| Summary: | Sperm DNA fragmentation compromises fertilization and early embryo development. Since spermatozoa lack the machinery to repair DNA damage, to improve the likelihood of establishing a healthy pregnancy, it is preferable to process ejaculates of stallions with a high sperm DNA fragmentation index (DFI) before artificial insemination or intracytoplasmic sperm injection. The aim of this study was to examine a modified flotation density gradient centrifugation (DGC) technique in which semen was diluted with a colloid solution (Opti-prep<sup>TM</sup>) to increase its density prior to layering between colloid layers of lower and higher density. The optimal Opti-prep<sup>TM</sup> solution (20–60%) for use as the bottom/cushion layer was first determined, followed by a comparison between a modified sedimentation DGC and the modified flotation DGC technique, using different Opti-prep<sup>TM</sup> solutions (20%, 25% and 30%) as the top layer. Finally, the most efficient DGC technique was selected to process ejaculates from Friesian stallions (<i>n</i> = 3) with high sperm DFI (>20%). The optimal Opti-prep<sup>TM</sup> solution for the cushion layer was 40%. The modified sedimentation technique resulted in two different sperm populations, whereas the modified flotation technique yielded three populations. Among the variants tested, the modified flotation DGC using 20% Opti-prep<sup>TM</sup> as the top layer yielded the best results; the average sperm recovery was 57%; the DFI decreased significantly (from 12% to 4%) and the other sperm quality parameters, including progressive and total motility, percentages of spermatozoa with normal morphology and viable spermatozoa with an intact acrosome, all increased (<i>p</i> < 0.05). In Friesian stallions with high sperm DFI, the modified flotation DGC markedly decreased the DFI (from 31% to 5%) and significantly improved the other semen quality parameters, although sperm recovery was low (approximately 20%). In conclusion, stallion sperm DFI and other sperm quality parameters can be markedly improved using a modified flotation DGC technique employing a 40% Opti-prep<sup>TM</sup> cushion and a 20% top layer.
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