Crypreservation of oocytes is recently being considered to be a valid choice in infertility treatments.Low survival and fertilization rates due to inefficient slow freeze protocols have been the outcome ofmany previous studies done in the field. However, introduction of the vitrification technique and itsapplication in reproductive medicine and to some extent new improved slow freeze protocols haveshown that oocytes can be cryopreserved with successful outcome. In this project the survival rate of oocytes after vitrification with MediCult Vitrification andWarming Media has been studied. Also, a comparison of the carriers Cryoloop (an open system) andCryopette (a closed system) has been performed. A total of 43 oocytes were vitrified and warmed according to MediCult's protocol, of which 21oocytes with Cryoloop and 22 with Cryopette. The cells were post-thaw incubated in a physiologicalenvironment for 24h. During that time the morphology and viability were observed and noted after 2h,over night and after 24h. No significant difference in survival rates of the oocytes' was observed usingthe two carriers, and the total survival rate of the oocytes was 83.7%. This indicates thatcryopreservation of oocytes is a valid choice in fertility treatments and that the new product Cryopettecan be used clinically with just as good results as the well established Cryoloop technique. This merits further studies on the applicability of vitrification on oocytes and the Cryopette technique.

Thesis (M.S.)--Virginia Polytechnic Institute and State University, 1991. / Vita. Abstract. Includes bibliographical references (leaves 111-116). Also available via the Internet.

An alarming number of anuran (frog and toad) species are facing the threat of extinction in the wild. In efforts to address this conservation crisis, captive breeding programs are rapidly being established at zoos and research institutions worldwide. However, the captive management of anurans can be challenging, as their reproduction is a tightly regulated hormonal response to environmental stimuli, often unknown or absent in captivity. Consequently, ex-situ breeding efforts tend to be greatly hindered by a paucity of knowledge in anuran reproductive physiology and, for many species on the brink of extinction, time is running out. Assisted reproductive technologies (ARTs), such as exogenous hormone induction of gamete release, artificial fertilization for population augmentation, and cryopreservation for the long-term storage of genetics, have the potential to greatly enhance captive breeding efforts in lieu of natural breeding. Broadly, research aims were to develop assisted reproductive technologies for captive populations of the declining Southern Rocky Mountain boreal toad (Anaxyrus boreas boreas), the critically endangered Mississippi Gopher Frog (Lithobates captio sevosa) and Puerto Rican crested toad (Peltophryne lemur). Specific objectives were to a) trial the efficacy of exogenous hormone treatments on sperm release in male target species by characterizing spermiation response across time; b) investigate methods for increasing sperm longevity through cold-storage and cryopreservation techniques; c) ascertain motility recovery rates and functional capacity of cold-stored and frozen-thawed spermatozoa through artificial fertilization techniques, and; d) apply successfully developed ARTs to determine the feasibility of genetically linking in-situ and ex-situ populations of A. b. boreas, through artificial fertilization of male and female gametes from wild and captive toads, respectively. Research outcomes from this study include the successful development of exogenous hormone protocols, spermiation profiles and sperm cryopreservation techniques for all target species. Additionally, these studies enabled validation of an alternative method for increasing genetic diversity in captive anurans through in-situ-ex-situ gamete linkage. Overall, this research emphasizes the potential value of assisted reproductive technologies as conservation tools for supporting the recovery of endangered frog and toad species worldwide.

The objectives of this study were to determine fatty acid composition and acrosome status from bull sperm with different freezabilities (n = 12). We hypothesized that lipid fractions had differentiated fatty acid compositions and such differences influence sperm freezability and the sperm acrosome. Fatty acids were extracted from fresh frozen sperm and fractionated by solid-phase extraction. Thirtyour fatty acids were quantified. Saturated fatty acids were predominant, accounting for 71 to 80% of fatty acids in both fractions. Differences in composition between fractions existed (P < 0.001). Branched chain fatty acid concentrations (15 to 18 µg) were almost twice that of polyunsaturated fatty acid concentrations found in the polar fractions (8 to 9 µg; P < 0.001). Sperm with differentiated freezabilities had few differences in 22:0, 18:1 cis 9, and 14:0 13-methyl fatty acids (P ≤ 0.011). Analyses of acrosome status of sperm revealed that acrosomes were affected differently among bulls.

