Investigations of mammalian sperm proteomes in our laboratory revealed enzymes involved in ß-oxidation of fatty acids to be present in ejaculated spermatozoa. Roles of fatty acids have been established in spermatogenesis, maintenance of sperm membrane fluidity and resistance to cryodamage, however potential utilisation of fatty acids in metabolism and energy production by ejaculated stallion spermatozoa has not been previously addressed. The aim of this study was to determine if ß-oxidation of fatty acids contributes to energy production by spermatozoa, by means of assessing motility following treatment with Etomoxir, an inhibitor of ß-oxidation.

Semen collected from pony stallions (n=9) was processed by dilution with Kenney’s extender and Percoll gradient centrifugation. Sperm at 20 million/mL in modified BWW were treated as follows: BWW only (Con), 10µM Etomoxir (Eto10) and 100µM Etomoxir (Eto100). Samples were incubated for 24 hours at 37˚C and assessed for motility (CASA), viability (Live-Dead stain, flow cytometry) and lipid peroxidation (anti-4HNE antibody, flow cytometry).

Total motility (TM) decreased in 10 µM Etomoxir (Con=22.4±2.4% vs Eto10=17.1±2.1%, p<0.01). Total, progressive (PM), and rapid (RM) motility were decreased in 100 µM Etomoxir (TM: Con=22.4±2.4% vs Eto100=14.6±1.5% (p< 0.01); PM: Con=8.6±2.3% vs Eto100=3.1±0.9% (p<0.05); RM: Con=15.2±2.1% vs Eto100=5.8±1.2% (p<0.01)). Sperm viability (61±2.0%) and lipid peroxidation (50.1±2.5%) were not affected by either treatment.

Inhibition of fatty acid ß-oxidation resulted in significantly decreased sperm motility without effects on viability or lipid oxidative damage, suggesting that ß-oxidation contributes to energy production by ejaculated stallion spermatozoa. Fatty acids may be able to provide an additional source of energy for supporting motility of stallion spermatozoa, and the effects of increased fatty acid utilisation on sperm survival and motility during in vitro storage should be examined in future studies.