The primary objective of the studies described in this thesis was to improve the cryopreservation success of koala spermatozoa for the purpose of establishing a genome resource bank for this species. A defining feature of the studies in this thesis was the implementation of an organelle-specific approach to better understand the causes of koala sperm cryo-injury. The functional attributes of spermatozoa, such as mitochondrial function, plasma membrane fluidity, membrane lipid asymmetry and DNA integrity were assessed as an indication of cryo-injury. Sperm mitochondrial function and plasma membrane integrity were examined by cryomicroscopy using the fluorescent probes JC-1 and propidium iodide (PI) respectively in a dual staining technique. Cooling and re-warming koala spermatozoa were more detrimental to mitochondrial function than to plasma membrane integrity. Mitochondrial membrane potential (MMP) was suppressed by freezing and thawing treatments; after thawing, MMP declined significantly during rewarming (from 5ºC to 35ºC). The distribution of GM1 ganglioside was examined using fluorescent-labelled cholera toxin B. No significant redistribution of GM1 was observed after chilling or cryotreatment. The externalisation of phosphatidylserine (PS) was examined using fluorescent-labelled annexin V. There was no significant increase in translocation of PS after chilling or cryopreservation. These observations imply that cryotreatment had little effect on plasma membrane lipid asymmetry. Koala spermatozoa were incubated in a range of anisotonic media to investigate whether nuclear swelling was caused by osmotic flux during the cryopreservation process. Although the most hypotonic solution tested (64 mOsm/kg) induced the highest incidence of nuclear relaxation (mean ± SEM; 12 ± 3%), this was not as severe as that previously documented following cryopreservation. Chromatin relaxation is a phenomenon observed in koala spermatozoa, where the sperm nucleus expands due to the result of structural changes in the natural conformation of the sperm DNA/protamine complex. DNA fragmentation was not a primary cause of cryopreservation-induced sperm chromatin relaxation, although in situ nick translation of putative DNA breaks indicated that these increased as the sperm head became progressively more relaxed. Using a Sperm Chromatin Dispersion test (SCDt) specifically developed and validated for koala spermatozoa, a continuum of nuclear morphotypes was observed, ranging from no apparent DNA fragmentation to spermatozoa with highly dispersed and degraded chromatin. A double comet assay was also developed to investigate DNA fragmentation in the koala spermatozoa. Conducted under neutral followed by alkaline conditions, this assay was able to differentiate between single- (SSB) and double-stranded (DSB) DNA damage in an effort to refine the interpretation of DNA damage in mature koala spermatozoa; the majority of the koala spermatozoa had nuclei with DNA abasic-like residues. The ubiquity of these residues suggested that constitutive alkali-labile sites are part of the structural configuration of the koala sperm nucleus. Spermatozoa with “true” DNA fragmentation exhibited a continuum of comet morphologies, ranging from a more severe form of alkaline-susceptible DNA, to nuclei that exhibited both SSB and DSB. Swelling of koala sperm chromatin following cryopreservation has largely been attributed to the absence of inter-molecular disulphide cross-linkages in the marsupial sperm nucleus. Fish spermatozoa also lack disulphide bonds within their chromatin, but nevertheless, have been successfully cryopreserved. To examine the hypothesis that the cryoprotectants used for fish sperm cryopreservation will confer a similar degree of protection on koala spermatozoa, various concentrations of five cryoprotectants (dimethyl sulphoxide, methanol, propylene glycol, ethylene glycol and dimethylacetamide) were evaluated. Each treatment was compared against an established koala sperm cryopreservation protocol that uses 14% glycerol. Dimethylacetamide at a concentration of 12.5% (v/v) was found to be comparable to glycerol in the successful cryopreservation of koala spermatozoa although high inter-male variability was observed. However, when the new protocol was subsequently validated for a larger population of captive koalas (n = 22), glycerol emerged the better cryoprotectant with respect to all sperm viability parameters assessed except for that of the incidence of chromatin relaxation, which was not affected by the cryoprotectant. Significant difference was also observed in the post-thaw survival of spermatozoa from different animals, which was independent of pre-freeze semen quality. Based on post-thaw semen viability parameters, the koalas could be divided into two distinct groups, where one group had significantly higher sperm viability compared to the other group, regardless of cryoprotectant used. Positive correlation between motility and MMP was observed before and after cryopreservation. However, cryopreservation significantly reduced the dependency between these variables (P < 0.001), suggesting that cryopreservation reduced the dependency between mitochondrial function and motility.

