Background: Quantitative measurements of sperm are essential for assessing their overall quality and viability. Whilst many putative tests of sperm function have been developed, none have been universally incorporated into routine clinical practice. We hypothesise that metabolites present within sperm could be used as biomarkers of sperm function, but measuring them in live sperm is challenging. Nuclear Magnetic Resonance (NMR) has previously been used to study cellular metabolism in a variety of live tissues. Here we validate its use to investigate metabolism in live sperm.

Aim: To use Carbon (¹³C) NMR to characterise sperm metabolites and metabolism of external substrates, with the hope of identifying signature biomarkers of sperm function.

Method: Boar spermatozoa of high quality and motility were prepared by density centrifugation. Sperm were then re-suspended in 1ml phosphate buffered saline with 200μl of 100mM ¹³C-1 or ¹³C-2 labelled sodium pyruvate. ¹³C NMR spectra were obtained using a Bruker Avance III 9.4T scanner and a ¹³C broadband probe. Spermatozoa were held at 39^oC and scanned every 4 minutes for up to 1 hour to monitor real time metabolite changes or overnight to identify all metabolites present.

Results: Metabolism of ¹³C₁ pyruvate generated CO₂, bicarbonate, and lactate, providing evidence of ATP production through aerobic glycolysis & oxidative phosphorlyation. This was further supported by the presence of glutamate (Tricarboxylic Acid Cycle metabolite) and alanine from ¹³C₂ pyruvate spectra. Overall this supports previous studies that suggest boar sperm use multiple metabolic pathways for ATP production.

Conclusions: We have shown that ¹³C NMR can be used to investigate metabolism in live sperm. Future experiments will use human sperm of varying quality and examine whether rates of ¹³C pyruvate metabolism differs between fertile and infertile men.