Thesis (Ph. D.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.

(Uncorrected OCR) Abstract of thesis entitled NOVEL ASPECTS OF AUTOCRINEIP ARACRINE REGULATION OF GROWTH HORMONE SECRETION AND SYNTHESIS IN GRASS CARP PITUITARY CELLS Submitted by ZHOUHONG for the degree of Doctor of Philosophy at The University of Hong Kong in March 2003 In this study, autocrine/paracrine regulation of growth hormone (GH) synthesis and secretion by local interactions of gonadotrophs and somatotrophs was examined in vitro in pituitary cells prepared from Chinese grass carp (Ctenopharyngodon idellus). Treatment with exogenous OH and gonadotropin (OTH) resulted in a dose-dependent increase in basal GH release, GH production, and GH mRNA levels. However, the opposite effects were observed by removing endogenous OR and OTH using immunoneutralization. Furthermore, GR and OTH immunoneutralizations at the pituitary level were effective in blocking the stimulatory influence on GH mRNA expression induced by GH-releasing factors in fish, including GnRH, dopamine, and PACAP38�Apparently" GH-induced GH gene expression was mediated by increasing the T1/2 ofGH mRNA in the cytoplasm and enhancing the production of GH primary transcripts in the nucleus. Since GH-induced OR mRNA gene expression could be blocked by inhibiting JAK2, P42144MAPK, P38MAPK, and PI3K, it is likely that the JAK/MAPK and JAK/PI3K pathways are involved in the GH receptor signaling. Similarly, exogenous GTH increased the production ofGH primary transcripts. However, it did not improve OR mRNA stability but rather enhanced the turnover of GH transcripts. GTR also increased cAMP production in carp pituitary cells. GTH-induced GH mRNA expression Was mimicked by activating cAMP synthesis and blocked by inhibiting adenylate cyclase (AC) and PKA.. GTH-induced OR mRNA expression was also sensitive to inhibition of JAKz, P42/44MAPK, P3SM.AP1C and PI3K. Similar inhibitions, except for PI3K, were all effective in blocking OR mRNA expression induced by activation of cAMP synthesis. These results indicate that GTH may induce GR gene expression through the AC/ cAMP/PKA pathway secondary coupled to JAK.2 andlor MAPK. Apparently, a cAMP-independent PI3K component is also involved in the post-receptor signaling. Using a colunm perifusion approach, the dynamic interactions between somaotrophs and gonadotrophs were examined. In this case, exogenous OTR induced a rapid rise in basal GH secretion, whereas exogenous GR was found to inhibit basal GTR release. In parallel studies, GTHinduced OR mRNA expression was abolished by OR immunoneutralization. Similarly, GTR immunoneutralization blocked GR-induced OR mRNA expression in carp pituitary cells. These results, as a whole, indicate that endogenously secreted OH and GTR, besides their functions as endocrine hormones, serve as novel autocrine/paracrine factors at the pituitary level to modulate GH secretion, OH production, OH gene expression, and somatotroph sensitivity to stimulation by hypothalamic regulators. These stimulatory influences of GH and GTR on OR gene expression axe exerted at the level of GR rnRNA stability and OH gene transcription, presumably via a direct coupling to the JAK/MAPK and JAKiPI3K cascades or an indirect coupling via the AC/cAMP/PKA pathway. Apparently, a local il1trapituitary feedback loop is present. In this case, GTH released from gonadotrophs stimulates GH secretion in neighboring somatotrophs. GR release from somatotrophs is essential to maintain basal GH synthesis and secretion and also exerts a negative feedback on basal GTB release. This intrapituitary feedback loop formed by local interactions between gonadotrophs and somatotrophs may represent a novel mechanism to control OR gene expression in lower vertebrates. / abstract / toc / Zoology / Doctoral / Doctor of Philosophy

Thesis (Ph. D.)--University of Hong Kong, 2007. / Also available online.

Thesis (Ph. D.)--University of Hong Kong, 2008. / Also available in print.

