Abstract
Metabolomic approaches allow the study of downstream gene expression events since metabolites are considered as the products of cell signaling pathways. For this reason, many studies in humans have already been conducted to determine the influence of the metabolites present in seminal plasma (SP) on sperm physiology, and to identify putative biomarkers. However, in livestock species, these relationships are yet to be uncovered. Thus, the present study aimed to explore: (i) if concentrations of metabolites in pig SP are related to sperm quality and functionality, and (ii) if they could predict the sperm resilience to liquid storage at 17°C. To this end, 28 ejaculates were individually collected and split into three aliquots: one was used for SP analysis through nuclear magnetic resonance (NMR) spectroscopy; another served for the evaluation of sperm concentration and morphology; and the last one was utilized to determine sperm functionality parameters using computer-assisted sperm analysis (CASA) and flow cytometry after 0 h and 72 h of liquid-storage at 17°C. NMR analysis allowed the identification and quantification of 23 metabolites present in pig SP which, except for fumarate, were not observed to follow a breed-dependent behavior. Moreover, specific relationships between metabolites and sperm variables were identified: (i) glutamate, methanol, trimethylamine N-oxide, carnitine, and isoleucine were seen to be related to some sperm quality and functionality parameters evaluated immediately after semen collection; (ii) leucine, hypotaurine, carnitine and isoleucine were found to be associated to the sperm ability to withstand liquid storage; and (iii) Bayesian multiple regression models allowed the identification of metabolite patterns for specific sperm parameters at both 0 h and 72 h. The identification of these relationships opens up the possibility of further investigating these metabolites as potential sperm functional biomarkers.
Highlights
Artificial insemination (AI) remains the most valuable and efficient reproductive biotechnology used for pig breeding, gathering more than 90% of cases in countries with intensive pork production (Roca et al, 2016; Yeste, 2017; Waberski et al, 2019)
Considering that fumarate could be influenced by the boar breed, this metabolite was excluded from further analyses in order to evaluate the effect of seminal plasma (SP)-metabolites on sperm physiology
As far as we know, this is the first report that performed an in-depth metabolomic profile of pig SP quantifying the identified metabolites. This is the first study conducted in a livestock species that explored the putative influence of SP-metabolites on sperm physiology and on the sperm resilience to liquid storage at 17 ◦C. This exploratory study reported that: i) a total of 23 SP-metabolites were present at measurable concentrations in pig SP; ii) except for fumarate, no differences in SP-metabolites concentration were identified between pig breeds (Duroc and Pietrain); iii) glutamate, methanol, trimethylamine N-oxide, carnitine and isoleucine concentrations in SP were related to specific sperm quality and functionality parameters evaluated right after semen collection; iv) leucine, hypotaurine, carnitine and isoleucine concentrations in SP were found to be related with the sperm ability to withstand liquid storage; and v) Bayesian multiple regression models allowed the identification of putative metabolite patterns for specific sperm parameters
Summary
Artificial insemination (AI) remains the most valuable and efficient reproductive biotechnology used for pig breeding, gathering more than 90% of cases in countries with intensive pork production (Roca et al, 2016; Yeste, 2017; Waberski et al, 2019). One of the main concerns is related to differences in the sperm ability to withstand liquid-storage between pig seminal AI-doses (Parrilla et al, 2012; Zakošek Pipan et al, 2014; Roca et al, 2015) This situation evidences that current sperm assessments used for ejaculate selection (which include sperm quantity, motility, and morphology) are not accurate enough to predict sperm fertilizing capacity after liquid preservation (Roca et al, 2016; Waberski et al, 2019). The heterogeneous and complex composition of SP, together with the fact that this fluid is able to interact with sperm, make it a valuable source of potential biomarkers (Rodríguez-Martínez et al, 2011)
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