Utvärdering av en ny selektionsmetod för hingstsperma : med avseende på membranintegriteten och membranstabiliteten
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...
| Main Author: | |
|---|---|
| Format: | L3 |
| Language: | Swedish Inglés |
| Published: |
SLU/Dept. of Anatomy, Physiology and Biochemistry (until 231231)
2007
|
| Subjects: |
| _version_ | 1855571991397924864 |
|---|---|
| author | Thorén, Josefina |
| author_browse | Thorén, Josefina |
| author_facet | Thorén, Josefina |
| author_sort | Thorén, Josefina |
| collection | Epsilon Archive for Student Projects |
| description | 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. |
| format | L3 |
| id | RepoSLU12054 |
| institution | Swedish University of Agricultural Sciences |
| language | swe Inglés |
| publishDate | 2007 |
| publishDateSort | 2007 |
| publisher | SLU/Dept. of Anatomy, Physiology and Biochemistry (until 231231) |
| publisherStr | SLU/Dept. of Anatomy, Physiology and Biochemistry (until 231231) |
| record_format | eprints |
| spelling | RepoSLU120542017-11-13T09:04:42Z Utvärdering av en ny selektionsmetod för hingstsperma : med avseende på membranintegriteten och membranstabiliteten Thorén, Josefina reproduktion spermie häst viabilitet single layer membranintegritet membranstabilitet kylförvaring 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. Idag avlar man framförallt på prestation och exteriör inom hästaveln. Detta medför att fertiliteten varierar mycket mellan avelshästar med kännbara ekonomiska förluster som följd. Hästaveln är därför i behov av metoder för att kunna diagnostisera sperman med avseende på dess kvalité och få fram de bästa spermierna att använda vid insemination. De vanligaste metoderna som finns idag för spermaselektion är enkel centrifugering, som innefattar spädning, centrifugering och återspädning, självmigrering (swim-up), adherensseparering och gradientcentrifugering. Ingen av dessa används dock ute på hingststationer med seminverksamhet. Syftet med studien var att utvärdera en ny selektionsmetod, Single Layer Centrifugering, SLC, med avseende på spermiens viabilitet. Detta har gjorts genom att analysera membranintegriteten och membranstabiliteten på ejakulerade spermier före och efter SLC. Syftet var också att se hur kylförvaring vid 5°C påverkade centrifugerad och ocentrifugerad sperma med avseende på membranintegriteten och membranstabiliteten under 48 timmar. SLC är en selektionsmetod som liknar gradientcentrifugering men endast ett lager kolloid används istället för två eller flera. De motila spermierna förflyttas genom kolloiden vid centrifugering och kan därför särskiljas från övrigt såsom eptielceller, immotila spermier, sädesvätska etc som fastnar och kan avlägsnas. I studien ingick tre (3) varmblodiga travhingstar. 7-12 år gamla. Totalt tio ejakulat, tre från två av hingstarna och fyra från den tredje, samlades under våren 2007. Hingstarna ingick samtidigt i den reguljära seminverksamheten. Ejakulaten analyserades med avseende på koncentration och motiliteten bedömdes subjektivt innan SLC. Därefter analyserades spermiernas membranintegritet och membranstabilitet, både i ocentrifugerade och centrifugerade prover, med hjälp av infärgningarna SYBR-14/PI respektive Annexin-V/PI i en flödescytometer vid tidpunkterna 0, 24 och 48h. Fyra grupper av spermier erhölls av Annexin-V/PIanalysen: levande spermier med stabilt membran (Annexin-V-negativa [AN-]/PInegativa [PI-]), spermier med instabilt men helt membran (Annexin-V-positiva [AN+]/[PI-]), spermier med skadat cellmembran men som ej fullständigt gått i nekros ([AN+]/ PI-positiva [PI+]) och de som hade helt skadat cellmembran (AN- / PI+). Tre grupper erhölls av SYBR-14/PI-analysen: levande celler (SYBR- 14+/PI-), döende celler (SYBR-14+/PI+) och döda celler (SYBR-14-/PI+). Resultatet visade att den centrifugerade sperman hade signifikant större andel levande spermier med avseende på membranintegritet än den ocentrifugerade sperman vid alla tre analystidpunkterna. För membranstabiliteten var skillnaden ej signifikant vid 0h och 24h. Vid 48h hade dock centrifugerad sperma signifikant fler levande spermier med stabilt membran än ocentrifugerad sperma. Resultaten visade också att ocentrifugerad sperma klarade sig sämre vid kylförvaring vid 5°C än centrifugerad sperma. Detta gör att man ej kan analysera ett ocentrifugerat spermaprov 24h efter samling och få ett representativt värde på viabilitet. SLU/Dept. of Anatomy, Physiology and Biochemistry (until 231231) 2007 L3 swe eng https://stud.epsilon.slu.se/12054/ |
| spellingShingle | reproduktion spermie häst viabilitet single layer membranintegritet membranstabilitet kylförvaring Thorén, Josefina Utvärdering av en ny selektionsmetod för hingstsperma : med avseende på membranintegriteten och membranstabiliteten |
| title | Utvärdering av en ny selektionsmetod för hingstsperma : med avseende på membranintegriteten och membranstabiliteten |
| title_full | Utvärdering av en ny selektionsmetod för hingstsperma : med avseende på membranintegriteten och membranstabiliteten |
| title_fullStr | Utvärdering av en ny selektionsmetod för hingstsperma : med avseende på membranintegriteten och membranstabiliteten |
| title_full_unstemmed | Utvärdering av en ny selektionsmetod för hingstsperma : med avseende på membranintegriteten och membranstabiliteten |
| title_short | Utvärdering av en ny selektionsmetod för hingstsperma : med avseende på membranintegriteten och membranstabiliteten |
| title_sort | utvärdering av en ny selektionsmetod för hingstsperma : med avseende på membranintegriteten och membranstabiliteten |
| topic | reproduktion spermie häst viabilitet single layer membranintegritet membranstabilitet kylförvaring |