Analysis of stallion spermatozoa metabolism using Agilent Seahorse XFp Technology

Sperm metabolism consists of a sophisticated network of biochemical reactions and varies between species, resulting in different metabolic strategies for ATP production to maintain sperm functionality. ATP can be produced through glycolysis or in the mitochondria by oxidative phosphorylation (OXPHOS). Since OXPHOS is the predominant metabolic pathway in horses spermatozoa, various assessments of mitochondrial activity are used to evaluate fertility, utilizing techniques such as fluorescent probes analysed via microscopy or flow cytometry, and polarographic electrode assays to measure current flow in response to an applied voltage. Though, these methods are limited by low throughput, as they assess mitochondrial activity at a single time point under a specific treatment condition. This study explores, for the first time, the application of the Agilent Seahorse XFp Technology to evaluate metabolism in stallion spermatozoa. This method enables real-time measurement of cellular metabolism across multiple samples or experimental conditions simultaneously. Ejaculates from eight different stallions were collected, and pools were prepared from three of them. Sperm viability and mitochondrial activity were evaluated by fluorescence microscopy, sperm motility by a computer-assisted sperm analysis system, and sperm metabolism was analysed via the Seahorse XFp analyser. Results confirmed a preference for OXPHOS over glycolysis in ATP production in stallion sperm, with mitochondria contributing significantly to total ATP generation. The Seahorse XFp Technology proved effective in evaluating equine sperm bioenergetics, offering insights into metabolic pathways critical for sperm function. In conclusion, this technology grants a new method for high-throughput analysis of sperm metabolism and quality, which could be applied to future reproductive studies in male equine fertility.