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

Simple Summary Sperm DNA fragmentation has a negative impact on reproductive success, because it compromises fertilization and embryo development. Since spermatozoa lack the machinery to repair DNA damage, the likelihood of establishing a healthy pregnancy can be improved by processing ejaculates with a high sperm DNA fragmentation index. The current study examined a modified flotation density gradient centrifugation technique in which semen was diluted with a colloid solution (Opti-prepTM) to increase its density, later layered between colloid layers of lower and higher density. This modified flotation technique was consistent in selecting viable, progressively motile, morphologically normal spermatozoa with intact acrosome and DNA. In reproductively normal stallions, sperm recovery was 57% and spermatozoa with damaged DNA decreased significantly (from 12% to 4%), with increase in the percentage of viable, progressively motile, morphologically normal spermatozoa with intact acrosome. In stallions with a high sperm DNA fragmentation index, this modified flotation technique markedly decreased the DNA fragmentation index (from 31% to 5%) and significantly improved the other semen quality parameters, although sperm recovery was low (approximately 20%). In conclusion, the stallion sperm DNA fragmentation index and other sperm quality parameters can be markedly improved using a modified flotation density gradient centrifugation technique employing a 40% Opti-prepTM cushion and a 20% top layer. Abstract 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-prepTM) to increase its density prior to layering between colloid layers of lower and higher density. The optimal Opti-prepTM 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-prepTM solutions (20%, 25% and 30%) as the top layer. Finally, the most efficient DGC technique was selected to process ejaculates from Friesian stallions (n = 3) with high sperm DFI (>20%). The optimal Opti-prepTM 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-prepTM 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 (p < 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-prepTM cushion and a 20% top layer.