Cryopreservation of gametes is a valuable tool for the fast-growing aquaculture industry in New Zealand. In the present study, research was aimed to improve the cryopreservation of Pacific oyster (Crassostrea gigas) oocytes. For this, two main approaches were used: the modification of an existing published (standard) cryopreservation protocol for oyster oocytes and the modification of the oocytes themselves prior to cryopreservation. The objectives in the chapters of this thesis were: (a) determination of the cryobiological characteristics of oyster oocytes; (b) assessment and reduction of intracellular ice formation (IIF) in oocytes; and (c) modification of the lipid fraction (cholesterol and fatty acids) of oocytes prior to cryopreservation. Knowledge of the membrane permeability parameters in response to concentrations of water and ethylene glycol (EG), the influence of temperature upon these parameters, and the osmotic tolerance limits of oyster oocytes were used to develop computer models that simulated the cellular volume changes that oocytes underwent during EG addition and removal. The models predicted that when one part of EG was added in one step to one part of oocyte suspension and equilibrated for 20 min at 20 �C, similar volume changes in oocytes would be obtained, compared to a more complicated multi-step addition method. This method of addition resulted in similar post-thaw fertilization rates to those obtained by using the multi-step addition method, thus reducing oocyte handling. Cryomicroscopy was used to assess the effect of cooling rates and EG concentration on the temperature at which oocytes underwent IIF. It was found that IIF occurred at higher subzero temperatures when fast cooling rates were used (30 and 5 �C min⁻�) and at EG concentrations ranged between 0 and 15%. At a relatively slower cooling rate of 0.3 �C min⁻� and with 10% EG, which are the conditions employed in the standard cryopreservation protocol, no IIF occurred. The steps of the standard protocol that were more likely to cause oocyte damage were identified by evaluating the fertilization rate of oocytes at each step. Results showed that oocytes were most damaged by cooling them to -35 �C and followed by plunging them in liquid nitrogen. Contrary to what had been observed under the cryomicroscope, transmission electron microscopy (TEM) analysis revealed that all oocytes cryopreserved by the standard protocol contained cytoplasmic ice. In addition, it was also observed that oocytes were at two developmental stages when frozen (prophase and metaphase I). These observations prompted the development of alternative cooling programmes aimed to reduce intracellular ice. The effect of cooling rate, plunge temperature and time held at the plunge temperature were thus evaluated, based on post-thaw fertilization rate of oocytes. Overall, neither the cooling rate nor the holding time had an effect on oocyte fertility. However, the plunge temperature had an effect, where oocytes plunged at -60 �C had lower post-thaw fertilization rates than oocytes plunged at -35 �C. Through the slowing of the cooling rate, lengthening of the holding time and lowering of the plunge temperature, it was possible to reduce the amount of ice in the cytoplasm. However, the reduction of intracellular ice did not improve the post-thaw fertilization rate of the oocytes; on the contrary, post-thaw fertilization decreased notoriously. From these results, it can be suggested that oyster oocytes are more likely to be damaged by exposure to high intra and extracellular solute concentration than IIF during cryopreservation. In an effort to modify the lipid content of oyster oocytes prior to cryopreservation and thus, making them more resistant during cryopreservation, oocytes were incubated in solutions that would add or remove cholesterol or in solutions rich in long chain fatty acids (EPA or DHA). Oocytes incubated in cholesterol-rich solutions showed a positive uptake of fluorescently labelled cholesterol and this effect was dose dependent. Nevertheless, this uptake did not improve the post-thaw fertilization rate nor did it increase the total cholesterol content of the oocytes. When oocytes were incubated in non-conjugated or conjugated EPA or DHA, no increase in the proportion of these fatty acids was identified in the fatty acid profiles of whole oocytes and no improvement of the post-thaw fertilization rate was recorded. Given that there was no uptake of fatty acids from the incubation media by the oocytes, a different approach was taken. This involved the supplementation of lipid-rich diets to the oyster broodstock during gametogenesis (cold-conditioning) and vitellogenesis (warm-conditioning). Despite results showing that lipid content and, indeed, fatty acid profile was altered through the diet, the results also showed that fresh oocytes from broodstock fed during cold-conditioning did not show any improvement in their fertilization rates, nor did they benefit from a lipid-rich diet during warm-conditioning. On the other hand, cryopreserved oocytes did have higher post-thaw fertilisation rates when broodstock were fed during cold-conditioning and, although no effect was found from feeding broodstock with either of the lipid-rich diets during warm-conditioning, trends indicated that a diet consisting of fresh microalgae or the commercial supplement Algamac would yield the highest post-thaw fertilization rates. This thesis has furthered the understanding of some of the factors that determine cryosurvival in oyster oocytes and has demonstrated that both physical and biological issues must be taken into consideration for cryopreservation. Specifically, the results in this thesis helped to modify an empirically developed cryopreservation protocol for Pacific oyster oocytes. In addition, the results also showed strong evidence of the survival of oyster oocytes to intracellular ice and highlighted the importance of supplying the broodstock with lipid-rich food during the periods of gamete formation and maturation in order to obtain oocytes that are more amenable to cryopreservation. These benefits could be of significant practical importance and may be extended for the development or refinement of cryopreservation protocols for other shellfish species of commercial importance to the aquaculture industry of New Zealand.

