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.

Metabolite Profiling of Pig Seminal Plasma Identifies Potential Biomarkers for Sperm Resilience to Liquid Preservation

“…The present work identified and quantified a total of 23 metabolites in pig SP, including amino acids, saccharides, salts, alcohols, and other compounds. Compared to our previous work (Mateo-Otero et al, 2020), the present study quantified SPmetabolites, as well as identified new molecules (i.e., formate, fumarate, hypotaurine, isoleucine, malonate, myo-inositol, and trimethylamine N-oxide). These new findings may result from improvements in sample handling and purification, and the higher frequency and resolution of the NMR spectrometer.…”

“…The NMR spectroscopy is one of the most used technologies in this field, because it provides a non-biased uniform detection of equal sensibility for all proton-containing molecules in samples, and requires little or no processing (Goodacre et al, 2004;Emwas et al, 2019). This analytical method has been widely used for SP-metabolite profiling of several mammalian species, including humans (Hamamah et al, 1993;Segalen et al, 1995;Gupta et al, 2011;Jayaraman et al, 2014;Bonechi et al, 2015;Mumcu et al, 2020), cattle (Kumar et al, 2015) and pigs (Kalic et al, 1997;Mateo-Otero et al, 2020). The present work identified and quantified a total of 23 metabolites in pig SP, including amino acids, saccharides, salts, alcohols, and other compounds.…”

“…In a previous study, we described, for the first time, the pig SP metabolomic profile and we suggested that certain SP-metabolites could play a key role in reproductive processes (Mateo-Otero et al, 2020). As far as we know, there are no data about the relationship between SP-metabolites and the sperm ability to withstand liquid preservation in any species.…”

Aldose Reductase B1 in Pig Seminal Plasma: Identification, Localization in Reproductive Tissues, and Relationship With Quality and Sperm Preservation

“…Three fractions/portions can be objectively collected with clear differences in the SP source: (a) the first 10 mL of the so-called sperm rich fraction (SRF-P1), rich in sperm and with SP mainly originating from the epididymis; (b) the rest of SRF (SRF-P2), also rich in sperm and with SP mainly coming from the prostate ( Rodríguez-Martínez et al, 2005 , 2009 ), and, (c) the post-SRF (PSRF), poor in sperm and with SP mainly arising from the seminal vesicles ( Einarsson, 1971 ; Rodríguez-Martínez et al, 2005 , 2009 ; Saravia et al, 2009 ; Rodríguez-Martínez et al, 2011 ). Interestingly, the SP from these ejaculate portions has also been found to vary in terms of proteomic, metabolomic, and antioxidant capacity ( Saravia et al, 2009 ; Rodríguez-Martínez et al, 2011 ; Barranco et al, 2015 , 2016 , 2017 ; Perez-Patiño et al, 2016 ; Mateo-Otero et al, 2020 ).…”

Seminal Plasma: Relevant for Fertility?

“…Markers for sperm dysfunction [ 218 ] and (in)fertility have been detected in spermatozoa [ 27 , 190 , 219 , 220 ] and in the SP [ 221 , 222 ] of many species, including humans [ 218 , 222 , 223 , 224 , 225 ] and livestock [ 70 , 226 ]. They vary from gene presence [ 227 ], markers of Sertoli cell function [ 228 ], proteins [ 36 , 229 , 230 , 231 ], anti-oxidative enzymes [ 223 , 232 , 233 ] and metabolites [ 234 , 235 , 236 ] to exosome-associated molecules [ 237 , 238 ] and microRNAs [ 181 , 239 , 240 , 241 ].…”