In the first part of the our studies we showed that whereas pre- and post-freeze membrane integrity and motility show a clear correlation, pre-freeze hypotonic resistance normalized towards the initial number of viable sperm does not correlate with sperm motility after cryopreservation. We thus conclude that pre-freeze hypotonic resistance cannot be used as a parameter to foresee sperm cryosurvival rates.
The second part of our studies showed that density centrifugation protocols can be used to select a subpopulation, which exhibits increased percentages of morphologically normal and progressively motile sperm with increased membrane and chromatin intactness.
Also, for use for delayed cryopreservation density centrifugation yielded increased numbers of high quality sperm when compared to diluted or ordinary centrifuged sperm. Performing density centrifugation directly after collection was found to result in higher cryosurival rates as compared to performing density centrifugation after storage just prior to cryopreservation.
Thus, for delayed cryopreservation it is best to perform density centrifugation processing directly after collection.
6 SUMMARY
Anna Heutelbeck (2013)
Cryopreservation of stallion sperm: correlating hypo-osmotic resistance and cryosurvival, and use of density centrifugation for delayed cryopreservation
Cryopreserved sperm from individual stallions display a high degree of variation in survival after thawing. During freezing and thawing, sperm are exposed to osmotic stress when the osmolality of the unfrozen fraction increases or decreases. We hypothesized that the ability of sperm to cope with osmotic stress correlates with its survival after cryopreservation.
The aim of the first part of the studies described in this thesis was to evaluate variation in osmotic properties of sperm from different stallions and correlate this with survival after cryopreservation. In addition, temperature effects on hypotonic resistance were studied. Pre- and post-freeze motility and plasma membrane integrity were determined using computer assisted sperm analysis and flow cytometry, and taken as a measure for freezability.
Hypotonic resistance was determined by evaluating percentages of plasma membrane intact sperm after exposure to saline solutions of decreasing osmolality, and the critical osmolality was calculated as the osmolality at which half of the sperm population survived hypotonic stress. Stallion sperm exhibited the greatest resistance towards hypotonic stress in the 15 to 30
oC temperature range. For incubations at 22 oC, the critical osmolality was found to vary between 55 to 170 mOsm kg-1 amongst individual stallions. Whereas pre- and post-freeze membrane integrity and motility showed a clear correlation, pre-freeze hypotonic resistance normalized towards the initial number of viable sperm did not correlate with sperm motility after cryopreservation. We thus conclude that pre-freeze hypotonic resistance cannot be used as a parameter to foresee sperm cryosurvival rates.
The infrastructure needed for cryopreservation of stallion sperm is not always available. In these cases, diluted semen needs to be shipped to a facility where cryopreservation can be done typically after 1 day storage at 5 oC. Centrifugation processing of semen, including density centrifugation, is generally used for selection of high quality sperm for use in artificial insemination. The aim of the second part of the studies described in
this thesis was to evaluate if selection of sperm via density centrifugation yields higher cryosurvival rates when performing delayed cryopreservation. Two-layer iodixanol as well as single-layer Androcoll density centrifugation were tested, and compared to ordinary centrifuged and diluted samples. Centrifugation processing directly after semen collection prior to refrigerated storage was compared with centrifugation of sperm after refrigerated storage for 1 d just prior to performing delayed cryopreservation. Sperm morphology and motility as well as membrane and chromatin integrity were evaluated before and after centrifugation processing. Sperm motility and membrane integrity were also assessed after cryopreservation. It was found that density centrifugation processing resulted in increased percentages of morphologically normal and motile sperm with increased membrane and chromatin intactness, as compared to ordinary centrifuged or diluted samples. For delayed cryopreservation, processing of sperm directly after semen collection prior to storage did yield higher post-freeze percentages of plasma membrane intact and motile sperm as compared to processing of stored sperm prior to cryopreservation. Two-layer iodixanol density centrifugation resulted in the highest percentages of plasma membrane intact sperm, whereas single-layer Androcoll density centrifugation resulted in the highest percentages of progressively motile sperm. Taken together, for delayed cryopreservation it is best to perform density centrifugation processing directly after collection.
