Effect of Density Gradient Centrifugation on Quality and Recovery Rate of Equine Sperm

Abstract

Density gradient centrifugation of sperm is a common assisted-reproduction procedure in humans used to improve semen quality. The technique allows sperm separation based on their isopycnic points. Sperm with morphologic abnormalities are often more buoyant, leading to their retention above centrifuged density gradients, with structurally normal sperm passing through the gradient. Three experiments were conducted to evaluate the effects of tube size, sperm number following centrifugation, and density gradient volume (height) on stallion sperm quality and recovery rate in sperm pellets following centrifugation. In all three experiments, equine semen was initially centrifuged to increase sperm concentration. In Experiment 1, one-mL aliquots were layered over EquiPure? Bottom Layer (1-Layer) or over-tiered EquiPure? Top and Bottom Layers (2-Layer). For Experiment 2, one-mL aliquots were layered over three different heights of EquiPure? Bottom Layer in 15-mL or 50-mL conical-bottom tubes. For Experiment 3, four different aliquots containing a sperm load of 1-4x were layered over a constant volume of EquiPure? Bottom Layer in 15-mL or 50-mL conical bottom tubes. The tubes were then centrifuged. Resulting sperm pellets were evaluated for morphologic quality, DNA integrity, motility and recovery rate. Sperm-EquiPure? centrifugation yielded improvements in motility, morphology and DNA integrity parameters (P<0.05), as compared to controls. The 1-Layer method resulted in a higher recovery rate than the 2-Layer method (P<0.05). Sperm processed in the 15-mL tubes yielded higher velocity and higher recovery rates than sperm processed in the 50-mL tubes (P<0.05). Within tube type, gradient volume did not impact parameters of semen quality or recovery rate. An increase in sperm number for density gradient centrifugation resulted in a decreased recovery rate (P<0.05) when 15-mL tubes were used.