Utvärdering av en ny selektionsmetod för hingstsperma : med avseende på membranintegriteten och membranstabiliteten

Sammanfattning: Today, equine breeding is based on performance and conformation, resulting in a vast variation in fertility among different horses, with noticeable economic losses as a consequence. The horse breeding industry is therefore in need of methods that diagnose the spermatozoa concerning their quality and provide the best sperm for AI. The commonly used selection methods for sperm today are: centrifugation, where extender is added, the sample is centrifuged and then resuspended; swim-up self-migration; adherence separation; and density gradient centrifugation. However, none of these methods are in used routinely in practice before inseminating mares with fresh or cooled semen. The aim of this study was to evaluate a new selection method, Single Layer Centrifugation (SLC), and its effect on sperm viability. The viability was analyzed with regard to membrane integrity and membrane stability of ejaculated spermatozoa. The aim was also to investigate how storage at 5ºC affected non-centrifuged and centrifuged semen regarding sperm membrane integrity and stability during 48 h. The SLC is a sperm selection method based on the principles of density gradient centrifugation but where only one layer of colloid is used instead of two or more layers of various densities. The motile spermatozoa traverse the colloid during centrifugation while epithelial cells, immotile spermatozoa, seminal plasma etc. are trapped i.e they do not pass and can thus be removed. This study included (3) Swedish warmblood stallions, 7-12 years old. Ten ejaculates were collected in May 2007, three ejaculates from each of two stallions and four from the third. Ejaculates were collected from the stallions regularly during the breeding season as part of a commercial enterprise. After collection, sperm concentration and subjective motility were assessed before SLC. The membrane integrity and stability were evaluated with the fluorescent dyes SYBR-14/PI and Annexin-V/PI respectively using flow cytometry at 0h, 24h and 48h. Four groups of spermatozoa were obtained by the Annexin-V/PI-assay: intact, living spermatozoa (AN-/ PI-), spermatozoa with unstable membrane (AN+/ PI-), spermatozoa with damaged membrane but not entirely necrotic (AN+/ PI+) and dead spermatozoa (AN-/ PI+). Three groups of spermatozoa were obtained by the SYBR-14/PI-assay: living spermatozoa (SYBR-14+/PI-), moribund spermatozoa (SYBR-14+/PI+) and dead spermatozoa (SYBR-14-/PI+). The results of the study showed that semen that had been centrifuged had a significantly greater proportion of living spermatozoa than non-centrifuged semen regarding membrane integrity at all three timepoints. The difference in results based on membrane stability was not significant at 0h or 24h. However, at 48h the proportion of living sperm was significantly larger in centrifuged semen than in non-centrifuged semen. This makes it unlikely that a non-centrifuged semen sample analyzed 24h after collection will get a true value regarding its viability.