Thesis (Ph. D. (Physiology)) -- University of Limpopo, 1993 / Refer to document

The strategy of producing expressed sequence tags (ESTs) is greatly used for gene mining and analysis of gene expression. An EST is a partial and single-pass sequence generated from a complementary DNA (cDNA) library by random selection, and is typically about 400 to 600 bases. Here, we constructed and analyzed a brain EST library from 10-day-old (10-day posthatching) tilapia, Oreochromis mossambicus, to assist in investigating the relation between brain neural development and neuroendocrine and brain sexual differentiation during the critical period of sexual dimorphism by understanding brain gene expression, and, furthermore, to find genes that are important in brain but are unknown nowadays. A total 1,124 ESTs were in the 10-day-old tilapia brain EST library, and 1,092 ones were readable, and after discarding one that is too short (<200bp) or is totally vector sequence from the readable ESTs, 1,084 ESTs were assembled and then analyzed. 108 contigs from 261 ESTs, 20 kinds of similar sequences from 40 ESTs, and 783 singletons from the 1,084 ESTs were found, and after aligning in turn with the non-redundant (nr) protein database, nr nucleotide database and EST database dbEST supported by the National Center of Biotechnology Information (NCBI) using Basic Local Alignment Search Tool (BLAST) programs BLASTx (translated nucleotide-protein alignment) and BLASTn (nucleotide-nucleotide alignment) respectively, the results are as follows: 57 and 16 contigs including 146 and 37 ESTs matched with sequences in the nr protein and nr nucleotide databases respectively, and the rest 35 contigs including 78 ESTs were novel sequences; nine, one and two kinds of the similar sequences including 18, two and four ESTs matched with sequences in the nr protein, nr nucleotide and dbEST databases respectively, and the rest eight kinds of the similar sequences including 16 ESTs were novel sequences; 309, 52 and 47 singletons matched with sequences in the nr protein, nr nucleotide and dbEST databases respectively, and the rest 375 singletons were novel sequences. As a whole, the most functions of proteins that the contigs, similar kinds of sequences, and singletons from the 10-day-old tilapia brain EST library matched with were binding and transport activity. Recently, many researchers provide reasonable explanations for evolution, relation between genomic polymorphism and drug effects, and isoforms presentation of known proteins by collecting ESTs from open EST databases or from EST libraries they constructed, and we believe that this 10-day-old tilapia brain EST library can promote our ability to resolve questions about the topic we are researching in.

The amylolytic activities of alpha-amylase extracted from Mirror carp (Cyprinus carpio) and Nile tilapia (Oreochromis niloticus) were significantly reduced by purified amylase inhibitor albumin of wheat when tested under in vitro conditions. The action of this inhibitor was rapid and maximum levels of inhibition were attained within 20 minutes. For both carp and tilapia, the enzyme residual activities after inhibition were found to be related inversely to inhibitor concentration and positively to the initial enzyme activity levels. The curvilinear relationships between these parameters were explained by deriving equations of the type: A2 = a+b A1 - c I + d I2 where a, b, c and d are constants, Ai = Initial amylase activity (mU/min), A2 = Residual amylase activity (MU/min), I= Inhibitor concentration as ug protein. Inhibitions were greatest for amylases from gut tissue and ýowest for amylases from gut fluids. 1ug of purified inhibitor was found to contain a potency, to reduce 298 Units of carp gut tissue alpha-amylase and 532 Units of tilapia intestinal tissue alpha-amylase, by 50%. When amylase inhibitor extracted from wheat was incorporated in the feed of carp in its active form for three weeks, it caused a significant reduction in the specific growth rate to only 0.16%/day, while in carp fed autoclaved inhibitort such reduction in growth was not seen and the SGR was maintained at over 1.00%/day. However, despite the presence of active inhibitor in the intestine, the fish were able to maintain alpha-amylase activities in the gut contents at a level similar to that in fish fed denatured inhibitor. This was achieved by hyperactivation of enzyme secretions in the tissues of hepatopancreas and intestine. Hepatopancreas from fish fed active inhibitor exhibited more than two-fold increase in amylase, activity compared to those fed denatured inhibitor. By the third week of the experiment this difference in enzyme activity levels was not apparent but there were also no indications of adaptation or improvement in growth rate. Degenerations in hepatopancreas were also not apparent. Feeding carp with diet containing wheat with its inherent content of inhibitor also caused pancreas hyperactivity and some reduction in growth rate for a short period in comparison to those fish fed autoclaved wheat. In carp, the alpha-amylase activity did not vary depending on the raw or gelatinized nature of starch, both forms elicited equal increases in enzyme activity. However, autoclaving wheat, though effective in inactivating the inhibitor, was found to lower the biological value and digestibility of wheat proteins. Contrary to the result of the carp trials in Nile tilapia, the growth was not significantly reduced by feeding on diet containing active inhibitor and a SGR of 1.57%/day was recorded in comparison to 1.81i/day in tilapia fed denatured inhibitor. Samples of stomach and intestinal contents collected 4 hours after feeding did not reveal the presence of active inhibitor. Apparently the acidic protease, pepsin, in the stomach of tilapia caused the total destruction of the inhibitor in the diet before the contents were passed into the intestinal region. The presence of active amylase inhibitor in tilapia feed did not affect the digestibilities of starch and protein in the diet. Both the groups were able to digest carbohydrates and protein to levels of over 90%. The implications of these results are discussed in relation to feed formulation and fish nutrition.