The present study focused on using food wastes and feed supplements, e.g. enzymes (bromelain and papain), baker’s yeast and Traditional Chinese Medicines (TCMs) for rearing freshwater fish (grass carp and grey mullet) in Hong Kong. Different types of food wastes, e.g. meats, bones, cereals, fruits and vegetables were collected from local hotels, mixed in different ratios and processed into feed pellets for feeding trials. The cereal dominant food waste feed (FW A) was more suitable for grass carp and grey mullet, with the best growth performance (e.g. feed conversion ratio (FCR), specific growth rate (SGR)) and higher protein digestibility (in grass carp), compared to FW B and FW C which contained higher proportions of meat products. The NBT (Nitroblue Tetrazolium) activities in blood and plasma protein levels were decreased in the grass carp, cultured with food waste feeds without any supplements, compared to the commercial feed, Jinfeng®, 613 formulation (Control). Upgrading FW A by the addition of 1% and 2% mixtures of bromelain and papain significantly increased the feed protein solubility and subsequent to growth (SGR and relative weight gain (RWG)) and feed utilization (e.g. apparent net protein utilization (ANPU), protein efficiency ratio (PER)) in both fish species. The protein and feed utilizations by grass carp were also promoted by the yeast supplements with the optimal dose of 2.5% yeast (S. cerevisiae) added to FW A upgraded by enzymes. This showed that yeast could further enhance nutrient utilization contained in feeds after addition of bromelain and papain. The in vitro study on the grass carp’s plasma treated with TCM extracts also showed that TCM extracts could stimulate plasma bactericidal activity (on Aeromonas hydrophila), possibly through enhancing plasma complement activity. The formulation with Radix scutellaria, Rhizoma coptidis, Herba andrographis and Radix sophorae flavescentis in the ratio of 1:1:2:3 was more effective in enhancing plasma bactericidal activity than single TCM extracts. Besides, R. coptidis and R. scutellaria possessed the strongest antimicrobial activity (in vitro) on fish pathogens (such as A. hydrophila, Lactococcus garvieae and Vibrio cholerae) among the 17 tested TCMs. In addition, TCMs were less likely for developing drug resistant pathogens than antibiotics. Grass carp immunity (NBT activity in blood, plasma bactericidal activity and total immunoglobulin level) was boosted by the addition of TCM formulation and baker’s yeast (S. cerevisiae). The disease resistance to pathogen (A. hydrophila) was also enhanced, with significantly lower mortalities observed in groups feeding with TCM (1 and 2% for 21 to 28 days) and baker’s yeast (2.5 and 5% for 28-56 days). The uses of yeast and TCMs led to positive effects on growth, immunity and disease resistance to pathogens in fish, but the effects (grass carp) were less effectual when both were supplemented in feed. The combined use of both supplements may impair the effects of TCM formulation or yeast in the modulation of gut mircoflora, and upset the balance of beneficial microbial communities. The present study demonstrated the feasibility of using feed supplements (TCM and baker’s yeast) to enhance fish immunity and enzymes upgraded food waste feeds for rearing fish, for the development of a more sustainable aquaculture in Hong Kong.

The overabundant proliferation of aquatic weeds in South African water conveyance systems cause a series of operational problems. Filamentous algae, such as Cladophora glomerata and pondweeds impede flow and reduce the capacity of irrigation canals to a significant extent. In worst case scenarios, irrigation scheme managers are faced with situations where they are unable to deliver water at the downstream ends of canal systems. This situation also contributes to water losses, crop losses and structural damage to concrete-lined canals. This dissertation is the product of a research project funded by the Water Research Commission to investigate the possible application of sterile (triploid) grass carp (Ctenopharyngodon idella) as biological control agent on aquatic weeds in concrete-lined irrigation canals. The aims of the project were firstly to investigate the suitability of a concrete-lined irrigation canal as grass carp habitat; secondly to test the efficacy of sterile grass carp as bio-control agent on filamentous algae; thirdly to evaluate the economic feasibility of this biological approach against the current chemical, physical and mechanical control methods; and fourthly to propose a management plan for the operational application of triploid grass carp as bio-control agent in irrigation canals. To achieve the goals set for the project, the Ramah-3 Canal near the town of Orania, was selected to serve as experimental canal. This canal can be regarded as a typical South African concrete-lined canal, and is located in the Orange-Riet River Canal System, downstream of the Vanderkloof Dam. As the majority of local irrigation canals are concrete-lined, skepticism existed amongst local scientists if the fish will be able survive in canals with a presumed high constant flow and low diversity of hydraulic biotopes. It was therefore a priority to establish the suitability of concrete-lined canals as habitat for this herbivorous fish specie. The study found that flow velocities in the Ramah Canal System never exceeded 1 m/s, even under high flow conditions (full capacity and 110% plus conditions) of more than 5 m3/s. It was found that grass carp thrived at flow rates ranging from 0.48 to 0.80 m/s, moving with ease upstream and downstream in a 16 km long experimental section of the Ramah-3 Canal. The sterile grass carp controlled the algal biomass in the Ramah Canal to significant levels. Cladophora was efficiently controlled at stocking rates of 3 to 7 fish per km canal. Triploid grass carp retrieved from the canal system after a six-month experimental period were found to be in an excellent physiological condition and displayed a mean weight increase of more than 300%. An ideal stocking protocol will probably be 10 individuals of 20 – 30 cm in length per kilometer canal, with a 10-15% annual supplementation figure. Civil structures, such as culverts, super-elevated canals and bridges will provide the fish with sufficient protection against possible predators. A few possible adaptations to the existing canal operation regimes should ensure that triploid grass carp could be managed as an effective biological control agent. This should be determined on a site-specific basis and could include additional civil structures such as sanctuary dams and small in-line fishways to ensure free migration throughout the target system. An attempt was made to conduct a brief economic analysis, based on information on expenditures of local irrigation schemes on aquatic weed control in their canals. The outcome of this brief investigation was in line with overseas findings in that biological control with sterile grass carp will be more economical than the currently applied control methods of herbicidal and mechanical or physical control. It can be concluded that the fish adapted to the artificial conditions experienced in a concrete-lined canal and perform their task as bio-control agent with ease. The author is of the opinion that this bio-control technique will contribute to the current Integrated Aquatic Weed Management Programmes (IAWMPs) of the Department of Water Affairs and Forestry. Site-specific conditions for each irrigation scheme will however, dictate a different approach to the aquatic weed problem. It is therefore strongly recommended that suitable qualified specialists should develop all aquatic weed management programmes on a site-specific basis. / Prof. G.J. Steyn

