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    ThesisItemOpen Access
    INTEGRATED CULTURE OF FISH WITH MICROPROPAGATED PLANTS IN A RECIRCULATORY SYSTEM
    (KUFOS, 2013-06-26) Navya, R; Devika, Pillai
    ABSTRACT An experiment was designed to study an integrated recirculatory sytem with micropropagated aquarium plants, fish and indigenous filter materials like coir, vetiver and bamboo as the filter media. The plants selected for the study were Bacopa caroliniana, Anubias minima, Aponogeton ulvaceus, Rotala rotundifolia and Nymphoides cristata. The first part of the work was to standardise the micropropagation techniques for the above said plants. Murashige and Skoog medium (half and full strength) was used as the basal medium for the establishment of cultures. The explants varied from nodal segments, rhizome buds, leaf petioles and lateral buds. Surface sterilization was carried out with a range of sterilants like mercuric chloride solution, ethyl alcohol, sodium hypochlorite solution etc. for varying durations and concentrations depending on the type of explant. Effect of growth regulators on explants were studied using auxin IAA and cytokinins, BA and Kn. A liquid culture media with 1.5mg l-1 BA and 0.1mg l-1 Kn concentrations was the best medium for Bacopa caroliniana. A combination of auxin cytokinin like 1.5 mg l-1 BA and 1.0 mg l-1 IAA in liquid culture was the best medium for Rotala rotundifolia. In Nymphoides cristata, hormone concentration of 1.0 mg l-1 each of BA and IAA in liquid culture gave the best results. In Anubias minima, a full MS solid medium with 6.0 mg l-1 of BA gave better results compared to lower levels of BA. In vitro trials for Aponogeton ulvaceus was not successful due to the exudation of phenolic extracts in all the treatments which eventually lead to the death of the tissues. Hardening success was 90 percent, 100 percent, 70 percent, 100 percent in the case of Anubias minima, Nymphoides cristata, Rotala rotundifolia and Bacopa caroliniana respectively. The micropropagated plants were incorporated in the recirculatory system along with fish and three different biofilter materials like coir, vetiver and bamboo splits. The study focused mainly on the filtering efficiency of the 155 three filters based on the nitrification curves. The plants were included in the system to be a part of biofilter and their exclusive role in nitrification was not studied since this is a preliminary work. A nitification graph was plotted with the observed values of ammonia, nitrite and nitrate in the three different biofilter systems and compared with a control. It was inferred that coir fibres were the best of filter materials tried in nitrification followed by bamboo and vetiver. The control system took more time (45 days) in stabilizing ammonia levels due to lack of a substratum for growing nitrifying bacteria.
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    ThesisItemOpen Access
    GENETIC CHARACTERIZATION, CONTROLLED BREEDING AND DEVELOPMENT OF TRANSGENIC VARIETIES OF PUNTIUS DENISONII (DAY, 1865).
    (KUFOS, 2010-12-10) MANOJ, C. K; Mohanakumaran Nair, C
    ABSTRACT Puntius denisonii, a beautiful ornamental fish indigenous to the Western Ghats, which has been indiscriminately exploited from the different rivers of Kerala has been recently declared to be vulnerable by the IUCN. The population structure and genetic diversity of P. denisonii has not yet been studied and documented. Many previous attempts to breed this fish in captivity have yielded negative results. The increasing demand for this fish to decorate aquariums worldwide could be satisfied only by developing controlled breeding techniques and larval rearing of its fry. In the present study, the present population structure of P. denisonii has been studied combining both phenotypic and genotypic techniques. Fishes were collected from Irrity, Chaliyar and Periyar rivers of Kerala. Truss network analysis was conducted and the size adjusted morphometric variables were subjected to Principal Component Analysis and Canonical Variance Analysis. Scatter diagram and Dendrogram was plotted using PCA and CVA loadings. The Irrity and the Chaliyar populations were grouped on the positive sector of the PC and CV component showing morphological similarities between the two populations while the Periyar population was placed in the negative sector of the component separated far from the other two. The PC scores were used to find out the variables showing maximum variation between fishes collected from different rivers. RAPD PCR was conducted after isolating DNA from the fins of different populations of P. denisonii. Universal random primers were screened and the primers that produced reproducible bands were selected. Popgene analysis of the binary data yielded the genetic structure of different populations of P. denisonii. Number and percentage of polymorphic loci, Nei's (1973) gene diversity, Shannon's Information index Lewontin (1972), Nei's Unbiased Measures of Genetic Identity and Genetic distance and Dendrogram Based Nei's (1978) Genetic distance using UPGMA --Modified from NEIGHBOR procedure of PHYLIP Version 3.5 were studied. The results obtained supports the truss analysis in that the Irrity and Chaliyar populations in Northern Kerala are genetically more similar while that of Periyar population in Central Kerala are distinct. P. denisonii was successfully induced bred under controlled conditions with synthetic hormone preparations Ovaprim and WOVA-FH. Stress during transport and handling was minimized and live feed was supplemented to enhance maturation of the broodstock. The whole developmental sequence starting from fertilized eggs to hatching was photographed and documented. It took 29-30 hours for the eggs to hatch at 280C. Rearing of fry was successfully accomplished under laboratory conditions. In an attempt to develop transgenic varieties of P. denisonii, pCMV-GFP was electroporated into newly fertilized eggs, maintained in hypoosmolar electroporation buffer. The electroporation parameters that yielded best results were 20V, 3 bursts at 1 second interval. Fin clips were taken from the transgenic individuals reared for a period of 6 weeks. Dot blot test was positive showing integration of the GFP gene in P. denisonii, eventhough expression was not detected under blue or UV light. The genetic and phenotypic data of P. denisonii populations in the present study will aid as a base line for formulating conservation procedures to protect the genetic diversity of wild ones. Stock identification studies are recommended for more concise information on each population. Moreover, the larval rearing and controlled breeding techniques along with the genetic diversity studies will help to design captive breeding programs and enhance the production of hatchery bred ones to meet increasing demand. Further research is recommended for generating transgenic lines with uniform GFP expression.