Recent advances in the fundamental theories in cryobiology using thermodynamic principles have created new opportunities for innovative methodologies in cryobiology. This thesis tested the hypothesis that calculated indicators of the two-factor hypothesis of cryoinjury, depending on cellular osmotic properties, will describe outcomes of cryobiological experiments. In addition, this thesis demonstrated that knowledge gained from improved descriptions of cellular osmotic parameters allows better understanding of cryoinjury and cryoprotection. The main objective of this thesis was to develop approaches using simulations that can be applied to development of cryopreservation procedures for cell types of interest for therapies. In order for this approach to be successful, a method to more accurately describe the osmotic solution properties of the cell (i.e. osmolality as a function of molality for the cytoplasm) was developed. Also, in-depth examination into the correlation between predictions of the two types of cryoinjury and measured post-thaw biological outcomes was required. The work presented in this thesis has shown that simulations, based on cell-specific osmotic characteristics, and coupled with interrupted cooling procedures can be used to determine conditions that minimize the two identified damaging factors in cryopreservation. Based on results from this research, both intracellular supercooling and osmolality, as indicators of intracellular ice formation and solution effects injury, respectively, should be calculated when attempting to compare simulations with biological experimentation. This thesis has also shown a novel method of obtaining the solution properties (i.e. osmolality as a function of molality) of the cytoplasm of living cells using equilibrium cell volume measurements. Using these newly calculated parameters, this research also demonstrated the magnitude of error introduced by making dilute solution assumptions of the solution properties in cellular responses to low temperatures, including simulations of interrupted freezing procedures. Overall, the research work presented in this thesis has extended the approach to cryopreservation to include the properties of the cell and the physical conditions of the freezing environment, which was only possible through the linkage between biological experimentation and simulations.

The increasing number of patients undergoing treatment with assisted reproductive techniques (ART) during the past years have led to the need of developing different methods for separation of spermatozoa that can be used for different fertilisation procedures and for freezing. Cryopreservation of spermatozoa includes preparation, freezing, storage and thawing. In this study two different cryomedia (Cryo Protec I and Cryo Protec II) for human spermatozoa were compared. The main outcome was spermsurvival rate for spermatozoa after freezing. Sperm viability was assessed using the Hypo-osmotic swelling test which is based on osmolality. A total of 86 samples of semen were used in this study (Cryo Protec I=38, Cryo Protec II=48). The survival rate between the two cryomedia did not differ much but Cryo Protectant I showed a small increase in survival for the spermatozoa after freezing. The Hypo-osmotic swelling test also showed similar values of viable spermatozoa for the two cryomedia both before and after freezing.

Cryopresevation is the process of cooling biological materials to low sub-zero temperatures for storage purposes. Numerous medical and technical applications, such as hematopoeitic stem cell transplantation and sperm banking, sometimes require the use of cryopreserved cells. Cryopreservation, however, can induce cell injury and reduce the yields of viable functional cells. Ice recrystallization is a mechanism of cryopreservation injury, but is rarely addressd in strategies to optimize cell cryopreservation. The results from this thesis demonstrate an association between the potency of carbohydrate-mediated ice recrystallization inhibition used in the cryopreservation of umbilical cord blood and recovery of viable non-apoptotic cells and hematopoietic progenitor function. Furthermore, increased numbers of apoptotic cells in hematopoeitic stem cell grafts were associated with reduced hematopoietic function and delayed hematopoietic recovery in patients undergoing blood stem cell transplantation. These findings provide a basis for pursuing further studies assessing ice recrystallization inhibition as a strategy for improving cell cryopreservation.