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.

Research Doctorate - Doctor of Philosophy (PhD ) / In Australia almost 40% of the carnivorous marsupials, or dasyurids, are threatened. Assisted reproductive techniques (ART), especially genome resource banking, have the potential to contribute to the conservation of these species by reducing the loss of genetic diversity. This project aimed to advance the knowledge of ART in dasyurids by focusing on the long term preservation of male and female gametes and establishing protocols for the production of mature oocytes for use in future ART. These studies used the fat tailed dunnart (Sminthopsis crassicaudata) as a model dasyurid and replicated many of the findings on threatened dasyurids. Dasyurid spermatozoa had a relatively unstable acrosome which lacked acrosomal membrane disulphide stabilisation. There was no evidence that S. crassicaudata spermatozoa were susceptible to high concentrations of cryoprotectants, but spermatozoa frozen with up to 40% glycerol using a rapid freezing protocol were not viable. Nonetheless the morphology and acrosomal integrity of frozen spermatozoa was normal and there was no evidence of DNA damage. The lack of success with cryopreservation is likely to be an artifact of cold shock, which was observed in S. crassicaudata and had not previously been described in any other marsupial. This susceptibility to low temperature can be overcome by slow cooling spermatozoa to 0 ºC at 0.5 ºC minute -1 with up to 20% egg yolk, and it is likely that this finding will result in successful sperm cryopreservation in the near future. Freeze drying spermatozoa represents an additional strategy for long term sperm preservation and freeze dried S. crassicaudata spermatozoa had normal morphology and nuclear integrity. In this study preserved dasyurid spermatozoa were immotile and non-viable but had no nuclear damage, suggesting that fertilisation may be achieved with intracytoplasmic sperm injection (ICSI). As ICSI requires a large number of mature oocytes to be collected, a reliable timed ovarian stimulation protocol was established in S. crassicaudata. This protocol enabled the collection of up to 28 oocytes which were either mature, or able to be cultured to the first polar body stage within 48 hours. Despite the success of induced ovulation, methods for preservation of the female gamete are essential to genome resource banking. This study also described a protocol for the enzymatic dissociation of dasyurid ovarian tissue allowing collection of high quality individual preantral follicles. The oocytes inside these follicles were able to be vitrified without any loss of viability and short term in vitro culture of immature follicles repaired the small amount of vitrification-induced damage to the surrounding granulosa cells. This collection of studies describes progress in genome resource banking for spermatozoa and oocytes from dasyurids and the development of protocols allowing the collection of a large number of oocytes for use in fertilisation experiments. These advances provide a solid and comprehensive framework for continuing the study of dasyurid ART which is timely due to the urgent need for genome resource banking in several threatened dasyurid marsupials.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 4 uppsatser.

Thesis (M. Phil.)--University of Hong Kong, 2008. / Includes bibliographical references (leaves 151-192) Also available in print.

Thesis (M. Phil.)--University of Hong Kong, 2008. / Includes bibliographical references (leaves 151-192) Also available online.

