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 enr ichment of viable, motile spermatozoa in three types of ram semen samp les: normospermic, asthenospermic, and frozen-thawed. Twelve normosper mic ejaculates from six rams and 12 asthenospermic ejaculates from fou r rams were used, Samples showing at least 70% motility were considere d normospermic. Asthenospermic samples showed 20-50% motility. Normosp ermic semen was frozen in 0.2-ml pellets using a Tes-Tris-based diluen t containing 20% (v/v) egg yolk and 4% (v/v) glycerol, and thawed at 3 7 degrees C. Immediately after collection or thawing, samples were fil tered through a 3-cm Sephadex column, centrifuged through a discontinu ous 45-90% Percoll gradient, or left at room temperature for the same time. After treatments, the samples were incubated for 3 h at 37 degre es C. Motility, membrane integrity and acrosomal stage were evaluated before and after treatments, as well as after 3 h at 37 degrees C, Bot h Percoll and Sephadex improved the three parameters analyzed, in froz en-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 degrees C, whereas Perco ll- and Sephadex-treated samples showed a significant decrease in moti lity 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 asthenosper mic samples. These results show that Percoll and Sephadex are effectiv e at increasing seminal quality in low-motility samples, due either to freeze/thawing or to asthenospermia. Despite the opposite effects fou nd (i.e. enrichment of high-quality spermatozoa on the one hand, and s hortening of their half-life on the other) by the use of both techniqu es, adjusting appropriate times for capacitation and insemination may allow its use in all types of semen samples.