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

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.

In vitro aging of stallion spermatozoa during prolonged storage at 5°C

“…After removal of the gel fraction by filtration through gauze, semen was diluted (1:1) with prewarmed INRA‐96® extender (IMV Technologies, l'Aigle, France) and transported to the laboratory in a Styrofoam box. Cushion centrifugation (1000 g , 20 min, room temperature) was performed in 50 ml conical centrifugation tubes using 1 ml of Opti‐prep™ 40% as a cushion [18]. After centrifugation, the supernatant and the bulk of the cushion fluid were removed by aspiration and the concentration of the remaining sperm pellet was determined using a Nucleocounter Sp‐100 (Chemometec, Denmark).…”

“…Semen was collected from 17 healthy warmblood stallions (5.6 Cushion centrifugation (1000 g, 20 min, room temperature) was performed in 50 ml conical centrifugation tubes using 1 ml of Opti-prep™ 40% as a cushion [18]. After centrifugation, the supernatant and the bulk of the cushion fluid were removed by aspiration and the concentration of the remaining sperm pellet was determined using a Nucleocounter Sp-100 (Chemometec, Denmark).…”