The world is currently facing an amphibian extinction crisis and wild salamanders and newts (Order: Caudata) are the most disadvantaged with 52% of species threatened. Captive breeding programs are been established to act as assurance colonies, but they are overwhelmingly failing due to the lack of environmental cues to stimulate reproduction and other factors. Therefore, assisted reproductive technologies (ART) are being developed to overcome these barriers. This project expands upon the limited existing information on caudate ART through studies in the model species, Ambystoma tigrinum, for application to threatened species. Specific objectives included the characterization of motility longevity in artificially collected sperm samples, investigation of cryoprotective agents and freeze rates in sperm cryopreservation, and application of ART in three at-risk newt species. This study informs needed future advances in caudate ART protocols, especially sperm cryopreservation, and demonstrates their transferability to threatened species across families.

Successful efficient whole cauliflower plant regeneration via somatic embryogenesis from root derived callus tissue was achieved. The research confirmed for the first time the capability of mass production of cauliflower somatic embryos through the indirect pathway. The best callus induction and proliferation was on semi solid Murashige and Skoog (MS) medium supplemented with 2, 4-D at 0.15 mg L-1 and Kinetin at 0.1 mg L-1 and 3% sucrose. The response of different explant types (cotyledon, hypocotyls and root) through callus induction and subsequent culture was determined. The best period for subsequent callus culture was 21 days. Continuous immersion in agitated liquid medium technique was subsequently used for primary somatic embryo production. The culture requirements were empirically optimized including: explants source and size of callus tissue, blending duration, plant growth regulator combinations and concentrations as well as carbohydrate type and concentration. The highest mean number of somatic embryos (30.9) per explant was achieved using root derived embryogenic callus tissue on MS medium provided with IAA 0.05 mgL-1 and Kinetin at 0.5 mgL-1 and 2% sucrose. Somatic embryos were developed and matured on this medium and germinated with the highest percentage (60%) on semi-solid MS medium devoid of growth regulators. The culture conditions that led to the formation of secondary somatic embryos were identified. The presence of activated charcoal in the culture medium had an effect on this process but some abnormality of secondary somatic embryos was observed. Artificial seeds were produced by encapsulating the somatic embryos with a sodium alginate gel (2%) and complexing with calcium chloride (100 mM) for 20 min. The ability of these artificial seed for germination was evaluated using various combinations of plant growth regulators that were either incorporated in the artificial matrix or in the germination semi-solid culture medium. It was confirmed that cauliflower root derived embryogenic callus tissue can be cryopreserved following a preculture-dehydration technique. Following cryopreservation, embryogenic cultures can proliferate in agitated liquid medium, and somatic embryos at the globular stage were formed. Also cold storage at 5 °C in the dark was used successfully to store cauliflower callus tissue for three months without diminution of the competence for somatic embryos formation. This ability for cold storage could have a positive effect in reducing costs and efforts that result from subsequent sub-culture. The encapsulation-dehydration technique was assessed for cryopreservation of somatic embryos but failed to lead to survival of any embryos. Somatic embryos that were produced in this study were able to be well acclimated using a reliable weaning procedure that achieved high rates of survival of plantlets and their subsequent growth to normal plants in the field was assessed. Morphological characteristics of somatic plants compared favourably with zygotic plants but although there was phenotypic similarity, some differences in plant height, curd size and time for curd maturity were observed.