In the last decade, there has been renewed interest in the use of granulocyte transfusions to treat infections in individuals with compromised immune systems. However, granulocytes only remain functional for about a day after isolation and this short shelf life is a significant drawback. Cryopreservation would allow long term storage of granulocytes, but an effective cryopreservation method is currently unavailable. The following study was performed to provide membrane permeability values for multiple cryoprotectants in hopes of aiding the optimization of cryoprotectant addition and removal and minimizing the detrimental effects of the process. The granulocytes were separated from whole blood using centrifugation with Polymorphprep as the separating agent. The cellular membrane permeability values were then measured using a Beckman Coulter Counter Multisizer 3 under custom setup conditions. The cryoprotectants studied were glycerol, DMSO, ethylene glycol, and propylene glycol at the respective total concentrations of 1, 2, 2, 1 Osm/kg at temperatures of 4, 21, and 37 °C. The resulting membrane solute permeability values at 20 °C reference temperature for DMSO, ethylene glycol, glycerol, and propylene glycol were respectively 5.96, 7.84, 0.950, and 3.45 um/min and the Arrhenius activation energies were respectively 60.4, 58.7, 68.2, and 62.3 kJ/mol. The resulting hydraulic permeability values in the same order and temperature were 0.196, 0.189, 0.259, and 0.113 um/(atm min) and the Arrhenius activation energies were respectively 56.3, 60.7, 68.5, and 47.1 kJ/mol. It is anticipated that these permeability values will aid in the development of successful cryopreservation procedures for granulocytes. / Graduation date: 2013

Die hier vorliegende Arbeit gehört zu eine der ersten im deutschsprachigen Raum durchgeführten Studien zu den Einstellungen von deutschen Kinderwunschpaaren im Umgang mit kryokonservierten Eizellen im Vorkernstadium und Embryonen sowie gegenüber der Legalisierung in Deutschland bisher unklar geregelter Verfahren. Insbesondere lag dabei der Fokus auf Einstellungen bezüglich der durch das EschG unklar geregelten Embryonenspende, der verbotenen routinemäßigen Kryokonser-vierung von Embryonen sowie dem weiteren Verbleib überzähliger Embryonen. Dabei wurden im Rahmen einer prospektiven Querschnittsstudie insgesamt 700 Kinderwunschpaare (in Behandlung im Fertility Center Berlin), die im Besitz krykon-servierter Eizellen im Vorkernstadium sind oder waren per standardisiertem Frage-bogen anonym befragt. Männer und Frauen wurden dabei getrennt voneinander befragt. Insgesamt sendeten 272 Patienten den Fragebogen korrekt ausgefüllt zu-rück. In der Mehrheit sprachen sich die befragten Kinderwunschpatienten für eine Legali-sierung der Embryonen- und Eizellspende aus. Außerdem sollte eine routinemäßige Kryokonservierung von Embryonen im Rahmen einer Kinderwunschbehandlung möglich sein. Die Paare würden dabei sog. überzählige Embryonen vorrangig für die eigene Kinderwunschbehandlung verwenden. Allerdings fand sich ebenso eine hohe Akzeptanz gegenüber einer Spende überzähliger Embryonen nach Beendigung des Kinderwunsches sowohl an andere Kinderwunschpaare als auch zu Forschungszwe-cken. Dabei machte es keinen signifikanten Unterschied, um welche Art der For-schung es sich dabei handeln würde (embryonale Stammzellforschung oder repro-duktionsmedizinische Forschung). Interessanterweise votierten jedoch mehr Patien-ten für eine Legalisierung der genannten Verfahren, persönlich dafür entscheiden, würden sich jedoch weniger Paare. Außerdem unterschieden die von uns befragten Patienten nach dem ethisch-moralischen Status gefragt nicht so rigoros wie das EschG zwischen Eizellen im Vorkernstadium und Embryonen. In der Mehrheit stell-ten diese frühen Formen menschlichen Lebens entweder Zellen mit hohem Schutz-anspruch dar oder beide hätten laut der Befragten sogar den Status eines Menschen mit vollem Schutzanspruch. Ob die Kinderwunschpaare dabei allerdings den Eizellen im Vorkernstadium oder Embryonen den höheren Schutzanspruch zugestehen, lässt sich anhand der Ergebnisse nicht zweifelsfrei belegen. Kryokonservierte Embryonen haben laut der Paare einen leicht höheren Schutzanspruch, in dem die Befragten diese eher als vollwertige Menschen gesehen haben. Insgesamt bleibt also festzuhalten, dass die Einstellungen der von uns befragten Kinderwunschpaare bezüglich früher Formen menschlichen Lebens nicht immer deckungsgleich sind mit denen des EschG. Die Paare sprechen sich mehrheitlich für eine Legalisierung verbotener Verfahren im Rahmen einer Kinderwunschbehandlung in Deutschland aus und unterscheiden in Bezug auf den moralischen Status nicht signifikant zwischen Eizellen im Vorkernstadium und Embryonen. Da diese Studie allerdings den Charakter einer Pilotstudie darstellt, konnten keine Einflussfaktoren ermittelt werden, die Daten stammen aus einem vorselektierten Kollektiv und müssen daher vorsichtig interpretiert werden. Nichtsdestotrotz können die Ergebnisse einen wertvollen Beitrag in der Diskussion um die Novellierung des EschG bzw. um die Notwendigkeit eines sog. „Fortpflanzungsmedizingesetzes“ leisten und damit zu einer Verbesserung der Ergebnisse, Effektivität und Sicherheit der Patienten im Rahmen der assistierten Reproduktion in Deutschland bieten.

