The concentration of glucose in the media influences the susceptibility of stallion spermatozoa to ferroptosis.

Stallion spermatozoa depend on oxidative phosphorylation as their major source of ATP, however, the metabolism of these cells is complex and a great degree of metabolic plasticity exists. The composition of the media in which the spermatozoa are extended, or exposed to in the mare's reproductive tract exerts a profound effect on sperm function and may even accelerate cell demise. Recent research indicates that high concentrations of glucose in the media, although common in commercial extenders, may be detrimental. To determine if supraphysiological glucose concentration may induce or predispose to ferroptosis (a caspase-independent form of programmed cell death, triggered by oxidative stress). aliquots of stallion ejaculates were incubated under different concentrations of glucose, 67mM (HG) or 1mM plus 10mM pyruvate (LG-HP), in the presence or absence of known inductors of ferroptosis. Furthermore, we developed a single-cell flow metabolic assay to identify different metabolic phenotypes in spermatozoa Storage and incubation of spermatozoa under high glucose concentrations led to an increase in the percentage of necrotic spermatozoa (P<0.0001). Moreover, ferroptosis was induced more intensely in sperm in media with high glucose concentrations (P<0.0001). Finally, we observed that induction of ferroptosis modified two proteins (oxoglutarate dehydrogenase and superoxide dismutase 2) in spermatozoa incubated in media containing 67mM glucose, but not in media containing 1mM glucose and 10 mM pyruvate The concentration of glucose, exerts a major impact on the functionality and lifespan of the spermatozoa. The results reported here may pave the way for improvements in existing semen extenders.