7 ZUSAMMENFASSUNG
Anna Heutelbeck (2013)
Kryokonservierung von Hengstspermien: Korrelation von hypo-osmotischer Resistenz und Überlebensrate nach dem Auftauen und Einsatz der Dichtezentrifugation bei der Kryokonservierung gelagerten Spermas
Die Gefriertauglichkeit der Spermien unterschiedlicher Hengste unterliegt einer großen Variabilität. Der Prozess des Einfrierens und Auftauens ist mit Veränderungen des osmotischen Druckes verbunden. Darauf basierend galt es zu überprüfen, ob die Reaktion der Spermien auf osmotischen Stress mit ihrer Gefriertauglichkeit korreliert. Das Ziel des ersten Teils dieser Dissertation war zum einen der Vergleich der osmotischen Eigenschaften der Spermien verschiedener Hengste und zum anderen die Korrelation dieser Eigenschaften mit der Überlebensrate der Spermien nach dem Auftauen. Zudem wurde der Einfluss der Temperatur auf die hypotone Resistenz der Spermien überprüft. Als Maß für Gefriertauglichkeit der Spermien wurde ihre Plasmamembranintegrität (ermittelt mit Hilfe des Durchflusszytometers) und Motilität (ermittelt mit Hilfe der computer-assistierten Spermienanalyse) nach dem Auftauen herangezogen. Zur Bestimmung der hypotonen Resistenz erfolgte eine Inkubation der Spermien in Salzlösungen mit absteigenden Osmolaritäten und eine darauf folgende Ermittlung des Anteils plasmamembranintakter Spermien. Die kritische Osmolarität, welche definiert ist als die Osmolarität, die 50 % der Spermien überleben, wurde als zusätzlicher Parameter ermittelt. Der Temperaturbereich, bei dem die Spermien hypotonen Stress besonders gut tolerierten, lag zwischen 15 und 30 °C. Bei einer konstanten Inkubationstemperatur von 22 °C zeigte sich, das die kritische Osmolarität zwischen den Hengsten von 55 bis 170 mOsm kg-1 variierte. Während ein deutlicher Zusammenhang zwischen dem Anteil plasmammembranintakter und motiler Spermien vor und nach dem Einfrieren bestand, korrelierte die hypotone Resistenz vor dem Einfrieren nicht mit der Motilität der Spermien nach dem Einfrieren. Daraus ergibt sich, dass die hypotone Resistenz vor dem Einfrieren kein geeigneter Parameter ist, um die Gefriertauglichkeit von Hengstspermien vorherzusagen.
Die für die Herstellung von Tiefgefriersperma erforderliche Ausrüstung ist nicht auf jeder Besamungsstation vorhanden. Sperma, welches der Tiefgefrierung zugeführt werden soll, muss somit gegebenenfalls gekühlt an ein Labor, in welchem die Tiefgefrierkonservierung innerhalb von einem Tag nach der Samengewinnung durchgeführt werden kann, versandt werden. Im Rahmen der künstlichen Besamung wird das Verfahren der Dichtezentrifugation zur Selektion qualitativ hochwertiger Spermien angewandt. Der zweite Teil dieser Dissertation dient der Evaluierung des Nutzens der Dichtezentrifugation im Hinblick auf die Herstellung von Tiefgefriersperma nach eintägiger gekühlter Lagerung.
Hierfür wurden die Verfahren der Zweischicht-Iodixanol-Dichtezentrifugation und Einschicht-Androcoll-Dichtezentrifugation mit den Verfahren der herkömmlichen Zentrifugation und der Verdünnung verglichen. Zusätzlich wurde der Einsatz der Dichtezentrifugation vor der Lagerung des verdünnten Ejakulates, im Vergleich zu deren Einsatz nach der Lagerung evaluiert. Die Qualität der Spermien vor und nach dem jeweiligen Verfahrensschritt wurde anhand ihrer Morphologie, Motilität, Membran- und Chromatinintegrität bestimmt. Nach dem Einfrieren wurden Motilität und Membranintegrität der Spermien bestimmt. Der Einsatz von Dichtezentrifugation-Verfahren resultierte im Vergleich zur Verdünnung oder herkömmlichen Zentrifugation, in einem erhöhten Anteil morphologisch normaler, motiler, membranintakter und chromatinintakter Spermien. Fand die Zentrifugation des Ejakulates direkt nach der Gewinnung statt, wurden höhere Überlebensraten nach dem Einfrieren erzielte als bei Zentrifugation des gelagerten Ejakulates unmittelbar vor der Tiefgefrierung. Unabhängig davon erzielte die Zweischicht-Iodixanol-Dichtezentrifugation die höchsten Anteile plasmamembranintakter Spermien und die Einschicht-Androcoll-Dichtezentrifugation die höchsten Anteile progressiv motiler Spermien.
Zusammenfassend erhöht der Einsatz von Dichtezentrifugation-Verfahren am Tag der Samengewinnung die Überlebensraten von Spermien, die nach eintägiger gekühlter Lagerung tiefgefroren wurden.
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