Ceftiofur sodium, a third generation cephalosporin, was studied to determine the potential of this drug as an alternative bacterial therapeutic agent for the aquaculture and ornamental fish industry. Grass carp, Ctenopharyngodon idella have been selected as the fish model for this study since they are a good representative for both foodfish and ornamental fish and are one of the major species grown worldwide. Pharmacokinetics of ceftiofur sodium after various routes of administration, histopathologic observations to detect possible toxic effects on the tissues involved in its metabolism and excretion, and the effects on the non-specific immune response were investigated in grass carp. For the pharmacokinetic studies, ceftiofur sodium was administered a single time to grass carp by four different routes : intracardiac (IC), intraperitoneal (IP), intramuscular (IM) and oral (PO) at a dosage of 8 mg/kg body weight. Serial blood samples were obtained and plasma samples were analyzed by high performance liquid chromatography for ceftiofur (as measured its metabolite, desfuroylceftiofur (DFC) and DFC-related metabolite concentrations). Disposition pharmacokinetic data were best described by a two compartment open model for IC and by a non-compartment model with no lag time for IP and IM administrations. Oral absorption of ceftiofur was not observed in this species. Following IC, IP and IM ceftiofur sodium administration, the final elimination half-lives, maximum plasma concentration, time to reach maximum concentration, volume of distribution and plasma clearance were 0.38, 0.45 and 13.86 hours ; 157.09, 31.54 and 8.86 mg/ml ; 0, 0.25 and 0.5 hours ; 0.09, 0.17, 0.53 l/kg ; and 0.21, 0.26, 0.26 ml/min.kg, respectively. Desfuroylceftiofur metabolite was highly bound with plasma protein at pH 7.0 and 8.0. For the histopathological studies, a single intramuscular dose of ceftiofur sodium at three different concentrations, 8 (1X), 40 (5X) and 80 (10X) mg/kg was administered to separate groups of grass carp for evaluation of the potential toxicity to major tissues involved in metabolism and excretion of this drug. These included the anterior kidney, posterior kidney, liver, and spleen. After 48 hours, lesions were seen in the posterior kidney at the highest dose of ceftiofur (10X). Morphological alterations observed microscopically included increased number of renal tubules, tubular necrosis and infiltration of inflammatory cells. No adverse effects on the glomeruli were observed at any concentration of the drug. For the immunotoxicity studies on the non-specific immune response, dosages of either 8 or 40 mg/kg body weight were administered intramuscularly. After 24 and 48 h, leukocyte number, phagocytic ability and H2O2 production were examined in the cells of the pronephros. The results showed that neither dosage had an effect on the number of leukocytes in the pronephros. Phagocytosis was also not significantly altered at either dosage in macrophages from the pronephros. Hydrogen peroxide production was not altered in the pronephros of fish dosed at 8 mg/kg, while at a dosage of 40 mg/kg, H2O2 production was significantly increased. In summary, ceftiofur sodium has potential as an efficacious chemotherapeutic agent for controlling bacterial infection in brood stock and ornamental fish at the recommended dose of 8 mg/kg. A dose as high as 40 mg/kg can be use with careful consideration. This dosage may not directly injure the posterior kidney but it may affect the non-specific immune response of the fish. / Ph. D.

published_or_final_version / abstract / toc / Zoology / Master / Master of Philosophy

published_or_final_version / Zoology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Zoology / Doctoral / Doctor of Philosophy