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    ThesisItemOpen Access
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    ThesisItemOpen Access
    BIOACTIVITY OF CAROTENOIDS FROM SHRIMP SHELL WASTE
    (KUFOS, 2010-08-30) SINDHU, S
    ABSTRACT Shrimp processing waste is the single largest industrial waste in the country causing diverse environmental problems. A study was carried out to assess the extractability of astaxanthin from shrimp waste in different organic solvents and vegetable oils. Extraction was tried using wet and dried waste, with and without deproteinisation. Waste was subjected to deproteinisation using alkali and enzyme (pancreatin). The different solvent systems tried were ether:acetone:water (15:75:10 v/v/v), acetone, hexane:isopropanol (3:2 v/v) and 90% acetone v/v. Astaxanthin in the extract was quantified by measuring the OD at 470 nm in hexane. Extraction was also done using vegetable oils viz. coconut oil, soybean oil and sunflower oil. Quantification of astaxanthin in pigmented oil was done by measuring the absorbance at 485 nm using 2155 as extinction coefficient. Astaxanthin yields from deproteinised samples were significantly lower than those from non deproteinised samples. The highest astaxanthin yield of 87.14 ± 4.55μg/g was obtained with non deproteinised wet waste extracted using acetone. The astaxanthin yield was significantly lower when oil was used as the extraction medium. Of the three oils coconut oil gave the highest yield. The results showed that acetone is the best solvent for extracting astaxanthin from shrimp shell waste in wet condition. The astaxanthin content in Aristeus alcocki shell waste is double that of Pandalus borealis shell waste, which is currently used as the commercial source of astaxanthin. The deep sea species Aristeus alcocki can thus be considered as a better source of astaxanthin for commercial exploitation than Pandalus borealis. TLC analysis of the shell waste extract showed that it contains free astaxanthin, astaxanthin monoester and astaxanthin diester in the ratio 1:1:2. GLC identification of the fatty acids esterified with astaxanthin revealed that saturated fatty acids, MUFA and PUFA are in the ratio 5:3:2 in monoester, whereas in diester they are in the ratio 4:3:3. The main fatty acids in monoester and diesters are palmitic acid, oleic acid, stearic acid and PUFAs: DHA and EPA. The in vitro antioxidant activity of the astaxanthin extract showed significant hydroxyl radical scavenging activity, superoxide anion scavenging activity and inhibition of lipid peroxidation. The IC50 values obtained were 56.43 ± 1.06 ng/ml, 27.91 ± 0.54 ng/ml and 26.54 ± 0.42 ng/ml, respectively. The antioxidant activity of astaxanthin from Aristeus alcocki was obtained at nanogram levels. This powerful antioxidant function may be due to the unique molecular structure of astaxanthin and synergistic effect of astaxanthin and PUFAs present in the astaxanthin monoester and diester fractions. The astaxanthin extract from shrimp shell waste significantly reduced carageenan induced paw edema in mice, percentage inhibition being 47.83 and 67.11 percent at astaxanthin concentrations of 0.5 mg/kg body weight and 1.0 mg/kg body weight, respectively. The inhibition of inflammation at 1.0mg/kg body weight was greater than that produced by the standard reference drug diclofenac. Cardioprotective effect of astaxanthin was examined in isoproterenol induced myocardial infarction in rats. Levels of diagnostic marker enzymes, LDH, CPK, GOT, GPT, CK, CK-MB in plasma, lipid peroxides, ascorbic acid, reduced glutathione and the activities of glutathione-dependent antioxidant enzymes GPx, GR, GST and antiperoxidate enzymes CAT, SOD and the membrane bound enzyme Na+ - K+ ATPase in the heart tissues of experimental groups of rats were determined. The prior administration of astaxanthin @ 10mg/kg feed for 45 days significantly prevented the isoproterenol-induced elevation in the levels of diagnostic marker enzymes in plasma, induction of lipid peroxidation and alterations in the level of reduced glutathione and in the activities of glutathione dependent antioxidant enzymes and antiperoxidative enzymes of experimental rats. Feeding astaxanthin caused a decrease in the inhibition of Na+ - K+ ATPase activity against isoproterenol induced myocardial infarction. The powerful cardioprotective effect of astaxanthin can be attributed to the multiple independent mechanisms viz. antioxidant effects, singlet oxygen quenching ability and inhibition of lipid peroxidation of membranes, increased functional gap junctional intercellular communication, anti-inflammatory effects etc. Immunostimulatory action of astaxanthin extract