Biochemical properties of bull spermatozoa separated in iodixanol density solution

A Modified Flotation Density Gradient Centrifugation Technique Improves the Semen Quality of Stallions with a High DNA Fragmentation Index

“…Three density gradient tubes (15 mL) were prepared for each of the modified sedimentation (Figure 1a) and the modified flotation DGC techniques (Figure 1b) by pipetting 2-mL layers of density gradient solution or diluted semen (DS vs. DS-Opti-prep TM ). The order of the solutions from bottom to top was: In comparison to the report on bull semen [8], the 1:1:1 mixture of stallion semen, INRA96 and Opti-prep TM 60% (DS-Opti-prep TM ) was less dense than pure 40% Optiprep TM and could not be placed on the bottom of the tube. Therefore, the DS-Opti-prep TM layer was used as the intermediate layer.…”

“…Density gradient centrifugation (DGC) is a sperm selection technique that separates spermatozoa into subpopulations based on their density. This is accompanied by a relative enrichment of viable and/or motile sperm with more uniform morphology in one of the subpopulations [8]. For equine assisted reproduction, single-or double-layer density gradient systems predominate among commercially available products (for a review, see reference [9]).…”

“…For equine assisted reproduction, single-or double-layer density gradient systems predominate among commercially available products (for a review, see reference [9]). In this context, Opti-prep TM (Iodixanol 60%), originally developed as an X-ray contrast medium, is one of the colloid solutions that has previously been used to separate spermatozoa from men [10,11], bulls [8] and stallions [12]. Opti-prep TM is nonionic, iso-osmotic and extensively tested for toxicity towards biological materials and can be used to generate isotonic solutions with densities up to 1.32 g/mL [13].…”

“…This property of Opti-prep TM makes it suitable as a bottom or "cushion" layer to prevent the overcompaction of the sperm pellet during centrifugation. Less-concentrated solutions of Optiprep TM can serve as the top layer in double-layer DGC, which separates spermatozoa into populations with different qualities [8,12]. Thus, discontinuous double-layer DGC using Opti-prep TM [12] to make both a cushion layer and a selective top layer has been proposed to be superior to ordinary centrifugation or density gradient centrifugation systems (single or double-layer) using Percoll [14], EquiPure [15] or Androcoll [16], because, in the latter, the selected sperm are collected as a pellet at the bottom of the tube.…”

“…However, in this classical approach, sperm recovery is low (33%) even for ejaculates from stallions with semen parameters within the normal range. An alternative method of DGC (modified flotation DGC) has been described for bull semen, where the semen is first diluted (1:1) with a gradient solution (Opti-prep TM ) to increase its density and then layered beneath two layers of Opti-prep TM with decreasing density [8]. Whether and to what extent this different DGC setup may improve sperm recovery and the selection of sperm with intact DNA is not clear.…”