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.

Thesis (Ph. D.)--Wageningen Universiteit, 2005. / Thesis propositions sheet inserted. Includes bibliographical references (p. [249]-262).

O Brasil é país detentor de grande biodiversidade, no entanto pequena parcela dessa já foi estudada e catalogada. Os recentes avanços antrópicos sobre os ecossistemas naturais tem levado a sua rápida fragmentação e eliminação de alguns biótipos ainda não estudados ou catalogados. Nessa realidade de erosão genética acelerada encontra-se também espécies de jabuticabeira (Plinia sp.) que são endêmicas do Centro Sul/Sudoeste do Paraná, no ecossistema Floresta com Araucária. Medidas de conservação de germoplasma para uso atual e futuro, bem como táticas de manejo e conservação dos recursos naturais são preponderantes para reduzir ao mínimo os danos causados a biodiversidade brasileira. Para tanto é necessário entender a diversidade genética existente nos ecossistemas naturais, que é o insumo básico para a sobrevivência e evolução entre os indivíduos frente as modificações ambientais. No presente trabalho foi realizado a caracterização genética de 110 jabuticabeiras que constituem o banco ativo de germoplasma desta espécie na UTFPR – Câmpus Dois Vizinhos. Foi possível identificar a transferibilidade de 9 marcadores moleculares microssatélites (SSR) com caráter polimórfico para a população estudada, e realizar a padronização de reação de PCR para cada um deles. Da análise das jabuticabeiras do banco ativo de germoplasma chegou-se a conclusão de que o mesmo abrigou valor considerável de diversidade alélica. No entanto tal diversidade está mal distribuída, já que 11 indivíduos sozinhos já são capazes de representar 59,2% de todos alelos da coleção de 110 plantas. Os valores encontrados de heterozigosidade observada e conteúdo informativo (PIC) nessa subpopulação de 11 indivíduos foram superiores aos valores encontrados para o conjunto integral do banco ativo. O agrupamento dos indivíduos mostrou a existência de 8 diferentes grupos, sendo que 89 indivíduos ficaram no grupo 1, demonstrando o seu possível grau de parentesco e baixo nível de diversidade genética. / Brazil is a country of great biodiversity holder, however small portion of that has been studied and cataloged. Recent advances human activities on natural ecosystems has led to its rapid fragmentation and elimination of some biotypes still, not it studied or cataloged. In this reality of genetic erosion is also accelerated jabuticaba tree species (Plinia sp.). That are endemic from South Central / Southeastern Paraná State, in Araucaria forest ecosystem. Germplasm conservation measures for current and future use, as well as tactics for management and conservation of natural resources are crucial to minimize the damage caused to Brazilian biodiversity. So it is necessary to understand the genetic diversity in natural ecosystems, which is the basic input for the survival and evolution among individuals facing environmental changes. In the present study was to characterize genetic of 110 jabuticaba tree fruit, that constitute the germoplasm bank of jabuticaba tree fruit from UTFPR - Câmpus Dois Vizinhos. It was possible to identify transferability of 9 microsatellite markers (SSR) with polymorphic character for the population studied, and it carry out standardization of PCR reaction for each of them. The analysis of plant germoplasm bank came to the conclusion that houses a considerable amount of allelic diversity, but this diversity is poorly distributed, since 11 individuals alone are already able to represent 59.2% of all alleles of the collection of 110 plant. The found values of, observed heterozygosity and information content (PIC) in this subpopulation of 11 individuals were higher than the values found for the full set of the germoplasm bank. The grouping of individuals showed the existence of 8 different groups, and 89 subjects were in group 1, demonstrating a possible relationship and low level of genetic diversity.