In order to improve production efficiency and profitability in tilapia aquaculture, further research is needed to develop methods to improve weight gain, feed utilization, and immune function of these fish. In this regard, numerous studies with several fish species have reported that dietary nucleotides can enhance growth performance, immune responses and disease resistance. Therefore, two feeding experiments were conducted to investigate the effects of a purified nucleotide mix on juvenile Nile tilapia, Oreochromis niloticus. A basal diet was formulated to contain 34% crude protein from fishmeal and soybean meal. A nucleotide mix containing salts of cytidine, uridine, adenosine, inosine, guanosine, and thymine was supplemented to the basal diet at 0.5, 1 and 2% of dry weight. In the first experiment, three replicate groups of 15 fish were fed the experimental diets. At the end of 8 weeks, weight gain, feed efficiency and survival were computed. Blood samples were analyzed for neutrophil oxidative radical production and plasma lysozyme activity. In the second trial, three replicate groups of 20 fish were fed the same experimental diets. At the end of 4 weeks, blood and kidney samples were analyzed for macrophage extracellular and intracellular superoxide anion production, blood neutrophil oxidative radical production, plasma lysozyme activity, and peripheral blood lymphocyte proliferation. Then, 12 fish per treatment were challenged with Streptococcus iniae, via intraperitoneal injection, and mortality was recorded for 21 days. Results showed that none of the nucleotide-supplemented diets induced significant (P < 0.05) effects on growth performance. On the other hand, the 0.5% treatment produced significantly (P < 0.05) higher intracellular superoxide anion (O2-) production and both the 0.5 and 1% treatments significantly (P < 0.05) increased lymphocyte proliferation. The disease challenge failed to show significant survival differences among treatments; however, the 2% nucleotide treatment tended to produce higher survivability. Results from both experiments lead to the conclusion that this particular nucleotide mix does not provide marked improvements in growth performance and disease resistance; however, dietary nucleotide supplementation did affect some components of the immune system of Nile tilapia.

Triploid rainbow trout produced by heat shock and control (diploid) siblings were raised separately at similar density, feeding and water quality regimes. No significant differences in body weight or condition factors were observed; however the weight of the eviscerated carcass was on average 20% higher (P&lt;0.05) in triploid fish compared to diploid fish at 20 and 44 months post-hatching. The effects of triploidy on males were most evident during the final stages of spermatogenesis; in contrast, the gonadal development of triploid females was affected during its early stages, with the majority of the oogonia (30-70%) remaining within the oogonial clusters. A major finding was the presence of male-differentiating areas in most triploid females examined, which by the end of the sampling period appeared as gonadal hermaphrodites. Testicular weight, gonado-somatic index, sperm cell density and spermatozoa motility were significantly lower in triploid than in diploid male siblings, although some triploid males produced viable progeny when crossed to normal (diploid) females. Characterisation of this progeny by image analysis of nuclear DNA revealed the presence of a near-triploid genome. A single 5 month-old juvenile had developed testes in meiotic phase, providing a first evidence for the generation of limited numbers of viable progeny by autotriploid rainbow trout males. A cytogenetic analysis was carried out on monosex diploid and triploid populations of Nile tilapia. Synaptonemal complex analysis in diploid genotypes revealed the presence of an incompletely paired segment in the terminal region of the longest bivalent in heterogametic (XY) genotypes, which was not observed in homogametic genotypes. This unpaired region provides cytological evidence for the chromosomal basis of sex determination in O. niloticus. Meiotic analysis in triploids revealed the presence of longer (P&lt;0.0001) synaptonemal complexes in heterogametic (XXY) than in homogametic (XXX) genotypes, with a significantly different (P&lt;0.0001) nature of pairing evident between both groups. A model to explain the different progress in gametogenesis observed between male and female teleosts is discussed.

