Current evidence suggests that 20-25% of young men have poor semen quality, and in 50% of cases the male factor is responsible for a couple’s infertility¹. In addition to adequate sperm count, sperm motility is essential for the successful fertilisation of the oocyte. The energy required for sperm swimming is provided by the metabolism of substrates to generate ATP². The two main energy pathways of metabolism, glycolysis and oxidative phosphorylation, produce different biomarkers, including lactate (glycolysis) and bicarbonate/CO₂ (Oxphos), and the rate of conversion of exogenous substrates could highlight the relative importance of different metabolic pathways. In this study we incubated ¹³C labelled metabolites with human spermatozoa to identify metabolic pathways and quantify rates of spermatozoa metabolism.Spermatozoa from 13 research donors (recruited with LREC approval) were washed using an 80:40% (v/v) Percoll/PBS gradient centrifugation followed by leukocyte depletion with magnetic CD45 Dynabeads. Sperm from the pellet and the 80:40% interface were re-suspended in 300-400μl PBS with 20μl D₂O, 100 units/ml Penicillin and 100 µg/ml Streptomycin antibiotics. Both were scanned at 37°C using a 9.4T MR scanner. 1.2-3.4mM of either ¹³C-labelled glucose, pyruvate, lactate or butyrate were added to the sperm and scanned immediately or placed in a water bath at 37°C for up to 24h before scanning. Spectra were acquired using a ¹³C{¹H} inverse-gated pulse sequence (SW=239ppm, NS=4096, AQ=0.5s, D1=2, flip angle=16°). The metabolic conversion of ¹³C substrate was followed with 4-5 sequential ¹³C spectra over a period of up to 20 hours. Integrals were fitted to an exponential equation to estimate the rate constants for the metabolic conversion of substrate to daughter product per million sperm.Incubating the sperm with either ¹³C_U-glucose or ¹³C_U-fructose resulted in the almost exclusive conversion to lactate, despite their potential to be converted into bicarbonate or other TCA cycle intermediates. In contrast ¹³C₁ or ¹³C₂ labeled pyruvate (pyr) incubation resulted in conversion to labeled lactate (¹³C₁-pyr or ¹³C₂-pyr), bicarbonate (¹³C₁-pyr) and glutamate (¹³C₂-pyr), showing that sperm can utilise both glycolysis and Oxphos. Sperm showed no metabolism of exogenous ¹³C₃-lactate or ¹³C₁-butyrate. By simultaneously administering a 1:1 ¹³C_u-glucose and ¹³C₁-pyruvate mixture to the sperm rate constants for metabolism in both the glycolytic and Oxphos pathways were estimated, see figure 1. The rate constants for glucose and pyruvate conversion to lactate were estimated as 1.1±0.5x10^-6s^-1 and 2.4±1.1x10^-6s^-1 per million sperm, respectively (mean±SD, n=4). Similarly, the pyruvate to bicarbonate rate constant was estimated as 1.7±1.2x10^-6s^-1 (mean±SD, n=3). The rate constant for ¹³C_u-fructose to lactate was estimated at 3.6±4.4x10^-6s^-1(mean±SD, n=2).Separating the sperm from seminal plasma allows active metabolic pathways used by live sperm to be assessed using ¹³C labelled substrates and ¹³C MR spectroscopy. By combining ¹³C metabolites (e.g. glucose and pyruvate), the relative flux through glycolysis and oxidative phosphorylation can be estimated. Further work is being done to estimate metabolic activity in sperm of normozoospermic and asthenozoospermic patients.