Comparison between Sephadex G-10 and Percoll for preparation of normospermic, asthenospermic and frozen/thawed ram semen

We have compared the efficacy of Percoll and Sephadex G-10 for the enrichment of viable, motile spermatozoa in three types of ram semen samples: normospermic, asthenospermic, and frozen-thawed. Twelve normospermic ejaculates from six rams and 12 asthenospermic ejaculates from four rams were used. Samples showing at least 70% motility were considered normospermic. Asthenospermic samples showed 20–50% motility. Normospermic semen was frozen in 0.2-ml pellets using a Tes-Tris-based diluent containing 20% (v/v) egg yolk and 4% (v/v) glycerol, and thawed at 37°C. Immediately after collection or thawing, samples were filtered through a 3-cm Sephadex column, centrifuged through a discontinuous 45–90% Percoll gradient, or left at room temperature for the same time. After treatments, the samples were incubated for 3 h at 37°C. Motility, membrane integrity and acrosomal stage were evaluated before and after treatments, as well as after 3 h at 37°C. Both Percoll and Sephadex improved the three parameters analyzed, in frozen-thawed and asthenospermic semen. However, non-treated semen (fresh normospermic, frozen-thawed or asthenospermic) maintained its initial characteristics after 3 h of incubation at 37°C, whereas Percoll- and Sephadex-treated samples showed a significant decrease in motility and membrane integrity at that time. Both procedures were equally effective at sperm recovery from normospermic and frozen-thawed semen, whereas Percoll appeared more effective than Sephadex in asthenospermic samples. These results show that Percoll and Sephadex are effective at increasing seminal quality in low-motility samples, due either to freeze/thawing or to asthenospermia. Despite the opposite effects found (i.e. enrichment of high-quality spermatozoa on the one hand, and shortening of their half-life on the other) by the use of both techniques, adjusting appropriate times for capacitation and insemination may allow its use in all types of semen samples.