was evaluated in experimental mice. Astaxanthin administration was found to enhance the proliferation of spleen cells and bone marrow cells. Esterase activity was found to be enhanced in bone marrow cells indicating increased maturation of cells of lymophoid linkage. Astaxanthin also enhanced number of antibody forming cells and circulating antibody titre. Thus astaxanthin exhibits strong immunomodulating properties. A significant reduction in the viability of ascites tumour cells DLA in vitro was noted in the current study. The % viability was reduced to 4.34 % at a concentration of 15μg astaxanthin/ml. The cytotoxic action of astaxanthin against DLA may be through induction of apoptosis or through a different pathway. Antitumour activity of astaxanthin was studied by ascite and solid tumour models in mice. An increase in life span of about 67 % was noted in DLA bearing mice administered with astaxanthin at 5 mg/kg body weight. The tumour volume and tumour weight were significantly lower in mice injected with 5 mg/kg body weight astaxanthin. In vitro studies revealed that astaxanthin from shrimp shell waste of Aristeus alcocki inhibited the proliferation of cervical cancer cells HeLa in a dose dependent manner.
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    ThesisItemOpen Access
    Biology of Horadandia atukorali Deraniyagala, 1943 (Cyprinidae: Teleostei), an ornamental fish of theWestern Ghats of India
    (Department of Fisheries Resource Management)
    ABSTRACT Horadandia atukorali, Deranyagala, 1943 is a small cyprinid (danionid) fish distributed in southern India and Sri Lanka, which is an indigenous ornamental fish of the Western Ghats of India. A total number of 1254 specimens were used for investigating the different aspects of biology. Sampling was done from January 2013 to December 2013 from two locations (location I, Panangad ponds and location II, Ezhupunna wetlands). The morphometric and meristic studies were conducted. 21 body measurements were expressed as percentage of standard length and 4 head measurements were expressed as percentage of head length and the relationships were significant. The percentages of morphometric characters show in two locations show slight variations, where as the meristic counts remained the same. Maximum Total length was 3.5 cm in location I and 3 cm in location II. Length weight relationship, condition factor and relative condition factor were studied for males, females, juveniles and pooled in both locations. They showed negative allometric growth. The b values of females were higher than the males which suggested that females gained weight at a faster rate in relation to its length than males. The overall condition of the fish was found to be in good condition. The oocyte development was classified into 9 oogenic stages. The dominance of females over males has depicted in the monthly sex ratio (1:3.4). The gonads were quantified into 5 maturity stages based on external morphology. The size at first maturity was found to be 1.5 cm TL for females and 1.3 cm for males. Ovaries of H. atukorali showed asynchronous development, in which oocytes at all stages of development were present in the same ovary at the same time. Ova diameter studies revealed that this species comes under the category C 299 of Karekar and Bal’s classification (1960), characterized by spawning more than once during a protracted spawning season. Based on spawning frequency study of the ripe ovary, H. atukorali was found to be a multiple spawner, with a protracted spawning season, with peak spawning activity in November. The absolute fecundity ranged between 22 (TL. 1.4 cm and 0.034 g body weight) and 172 eggs (TL. 3.5 and 0.15 g body weight). They did not exhibit clear cut sexual dimorphism. The possession of a terminal mouth with moderate gape suggested that it is a column feeder. They belonged to the group of stomach-less fishes in which the digestive tube consisted of mouth, pharynx, oesophagus, intestine and anus. It is a column feeder, mainly a carni-omnivore as well a micropredator feeding on small insects, worms, crustaceans and other zooplanktons along with small amount of phytoplankton. The relative gut length were calculated in males, females and juveniles and it ranged between 0.6 and 0.95 in males, 0.5 and 0.7 in females and 0.5 and 0.7 in juveniles respectively. Complete information on its reproductive biology and food and feeding habits will definitely help in the commercial production and selective breeding under captive condition. This will increase the domestic and export market of this indigenous ornamental fish. It will also help in the conservation and management of the species and their habitat.