Maintenance of sperm motility is central to the continuing capacity of spermatozoa to fertilise an oocyte. Motility decreases over time, and most in vitro stored samples will contain a proportion of live but immotile spermatozoa. Whilst providing appropriate substrates for ATP production to support sperm motility is important, it is also vital to understand and exploit the molecular mechanisms that activate ATP production when sperm have depleted their energy stores. The AMP-activated protein kinase (AMPK) system is a cellular energy ‘fuel gauge’, responding to ATP depletion by shifting the cell’s metabolic balance towards ATP production rather than consumption. 5-Aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside (AICAR) activates AMPK by mimicking the cellular effects of 5’-AMP. We hypothesised that stimulating sperm AMPK phosphorylation with AICAR would improve motility parameters of stallion spermatozoa via increased ATP production.

 Semen was collected from pony stallions (n=3), transported in Kenney’s extender and processed by colloid gradient centrifugation immediately upon arrival at the laboratory. Spermatozoa were resuspended in modified BWW medium at 20 million/mL and treated as follows: BWW only (Con) and BWW with AICAR at concentrations of 1µM, 10µM, 50µM, and 100µM (A1, A10, A50, A100).  Samples were incubated at 37˚C and motility was assessed at 1 and 24 hours of incubation using a CASA system. Sperm viability was assessed at 24 hours by staining with Sytox Green and flow cytometry.

Total (TM), progressive (PM) and rapid motility (RM) were notably increased at all doses of AICAR versus BWW-only control at 24 hours. Treatment with 1, 50, and 100µM AICAR was associated with significant increases (p<0.05) in total motility (TM: Con=27.3±3.0%, A1=53.7±4.7%, A50=44.7±5.7%, A100=39.7±4.3%) and rapid motility (RM: Con=21±1.5%, A1=45±3.6%, A50=38±3.1%, A100=33.3±1.9%). Sperm viability was unaffected except for a slight increase with 100µM AICAR (Con=73.8±0.2% vs A100=76.1±0.4%, p<0.05).

We conclude that in vitro supplementation of media with AICAR can improve stallion sperm motility and may be useful in maintaining the fertilising capacity of stored spermatozoa.