The NDSU EarlyGEM or the Early Germplasm Enhancement of Maize (Zea maize L.) is a long term incorporation program designed to increase the genetic diversity of short season hybrids. Starting in 1999, exotic GEM breeding crosses derived from temperate accessions: BR52051, CH05015; tropical accessions: SCR01, CUBA17, FS8B; and tropical hybrid DKB844 along with late checks: B73, Mo17, and Iowa Stiff Stalk Synthetic (BSSS), were adapted to short-seasons and incorporated via a modified backcross (BC) procedure. This study was designed to assess the genetic diversity in exotic derived BC1:S1 lines and their competitive potential as sources of new and unique hybrids. Useful genetic diversity was evaluated with testers belonging to opposite heterotic groups, LH176 representing a non stiff stalk and TR3026 x TR2040 a stiff stalk testers and were tested in five North Dakota environments over two years (2009 and 2010). All the traits showed highly significant (P<0.01) differences across genotypes except root and stalk lodging. Among 236 experimental testcrosses, 64 were statistically not different (LSD, 0.05) to industry hybrids for grain yield. BC derived lines from BR52051, CHO5015, DKB844 showed diverse alleles for low grain moisture (below 87 relative maturity days) at harvest and high grain yield. SCR01, BR52051, CHO5015 and CUBA117 derived lines produced hybrids with high grain oil (4. 9% vs. 4.1%) and grain protein (10.4% vs. 9.1%) contents compared to top checks. The results showed that the exotic incorporations are the sources of unique new alleles for early maturing maize not present in existing US germplasms (e.g. B73, Mo17, and BSSS). Even though each exotic cross was unique to integrate diverse alleles, utilizing multiple unique exotic crosses for incorporation showed large variation for specific traits. Phenotypic correlations of traits showed grain moisture played the most important role for short season hybrid development. Exotic incorporation through NDSU EarlyGEM has shown a new way of breeding early maturing maize keeping the breeding program open and genetic diversity high.

No description available.

Gray leaf spot (GLS) of corn (Zea mays L.), caused by the fungus Cercospora zeaemaydis (CZM) (Tehon and Daniels) has increased in incidence and severity with increasing use of no-tillage and continuous corn practices. This disease can be yield limiting. Corn hybrids were evaluated under natural disease pressure for three years (1989, 90, and 91) at two locations (Montgomery and Wythe Co., VA). Yield losses ranged from 2127.4 kg/ha (Wythe Co., 1991) to 4242.2 kg/ha (Wythe Co., 1990). It was estimated that 77% of the variability in yield was due to GLS. Fungicides were evaluated for the control of GLS over three years on a susceptible hybrid, Pioneer Brand 3320. All fungicides, with the exception of mancozeb, provided significant control over nontreated check in all years. Benomyl, propiconazole and terbutrazole were the most effective fungicides. As much as 93% of the variablilty in yield was attributed to blighting. Reduction in blighting also increased the kernel weight. The toxin, cercosporin, produced by CZM was evaluated for its ability to elicit differential responses in corn germplasm by three methods, ie., vein inoculation, root, and shoot uptake. No consistant differential reponses were found with vein inoculation, but 31-day old plants were significantly more sensitive to the toxin than 21-day old plants, as measured by lesion width. Root and shoot uptake of the toxin by inbred germplasm produced lesions that resembled those produced by CZM in the field. Microscopic, yellow fluorescing crystals were found associated with necrotic tissue from toxin-treated inbreds. Significantly more injury occurred to toxin-treated inbreds exposed to light than to darkness. By chromatographic analysis, 407.1-1076.7 ng of toxin/g of tissue was recovered from leaf lesion extracts of plants exposed to light. Five inbreds (B73, H99, Va59, NC250a, and NC264) showed consistent and differential responses to the toxin. H99 and NC250a showed differential responses to the same concentration of toxin, thus suggesting that some germplasm are more sensitive to the toxin than others. Tests using the toxin as a means to identify resistant germplasm did not provide reliable predictions of germplasm response to CZM in the field. / Master of Science

MSCAGR ( Plant Production) / Department of Plant Production / See the attached abstract below

