Gln); Lysine (Lys); Alanine (Ala); Valine (Val); Leucine (Leu).

Figure 2

Selected metabolites’ spectral bin data normalization to DSS of pig seminal plasma, generated using MetaboAnalyst. Adenosine Triphosphate (ATP), Alanine (Ala), Choline (Cho), Citrate (Cit), Creatinine (Cr), Glutamic acid (Glu), Glutamine (Gln), Glycine (Gly), Glycerophosphocholine (GPC), Lactate (Lac) and Valine (Val). Normalization was performed with pareto-scaling and is shown on the left side. Empty dots in box-plots represent the outlier values both before and after normalization. Values were classified as outliers when their value was lower than 1.5 times the 25th percentile or higher than 1.5 times the 75th percentile.

Figure 3

Comparison of the relative areas under each metabolite peak from the pig seminal plasma (SP) of different ejaculate-portions. (A) Graphical representation of one-way ANOVA, generated using MetaboAnalyst [48]. Red points represent metabolites whose relative values differ (p < 0.05) between at least two ejaculate-portions. Green points represent metabolites whose relative values do not differ (p > 0.05) between at least two ejaculate-portions. (B) Box plot representations of the original and the normalized relative abundance for the metabolites presenting differences between ejaculate-portions (first 10 mL from the sperm-rich fraction [SRF-P1], the rest of the sperm rich fraction [SRF-P2], the post-sperm-rich fraction [PSRF] and the entire ejaculate [EE]). Different superscripts (a–c) indicate differences (p < 0.05) between ejaculate-portions.

Figure 4

Correlation plot of the eleven analyzed metabolites identified in boar seminal plasma, generated using MetaboAnalyst. The color saturation of red to blue represents the Pearson correlation coefficients (r) between metabolites, from 0 to 1, respectively. Adenosine Triphosphate (ATP), Alanine (Ala), Choline (Cho), Citrate (Cit), Creatinine (Cr), Glutamic acid (Glu), Glutamine (Gln), Glycine (Gly), Glycerophosphocholine (GPC), Lactate (Lac) and Valine (Val).

Figure 5

Intra-ejaculate analysis of the variance of pig seminal plasma. (A) Scree plot showing the individual variance contribution for each principal component (PC) in blue and the cumulative variance explained along the PCs in green. (B) PCA scores plot showing the distribution of the samples in PC1 and PC2. The colored areas represent the 95% confidence interval, depicting which ejaculate-portion each sample belonged to (the first 10 mL from the sperm-rich fraction [SRF-P1], the rest of sperm rich fraction [SRF-P2], the post-sperm-rich fraction [PSRF] and the entire ejaculate [EE]). (C) Representation of the weighted sum of PCA loadings from the first three PCs. (D) Abundance plot representing the relative levels of each metabolite for every ejaculate portion. Color variation from red to blue correspond to high and low relative levels, respectively. Adenosine Triphosphate (ATP), Alanine (Ala), Choline (Cho), Citrate (Cit), Creatinine (Cr), Glutamic acid (Glu), Glutamine (Gln), Glycine (Gly), Glycerophosphocholine (GPC), Lactate (Lac) and Valine (Val).