Cryopreservation of fish embryos has proven to be a difficult problem in cryobiology. Three main difficulties have been identified or suspected: 1) embryo membrane permeability barriers to cryoprotectants and water; 2) high chilling sensitivity of the embryos; and 3) the twocompartment nature of the embryos with a large yolk. Using the zebrafish embryo as a model system, these limiting factors and possible approaches to overcoming them were investigated with a view to developing an effective procedure for fish embryo cryopreservation. Compared to previous studies, vitrification of zebrafish embryos on gold electron microscope grids using methanol as the cryoprotectant resulted in improved morphological survival, being -50% for early stage (I-cell and 64-cell) and ~ 80% for late stage (50%epiboly, 6-somite and prim-6) embryos, but no embryo showed viability. Poor cryoprotectant permeation and embryo dehydration, and consequently intraembryonic ice formation, remained as the main problem for vitrification. Embryo chilling sensitivity studies suggested that later stage zebrafish embryos were sensitive to cold shock injury arising from rapid cooling followed by being held for an extended exposure period (1 h) at 0 or -5°C. Studies on embryo developmental arrest by anoxia showed that chilling injury in zebrafish embryos was probably not associated with their high development rate. However, the chilling sensitivity of zebrafish embryos was found to be related to the amount of yolk present. Yolk-reduced embryos at prim-6 and high-pec stages became less sensitive to chilling at O°C. Differential scanning calorimetry studies on the depression of intraembyonic nucleation temperatures by cryoprotectants revealed that multi-punctured embryos at 6-somite and prim-6 stages became significantly more permeable to methanol and propylene glycol when compared with their nonpunctured controls. Puncturing of the yolk-sac of fish embryos to reduce yolk content, and the increased permeability to cryoprotectants that this promotes, may offer a new approach to surmounting the difficulties confronting the cryopreservation of fish embryos.

Cryopreservation of fish embryos was studied using zebrafish embryo as a model system. Cryoprotectant toxicity, chilling sensitivity and embryo membrane permeability were investigated with a view to developing optimum protocols for cryopreservation of the embryos using controlled slow cooling, non-freezing storage and vitrification. Methanol was found to be the most effective cryoprotectant for controlled slow cooling and non-freezing storage of zebrafish embryos with 11 %heart beat stage embryos surviving after controlled slow cooling to -25°C. Zebrafish embryos were found to be very chilling sensitive with early developmental stages being the most sensitive to chilling injury. Embryo developmental stages after closure of the blastopore (>12-h), especially post heart beat stages were much more resistant to cryoprotectant toxicity and chilling injury. Heart beat stage (27-h) embryos proved to be the best embryo developmental stage for controlled slow cooling and non-freezing storage. Dechorionated embryos are more sensitive to cryoprotectant toxicity and chilling injury. The sensitivity to chilling injury of zebrafish embryos limited the use of controlled slow cooling and non-freezing storage for long term cryopreservation. The attempts at cryopreservation of zebrafish embryos using vitrification produced no embryo survival, although up to 32 % embryos remained morphologically intact immediately after vitrification. Poor cryoprotectant permeation, dehydration and consequently ice formation within the egg are probably the main factors on effecting embryo survival. The results of zebrafish embryo permeability studies demonstrated that the chorion of the embryos was permeable to water and cryoprotectants, whilst the vitelline (plasma) membrane was an effective permeability barrier. The inability to achieve sufficient penetration of the vitelline membrane by cryoprotectants poses severe problems for long term cryopreservation, which need to be overcome, possibly by permeabilisation of the vitelline membrane, before successful cryopreservation can be achieved.

Master of Science / Department of Anatomy and Physiology / Mark Weiss / Artificial insemination and cryopreservation of sperm are important components of any transgenic animal facility because they allow for the reduction in animal colony size and the safe storage of germplasm from valuable strains. In addition, they allow long-term storage of these strains and easy transportation of the genetic material to other research facilities internationally. Thus far, only one laboratory has created live rat pups after sperm cryopreservation and intrauterine insemination. Another laboratory made advances in cryopreservation media that improved sperm motility post-thawing, but no pups resulted from this work. In my study, these two cryopreservation media were utilized to perform intrauterine inseminations with both fresh samples of rat sperm as well as samples that were cryopreserved in liquid nitrogen to replicate and extend these studies. Pharmacoejaculation was tested as a means to obtain spermatozoa without euthanizing the male to collect the epididymis, but results were inconsistent and the samples were not useful for intrauterine inseminations or cryopreservation. Epidiymal sperm was then collected into the various media and frozen in liquid nitrogen. In my hands, the frozen/thawed rat sperm achieved motility of less than 1%. Next, the impact of altering the freezing rate on sperm motility was evaluated. Epididymal sperm was collected and processed using a modified protocol and were then frozen at 2, 4 or 6 cm above the level of liquid nitrogen. Four to six days after freezing, samples were thawed and post-thaw sperm motility was evaluated. Sperm motility was measured prior to freezing as well as after-thawing. The sperm motility was correlated with LIVE/DEAD® staining. Sperm motility did not differ between the groups as a result of the freezing rate (Friedman test p=0.23). The published techniques are not robust and require further development to improve the motility of rat sperm after cryopreservation and achieve pregnancy via intrauterine insemination.

Dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science. School of Animal, Plant and Environmental Sciences, University of the Witwatersrand, Johannesburg, South Africa / In the forestry industry the requirement for the maintenance of a broad genetic base is integral to the success of breeding programmes such as those for Eucalyptus grandis, an important species to the South African forestry industry. Plant cryopreservation is an economical option to maintain such a genetic base, as it allows storage of vegetative materials at sub-zero temperatures, while maintaining juvenility. However, successful cryopreservation of this sub-tropical species has been restricted by its sensitivity to cryopreparative drying. As a consequence, the viability of material is reduced even before reaching the freezing stage. Despite this abiotic stress restriction, evidence of upstream ‘cross-talk’ implying downstream ‘cross-tolerance’ has suggested the possibility that cold acclimation may improve the tolerance responses towards dehydration stress by means of ‘cross-acclimation’. It was therefore the aim of the study to understand some of the physiological and biochemical responses of in vitro E. grandis shoots to different non-freezing low (chilling) temperatures and exposure periods, and to establish an appropriate ‘cross-acclimation’ regime for the physical drying pre-treatment. E. grandis shoot clusters (4-8 leaves and 2-5 axillary buds) were exposed to the chilling temperatures of 5°C, 10°C or 15°C for 1 or 3 days. The physiological and biochemical responses were evaluated, and thereafter the appropriate cold acclimation (or ‘cross-acclimation’) regime selected. The appropriate physical drying time was also selected for shoot clusters according to their physiological responses. When the appropriate regimes had been determined, the physiological and biochemical responses of shoot clusters treated consecutively to cold acclimation and then physical drying were evaluated. The physiological responses evaluated were water content, viability, and vigour (i.e. the number of visible axillary buds and shoots produced over 2 weeks). The biochemical responses measured were the concentrations of: 1) total soluble sugars, 2) starch, 3) phenolic acid, and 4) superoxide. The data suggested that the appropriate cold acclimation regime was treatment at 10°C for 3 days. This was based on the accumulation of the high levels of phenolic acid (3.05 ± 0.09 mg GAE.g-1 FWS) and positive vigour responses (11.90 ± 0.60 visible axillary buds/week and 3.10 ± 0.20 visible shoots/week), compared with the other chilling temperature treatments. The appropriate drying time selected for shoot clusters was 80 min over activated silica gel to achieve a water content of 0.32 ± 0.04 g water.g-1 FWS. In the dried material there were high levels of soluble sugars (47.65 ± 1.90% of the fresh weight of shoots) and unknown components that accounted for 48.10 ± 1.86% of the fresh weight, followed by phenolic acid (3.09 ± 0.05%) and proline (0.490 ± 0.011%). Despite these measured responses, viability of the shoots was impacted by drying, dropping to 88.9 ± 3.9%. When shoot clusters were pre-treated at 10°C for 3 days and then physically dried, viability of all (100%) the material was retained and the water content did not drop as low as with physical drying alone, dropping to 0.52 ± 0.05 g water.g-1 FWS. The biochemical responses showed that tolerance was strongly dependent on a high proportion of soluble sugars (83.66 ± 1.48% of the fresh weight of shoots) and phenolic acid (3.77 ± 0.12%), followed by proline (0.406 ± 0.018%). The study had confirmed that ‘cross-acclimation’ through means of cold acclimation (chilling pre-treatment at 10°C for 3 days) can induce ‘cross-tolerance’ towards physical drying, where osmotic adjustments and osmoprotection appeared to have been improved. It is therefore possible that this may have the potential to improve survival during the latter stages of the cryopreservation procedure, despite the higher retention of water in shoot clusters after drying.