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After epididymal maturation, sperm capacitation, which encompasses a complex series of molecular events, endows the sperm with the ability to fertilize an egg. It is well established that capacitation requires Na+, HCO3-, Ca2+ and a cholesterol acceptor; however, little is known about the function of Cl- during this important process. In this study, it is shown that Cl- is essential for capacitation and that Cl- act upstream of the cAMP/PKA signaling pathway. To investigate the Cl- transporters involved, sperm incubated in complete capacitation medium were exposed to a battery of anion transport inhibitors. Among them, bumetanide and furosemide, two blockers of Na+, K+, Clcotransporters (NKCC), inhibited all capacitation-associated events suggesting that these transporters may mediate Cl- movements in sperm. Consistent with these results, western blots using anti NKCC1 antibodies showed the presence of this cotransporter in mature sperm. Other key issue in sperm capacitation that remains unsolved despite many years of research is the possibility that transmembrane adenylyl cyclases (tmACs) have a role in sperm physiology. If tmACs were involved in the increase of cAMP in sperm, a role of G proteins in these responses would be plausible. Uncertainties exist about the activation of sperm adenylyl cyclase(s) by heterotrimeric G proteins. In this regard, there is some debate as to whether Gα s is present in spermatozoa. In this work, Gαs was detected on membranes purified from mouse sperm by [32 P] ADP-ribosylation with cholera toxin followed by immunoprecipitation with a specific antibody. In addition, immunofluorescence studies identified a specific signal in the sperm acrosomal region. Moreover, forskolin was able to induce acrosome reaction after incubating sperm in capacitating conditions and a higher accumulation of cAMP was observed after incubating sperm with forskolin. In this context, the clarification of the role of the ACs and G proteins will constitute a pivotal step for further investigation on sperm capacitation. Forskolin induces acrosome reaction only on capacitated sperm. These data suggest that a tmAC might be involved in the acrosome reaction and that capacitation is needed to couple this signal to other pathways related to the exocytic process.

This study evaluated procedures to improve culture and cryopreservation of in vitro produced bovine embryos. Successful in vitro development of early embryos is crucial for areas of biotechnology such as transgenics and cloning. The limiting factor in culturing in vitro matured and fertilized (IVM-IVF) embryos is the media in which the embryos develop in vitro. An optimal culture system which would support development equivalent to that found in vivo has yet to be found. If such a system were developed, studies could be conducted to elucidate the factors responsible for normal embryonic development and then apply these factors toward the development of defined media. The mitogenic capabilities of bovine (BVH) and rabbit (RVH) vitreous humor were tested on the in vitro development of IVM-IVF bovine embryos. Embryos were cultured from day 2, 3, 4, 5 or 6 until day 8 in different concentrations of BVH/RVH. Bovine embryos are inconsistent in their ability to develop in these fluids until the late morulae or early blastocyst stage. The activity of vitreous humor is not species-specific as bovine embryos developed in RVH as well or better than BVH. Blastocyst development was not improved with culture in BVH or RVH, although there is evidence to suggest that RVH enhances mean cell number in developing embryos. Delaying culture in BVH or RVH did improve mean cell number and blastocyst development. A simple and reliable method was developed to cryopreserve in vitro produced bovine embryos. The developmental capability of IVM-IVF derived morulae/early blastocysts was evaluated following different methods of diluting cryoprotectants after vitrification. One-step 0.1M or 0.3M sucrose dilution was optimal for removing the high concentrations of intracellular cryoprotectants in the vitrification medium as post-treatment survival was no different than culture controls. Step-wise dilution/gradual rehydration of vitrified embryos does not improve development following vitrification. Freezing and vitrification are equivalent for in vitro development of IVM-IVF bovine embryos following cryopreservation, although blastocyst development is approximately 1/2 the rate of their culture controls. These data are the first to indicate that IVM-IVF generated bovine embryos can be cryopreserved by vitrification.

