Exposure to seminal plasma reduces the duration that epididymal ram sperm exhibit mass motility by increasing glycolytic flux and driving a reduced redox state — ASN Events
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Exposure to seminal plasma reduces the duration that epididymal ram sperm exhibit mass motility by increasing glycolytic flux and driving a reduced redox state (#471)

Madeleine Van de Hoek 1 , Jessica P Rickard 1 , Simon P de Graaf 1
  1. School of Life and Environmental Sciences, Faculty of Science, The University of Sydney, Sydney, NSW, Australia

Mass motility is widely used to assess ram semen quality following ejaculation, yet the mechanisms regulating its longevity remain poorly understood. This study examined the effects of seminal plasma (SP) on the intensity and duration of mass motility, metabolic regulation, and redox balance of epididymal ram sperm.

Epididymal ram spermatozoa were incubated 1:1 (v/v) in saline or SP at 23°C. Mass motility and extracellular pH were assessed, and intracellular and extracellular metabolites associated with glycolysis, oxidative metabolism and NAD⁺/NADH redox status were quantified using targeted mass spectrometry.

Epididymal ram spermatozoa maintained mass motility for >24h under saline incubation, whereas exposure to SP reduced motility longevity to approximately 3h. This functional decline was accompanied by progressive extracellular acidification (−0.69±0.02 pH units; p=0.006), contrasting the gradual increase observed in saline-treated sperm.

Targeted metabolomics provided evidence that SP accelerated glycolytic flux over 3h of incubation. This was characterised by rapid extracellular fructose depletion (−2.58±0.13×10⁶ AU; p<0.001) and lactate accumulation (+21.45±3.05×10⁶ AU; p<0.001), accompanied by declining NAD⁺/NADH ratios (−2.64±0.62) and accumulation of multiple Krebs cycle intermediates (all p<0.001), indicating that oxidative metabolism became increasingly unable to regenerate NAD⁺ during sustained glycolytic activity. This progressively reduced intracellular redox state likely contributed to Krebs cycle bottlenecks and promoted increased conversion of pyruvate to lactate, ultimately accelerating extracellular acidification and motility loss. In contrast, SP-naïve spermatozoa maintained mass motility (+1.17±0.36; p=0.07), stable extracellular fructose concentrations (p=1.000) and a slight increase in NAD⁺/NADH ratio (+1.85±0.17), consistent with balanced glycolytic flux and continued oxidative metabolism. 

These findings demonstrate that SP shifts ram spermatozoa towards a highly glycolytic metabolic strategy that exceeds the capacity of oxidative metabolism, progressively disrupting intracellular redox homeostasis and accelerating motility loss. Targeting this pathway may facilitate the development of strategies to improve liquid semen storage and extend the time available for semen processing and transport.