172 Untargeted metabolomic analysis of Zulu ram seminal plasma metabolome following selenium supplementation and their possibility as fertility biomarkers

Kwa-Zulu Natal province in South Africa is home to the indigenous Zulu sheep kept mostly by communal farmers due to their good adaptability traits and resistance to tick-borne diseases. Heat stress resulting from high ambient temperatures in those regions suppresses the ram’s testicular functions through oxidant damage, which causes an imbalance in reactive oxygen species (ROS), DNA damage, and apoptosis. The Kwa-Zulu Natal province has soils and pastures that are deficient in selenium, due to leaching of minerals by high rainfalls. Selenium is an antioxidant used to scavenge and suppress ROS activity. However, it is unknown whether selenium supplementation has an influence on the SP metabolome, and therefore, it is hypothesised that it changes sperm metabolite activity, which is the end point of enzyme/protein activity. The purpose of the study was to investigate the effect of selenium on the seminal plasma (SM) metabolome of Zulu rams and their association with fertility. Seminal plasma of 10 rams aged between 2–5 years was harvested by centrifugation from semen and stored at −80°C for metabolomic analysis. The rams were randomly allocated into 2 treatment groups (G1: selenium; G2: control); the selenium group received sodium selenite orally on a bi-weekly basis for 6 months. Selenium was supplemented for 2 months before semen collection to cover the duration of spermatogenesis. Pegasus 4D GCxGC-TOFMS coupled to a time-of-flight mass spectrometer (TOFMS) equipped with a Gerstel multi-purpose sampler system with a cryogenic cooler was used to generate mass fragmentation patterns and their respective retention time. Normalization was carried out to ensure reproducibility and reliability of data. Quality control samples were generated by mixing a small aliquot of each analytical sample to determine the variance of metabolite features. Peak identities were determined by comparing them with commercially available NIST spectral libraries, and all statistical analyses were done on quantified data sets using MetaboAnalyst 4.0, a web server for metabolomics data analyses. A total of 536 metabolites were discovered; however, over half of the metabolites were unknown. Top 15 distinct metabolites identified by partial least-squares discriminant analysis that could be potential fertility biomarkers were unknown. The study findings indicate that most of the metabolites found in the SM were involved in energy metabolic substrates and antioxidant activity. The top four metabolic pathways in the SM were glutathione, glutamine, malate-aspartate, and propanoate metabolism. Hydroxybutyric acid, D-fructose, and L-glutamic acid were the most abundant metabolites found in the selenium-treated group and they are involved in the energy and antioxidant metabolic pathways. Many of the SM metabolites in the study were unknown; therefore, there is a need to annotate and build libraries for fertility biomarker discovery. Selenium did not affect the metabolite concentrations. However, results also suggest that selenium had an influence on certain metabolic pathways including glutathione, glutamate, malate-aspartate, and propanoate metabolism which led to an increased antioxidant and ATP activity.