The effect of calcium, pH and other regulators of motility on the axoneme of demembranated stallion spermatozoa

Abstract

Despite being a standard procedure in most other domestic species, in vitro fertilization (IVF) has had poor success in the horse. This appears to be due to the failure of equine sperm to spontaneously initiate hyperactivated motility in vitro, as pharmacological stimulation of hyperactivation has been shown to increase equine IVF rates. To investigate the control of hyperactivated motility in equine sperm, we evaluated factors affecting motility in a demembranated sperm model. Exposure of sperm to 0.02% Triton X-100 for 30 seconds maximized membrane permeability as shown by transmission electron microscopy and staining with propidium iodide, while maintaining motility as determined by computer-assisted sperm motility analysis. Using this model, we evaluated the effects of medium pH, free calcium, ATP and dibutyryl-cAMP on motility of demembranated sperm. We also assessed the effects of known physiological stimulators of hyperactivation, 4-aminopyridine and procaine, as well as motility inhibitors nickel and cadmium. A minimum of three replicates was performed for each experiment, and data was analyzed by analysis of variance, utilizing the Holm-Sidak method for multiple comparisons. Increasing concentrations of ATP were associated with an increase in both curvilinear velocity and amplitude of lateral head movement. Cadmiumwas also associated with increases in these parameters, in contrast to the significant decline in motility seen in other species; however, nickel decreased motility as expected. Sperm motility was not affected by dibutyryl-cAMP. At a standard free-calcium concentration of 100 nM, maximal motility was induced at pH 6.5, a