The Smyth line chicken (SL), an avian model for autoimmune vitiligo, is characterized by a postnatal loss of melanin pigmentation in feathers and the choroid of the eye. Earlier studies supported a hypothesis that an inherent pigment cell defect is necessary for the amelanosis which is the consequence of the selective destruction of melanocytes by the immune system. Data collected during the last decade has demonstrated that the MHC plays a major role in the development and severity of the SL vitiligo. The major objective of this study was to determine the factors contributing to the variable expression of the disease in the SL102 subline, selected by serotyping to be homozygous for the 102 MHC haplotype. The homogeneity of the MHC loci among SL102 and BL (parental control) birds was evaluated by serological, mixed lymphocyte response (MLR) and restriction fragment length polymorphism (RFLP) techniques. The results of these analyses were then studied in respect to their effects on the variation in incidence and severity of autoimmune vitiligo. All SL102 birds used in these studies appeared to carry the same, or nearly identical Ea-B haplotype as determined by serology as well as by MLR. Surprisingly, only 15 out of 22 BL birds were homozygous for Ea-B102, possibly due to a previous pedigreeing error. RFLP genotype assignment based on 4 different restriction enzymes and MHC class I and class II probes did not appear to have any influence on the incidence of vitiligo. No polymorphisms were found among SL102 birds using class II probe, while 2 RFLP genotypes were revealed with the class I probe. One of these loci was shown (P $<$.05) to be associated with the severity of the vitiligo. Peripheral blood lymphocytes obtained from the serologically haplotype matched SL, BL and an unrelated control (LBL) birds were analyzed for their mitogenic responses as well as their lymphocyte subset populations. SL102 birds had a higher proportion of CD4+ and CD8+ T-cells and a lower proportion of TCR1+ T-cells; but, they were also the poorer responder to a T-cell mitogen, ConA. One of the variant B-F RFLP polymorphisms was associated with a low ConA response, although these birds had a higher proportion of CD8+ T-cells than controls. From this study it is hypothesized that MHC class II locus is involved in the incidence of autoimmune vitiligo where as the severity of the disease is associated with MHC class I genes. The variation in mitogenic response and lymphocyte subset populations are possibly associated with subline differences that may not be MHC-related.

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The marketing of professional services is a relatively recent discipline driven by a changing political and social environment. The services marketing literature has evolved within this changing environment from an initial focus upon the distinction between products and services; to the importance of quality, relationships, interfunctional coordination and processes; and most recently to the contemporary marketing practices framework and the service dominant logic. In this thesis, secondary data were analysed to understand trends within the major forces affecting the attractiveness of the companion animal veterinary services industry in Australia. Case studies complemented this analysis by describing individual organisations operating within local environments, their marketing practices and performance. A survey of the industry was subsequently administered and analysed to describe the relative importance of industry forces upon incumbents, the range of contemporary marketing practices adopted, and to test relationships between environment, marketing practices and marketing performance. Whilst secondary data revealed that pet ownership and household demographic trends were leading to stabilisation of the number of potential buyers for companion animal veterinary services at a national level, this research has found that changing attitudes towards pets, pet care and the human animal bond, together with advances in technology and individual veterinary hospital marketing practices, had generally resulted in a positive impact on veterinary revenue within the last 10 years. For individual veterinary hospitals competing within this industry, external environmental and industry structural forces were of varying importance and both functional and process approaches to marketing were evident within the industry. Specifically with respect to the contemporary marketing practices framework, companion animal veterinary hospitals in Australia mostly related to their markets through interaction marketing but also employed transaction, database and network marketing practices. Network marketing practices were found to be effective for acquiring clients and interaction marketing practices were effective in driving revenue growth. This research has provided potential contributions to extant literature in the areas of industry analysis, services marketing and more specifically within the discipline of veterinary business management. Firstly, the framework combining external forces (political, economic, social, technological and natural) with industry structural forces analysis was an effective method for analysing the marketing environment affecting the companion animal veterinary services industry in Australia. Secondly, industry attractiveness was significantly related to industry profitability and transaction marketing practices were associated with less attractive environments. Thirdly, whilst secondary data defined the industry as mature, marketing practices adopted by industry incumbents have resulted in significant revenue growth in recent years. Finally, whilst the marketing literature has emphasised the distinction between transactional and relational perspectives, this research has highlighted the possibility of considering a functional and process approach to the marketing of professional services. Linked to this latter finding, this research has highlighted the value of the contemporary marketing practices framework for understanding the marketing of professional, high-involvement services, and integrating theoretical and empirical perspectives in marketing. / Doctor of Philosophy (PhD)