M.Sc. / Climate change, loss of habitat and over-exploitation of natural resources as well as the introduction of invasive alien species through human activities are resulting in an ever increasing risk of extinction of many plant and animal species. There are two major approaches to conserving threatened and endangered species. Firstly the large scale preservation of natural habitat and ecological processes, thereby protecting the species inhabiting the habitat. The second approach involves the ex-situ breeding of rare and endangered species. It is estimated that in the next 200 years approximately 800 mammalian species will require the assistance of breeding programs to ensure long term genetic viability. Biological Resource Banks (BRB) can potentially contribute to this challenge by providing a source of genes that can be used to counter the effects of external selection pressures, genetic drift and inbreeding depression in small or fragmented populations. These banks commonly contain biological materials such as cryopreserved sperm, embryos and cell cultures mainly as genetic and research resources. . Biological resource banks can potentially use these cryopreserved gametes together with assisted reproductive technologies (ART), such as artificial insemination (AI), in vitro fertilisation (IVF), embryo transfer (ET), intracytoplasmic sperm injection (ICSI) and nuclear transfer (NT) to maintain genetic heterogeneity in ex-situ and wild populations. Ascertaining the appropriate protocols for developing the ARTs necessary for non-domestic species is one of the major challenges faced by reproductive physiologists. Typically, there is very little available information about the processing of semen, the effects of diluents, concentration and type of cryoprotectants and freeze-thaw methods for sperm samples of non-domestic species. Procedures proven to be highly effective in humans and laboratory or domestic species, are frequently adopted and modified for use in related wildlife species. It is thus necessary to gain knowledge of the reproductive physiology of wildlife species in order to define effective protocols for the cryopreservation of biomaterials which assists in the conservation of South Africa‘s diverse wildlife species. Sperm quality assessment is a useful tool for assessing the reproductive health of free-ranging populations as well as for selecting individuals for future assisted reproduction programs.

Indigenous communities have developed a wealth of knowledge, which plays a crucial role in providing leads for the use of genetic resources and bioprospecting. However, such knowledge is under increasing threat due to the misappropriation of the biological resources and associated traditional knowledge of indigenous communities, through both bioprospecting, as well as the inappropriate exercise of intellectual property rights. The internationally agreed Convention on Biological Diversity (CBD) attempts to provide a bulwark against biopiracy and although it assists indigenous communities to regain some control, the CBD has proven inadequate in the protection of the traditional knowledge of indigenous communities. The subsequent Nagoya Protocol on Access to Genetic Resources and the Fair and Equitable Sharing of Benefits Arising from their Utilization to the Convention on Biological Diversity (Nagoya Protocol) attempts to address some of these limitations but unfortunately has its own shortcomings, as it was largely concluded on the basis of a compromise between developed and developing countries. This dissertation will undertake a critical analysis of the provisions of the CBD and Nagoya Protocol, with a view to establishing the level of protection these instruments afford indigenous communities. It will be shown that notwithstanding the drawbacks of both the CBD and Nagoya Protocol, they nevertheless represent major achievements in the journey to protect the genetic resources and associated traditional knowledge of indigenous communities. It is in this context that this dissertation will analyse South Africa’s Access and Benefit Sharing (ABS) regime in relation to the protection it affords indigenous communities and in the light of the implementation challenges that such legislation presents. A particular focus will be on whether South Africa’s ABS legislation complies with the country’s international obligations relating to the protection of indigenous communities and whether South Africa’s approach to the protection of the genetic resources and associated traditional knowledge of indigenous communities, in the context of bioprospecting, is adequate or whether there exists potential for its enhancement. / Thesis (LL.M.)-University of KwaZulu-Natal, Durban, 2013.

Glochidia (larvae) of freshwater mussels are obligate parasites which attach to and become encysted in the gills or fins of host fish species. The immune responses of the host fish to the parasite affects the susceptibility of the fish to glochidia of different mussels. The immune response provides an opportunity to identify which fish species are hosts. The number and variety of mussels in rivers and lakes has sharply declined since the last century due to various anthropogenic factors, and some mussels species are facing extinction. It is an urgent task to preserve these vanishing mussels, or extinction will be inevitable. An attempt was made to develop an assay, using the immunological response to glochidia, to screen fish species for appropriate hosts. This would facilitate the production and rearing of juveniles. In order to design these assays, reagents such as anti-immunoglobulins which can react with antibodies from many different fish species have to be developed. This work was carried out to develop such reagents. Host and non-host fish were immunized with killed bacteria (Brucella abortus) to study their humoral immune response to an antigen. All fish were able to respond well, as measured by agglutination and Western Blot assays. Antibodies produced by the Brucella injections were used to stimulate anti-fish immunoglobulins in goats, and the antisera were tested for their ability to recognize immunoglobulins from different host fish species. The specificities of these reactions were compared to the reactivity of Protein A. Goat antisera were able to cross-react with different fish antibodies, but it was found that Protein A was a more suitable reagent. Protein A is seemingly suitable to identify the host-fish species and could be used as a reagent for the serological diagnosis of various fish diseases. / Master of Science