Cryopresevation is the process of cooling biological materials to low sub-zero temperatures for storage purposes. Numerous medical and technical applications, such as hematopoeitic stem cell transplantation and sperm banking, sometimes require the use of cryopreserved cells. Cryopreservation, however, can induce cell injury and reduce the yields of viable functional cells. Ice recrystallization is a mechanism of cryopreservation injury, but is rarely addressd in strategies to optimize cell cryopreservation. The results from this thesis demonstrate an association between the potency of carbohydrate-mediated ice recrystallization inhibition used in the cryopreservation of umbilical cord blood and recovery of viable non-apoptotic cells and hematopoietic progenitor function. Furthermore, increased numbers of apoptotic cells in hematopoeitic stem cell grafts were associated with reduced hematopoietic function and delayed hematopoietic recovery in patients undergoing blood stem cell transplantation. These findings provide a basis for pursuing further studies assessing ice recrystallization inhibition as a strategy for improving cell cryopreservation.

The Amyloodinium ocellatum was collected from cobia ( Rachycentron canadum ) gill and four tests including 4 ¢J storage, toxicity of cryoprotectant, cryopreservation and in vitro cultivation on fish cell line were conducted to establish the methods of preservation of Amyloodinium ocellatum. Survival of trophont, morphology and division of tomont and number of dinospore released were evaluated the effects of this study. The results showed that division irregulated, delayed and stopped of the tomont were found after stored at 4 ¢J over 48 hours. It was produced 1.08 x 10 4 cell/ml dinospores from 1 x 10 3 trophont at 4 ¢J, 24 hours storage group and significant higher ( p¡Õ0.0001 ) than other storage groups. For the toxicity of cryoprotectant, the concentration of DMSO 3~10¢M, Glycerol 3~10¢M, Methanol 3~10¢M, Ethanol 3~5¢M, PrOH 3~5¢M, DMAc 3~5¢M, Sucrose 3~15¢M, Trehalose 3~15¢M, Dextran 3~5¢Mand Ficoll 3~10¢Mwere safety to use on A. ocellatum trophont preservation. It was unsuccessful to cryopreserve the trophont of A. ocellatum when stored at direct liquid N2 freezing, different -20 ¢J freezing time, -1 ¢J min-1 freezing container and different cryoprotectant equilibration time contain 10¢MGlycerol and DMSO, respectively. Using the U-shaped tube of sigle and double loop could gain pure and bacteria-free dinospores. The results of in vitro cultivation of A. ocellatum showed that eel epidermis and cobia fin cell line with different culture mediums were unable to grow the trophont and tomont of A. ocellatum.

Thesis (M.S.)--University of Missouri-Columbia, 2008. / "May 2008" The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Includes bibliographical references.

Thesis (Ph. D.)--University of Alberta, 2010. / Title from pdf file main screen (viewed on Jan. 15, 2010). A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Chemical Engineering and Medical Sciences, Departments of Chemical and Materials Engineering and Medical Sciences - Laboratory Medicine and Pathology, University of Alberta. Includes bibliographical references.