Thesis (M.S.)--University of North Texas, Dec., 2009. / Title from title page display. Includes bibliographical references.

Thesis (M. S.)--Biomedical Engineering, Georgia Institute of Technology, 2008. / Committee Chair: Karlsson, Jens; Committee Co-Chair: Nerem, Robert; Committee Member: Meda, Paolo; Committee Member: Prausnitz, Mark; Committee Member: Sands, Jeff; Committee Member: Zhu, Cheng.

Thesis (MScMed)--University of Stellenbosch, 2008. / Bibliography. Also available via the Internet.

Made available in DSpace on 2014-08-13T14:50:33Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-05-02Bitstream added on 2014-08-13T18:01:15Z : No. of bitstreams: 1 000732142.pdf: 1071161 bytes, checksum: 2cb970afdbdc6d295fe573aeb86e30a8 (MD5) / Apesar dos benefícios proporcionados pelo plasma seminal durante a cobertura, quando biotecnologias são utilizadas, como a inseminação artificial, sua função é questionável, visto que trabalhos utilizando espermatozoides da cauda do epidídimo acrescido ou não do plasma seminal não apresentaram diferença nas taxas de concepção. Além disso, estudos relataram efeito deletério das secreções das glândulas anexas sobre a viabilidade espermática e taxa de concepção, principalmente em garanhões com baixa qualidade seminal. Neste sentido, o objetivo do presente estudo é: 1) comparar os parâmetros de cinética espermática, viabilidade estrutural e fertilidade dos espermatozoides recuperados do ejaculado e da cauda do epidídimo de garanhões subférteis; 2) avaliar diferenças na composição eletrolítica do plasma seminal de animais com alta e baixa fertilidade. 3) comparar o efeito da adição de plasma seminal de garanhões de alta e baixa fertilidade sobre a congelabilidade e viabilidade dos espermatozoides do ejaculado e da cauda do epidídimo de garanhões subférteis. As avaliações das amostras seminais consistiram na análise computadorizada das características da motilidade (CASA, Thorne Research - HTM IVOS) e nas análises de citometria de fluxo da integridade da membrana plasmática, da integridade acrossomal, da peroxidação das membranas espermáticas, da fragmentação do DNA, da capacitação espermática, da translocação de fosfatidilserina da membrana e da ativação de caspase. Os espermatozoides do epidídimo após descongelação apresentaram superioridade nos parâmetros de cinética e viabilidade espermática, além de melhor fertilidade, demonstrando que estes espermatozoides são mais resistentes ao processo de congelação quando comparado aos espermatozoides do ejaculado. O plasma seminal de garanhões subférteis apresentou maiores concentrações de sódio ... / Despite the benefits provided by seminal plasma during natural cover when biotechnology are used as artificial insemination, its function is questionable, since studies using spermatozoa from cauda epididymis with or without the addition of seminal plasma showed no difference in conception rates. Furthermore, studies have reported deleterious effects of secretions from accessory glands on sperm viability and conception rate, especially in stallions with poor semen quality. Thus the aims of this study were: 1) compare the kinetic parameters, structural viability and fertility of sperm recovered from ejaculate and cauda epididymis of subfertile stallions. 2) evaluate differences in electrolyte composition in the seminal plasma of animals with high and low fertility. 3) compare the effect of seminal plasma addition from stallions with high and low fertility on freezability and viability of sperm from the ejaculate and cauda epididymis of subfertile stallions. The assessment of each samples was determined by computer-assisted sperm analyses of motility (CASA, Thorne Research - HTM IVOS) and flow cytometry analyses of plasma membrane integrity, acrosome integrity, peroxidation of sperm membranes, DNA fragmentation index, degree of protein tyrosine phosphorylation in sperm surface and caspase activity. The epididymal sperm after thawing showed superiority in kinetic parameters and sperm viability, as well as better fertility, demonstrating that these spermatozoa are more resistant to freezing process when compared to sperm from the ejaculate. The seminal plasma of subfertile stallions has higher sodium concentrations in the seminal plasma than fertile stallions, what makes this finding a possible way to explain the deleterious effect of seminal plasma in these animals. Moreover, it was found that this deleterious effect is dose-dependent since the addition of 20% of seminal plasma from fertile and ...