Reproductive traits, age of female broodfish and aspects of parental behaviour influencing the production and quality of fry in the mouthbrooding tilapia species Oreochromis niloticus and O. mossambicus were investigated. Two incubation containers (conical and round-bottomed) and rearing temperature were first studied to ascertain their effects on egg and fry performance. Partial agitation of eggs in round-bottomed containers significantly (P < 0.05) improved hatchability and success rate of fry production, by 17% and 25%, respectively, compared with conical containers. For the temperature trials only O. niloticus were studied. Two egg acclimation conditions were tested; these influenced the temperature range of hatching, hatching success and the upper and lower median temperature tolerance limits of artificially reared eggs. Thermal tolerance of eggs and fry decreased with progressive development and optimum (> 90%) survival and growth of swim-up fry occurred at 28°-3QoC. Hatching times were inversely related to temperature (P < 0.01) and rates of development to hatching were best described by a curvilinear relationship (P < 0.01). Growth rates, gross yolk utilization efficiency to maximum body weight, age at maximal body weight, onset of exogenous feeding and 50% irreversible starvation (point-of-no- return, PNR) were temperature-dependent. At 24•, 28' and 30' c (xi) maximum body weight occurred on days 18, 9 and 6 post-hatching, respectively, four days earlier than fry at 24' C. Similarly, PNR occurred on days 23, 20 and 18 at 24•, 28• and 30•C, respectively. Reproductive traits of 0+, 1+ and 2+ age-classes of broodfish were investigated. In O. niloticus mean dry egg weight and clutch weight were significantly (P < 0.05) different between all three age-classes, and yearlings produced the smallest eggs, whereas for total and relative fecundity only yearling females were significantly (P < o. 05) different to older broodfish. In both species broodfish age-class had no effect (P > 0.05) on egg:body weight ratio. In both species all reproductive traits were significantly related (P< 0.01) to female age, length and weight. The strongest influences were maternal age on egg size and maternal length and weight on total fecundity and clutch weight. The influence of maternal age and hence egg size on hatching time, and growth, survival, onset of feeding and PNR of fry developing solely on their yolk reserves was investigated. Larger eggs produced longer (P < 0.001) and heavier (P < 0.001) fry which sustained starvation stress longer (P < 0.001). Initial advantages of egg size on growth persisted through to 60 days post-hatching (P <0.05). Feeding success was improved by using of yearlings. For fry from fry from 1+ and 2+ females instead 0+, 1+ and 2+ O. niloticus and O. mossambicus females, PNR was reached on days 9, 12 and 12, and 12, 15 and 18, respectively. Delaying initial feeding beyond six days post-hatching significantly (P < 0.05) reduced the growth of fry. (xii ) Overall survival (between 6-20 days post-hatching) was improved by using older females. The effects of parental breeding behaviour on fry production and quality were investigated. Egg fertilizing capacity of males was inversely related to their number of spawnings in a day. During oral rearing cumulative fry damage increased linearly during the first eight days after spawning and plateaued at 25%-29%. Possible reasons for fry damage are discussed. Naturally reared fry were lighter (P < 0.05) than artificially reared 'siblings'. The implications of broodfish age and size and parental breeding behaviour for mass production of high quality tilapia fry and the need, advantages and feasibility of artificially rearing Oreochromis eggs and fry are discussed.

This study investigated the reproductive biology of Oreochromis niloticus broodstock of known age structure and spawning history with the aim of synchronising and controlling their spawning for mass fry production. Hatchery reared stock was subjected to a constant photoperiod of 12L:12D and maintained at 27 ± 1°C. All stock was fed on commercial trout pellets. The feeding frequency and protein content of the diet varied depending on fish size. Oocyte development was classified into 6 stages including that of atresia based on histology. In order to quantify ovarian maturity, three stereological methods were compared. The ovarian volume fractions of different oocyte stages estimated by the mass, graphical and intersection methods showed homogeneous results. The intersection method required less time (2.6 mins/sample) whereas the others needed 11-12 mins/sample. In addition, the numerical density technique employing the intersection method was used and yielded similar oocyte estimates to those derived from the Gilson's fluid method. Onset of sexual differentiation was influenced by the stocking densities. At 10 and 20 fry/l, 30 and 45% of those fry, respectively, were sexually differentiated by day 11 post-hatch, whereas those held at 2 fry/l were not. Gonadal development was monitored in fish of known age. Fry were randomly sampled after hatching at two week intervals until 24 weeks. Total body length and weight were recorded and gonads were fixed for maturity determination. Serum samples were analyzed for total calcium (Ca2+), testosterone (T) and oestradiol-17ß (E2). The males grew faster than the females of the same age and showed secondary sexual characteristics and attained maturity with significantly (P<0.05) higher T levels by 16 and 22 weeks, respectively. Females in comparison showed a significant (P<0.05) increase in GSI during 18-24 weeks (0.5-3.6%). The volume fraction of stage 6 oocytes, which were positively correlated to GSIs (r2=0.84; P<O.05), increased from 46.7% (20 weeks) to 71.8% by 22 weeks and then declined to 67.5% by 24 weeks. These results coincided with the mean levels of E2 whereas the Ca2+ and T levels showed high average levels through 24 weeks. These trials suggested that the females attained sexual maturity by 22 weeks. Ovarian recrudescence and average levels of Ca2+, T and E2 over 2 to 3 spawning cycles were studied. Within each spawning cycle the volume fraction of stage 6 oocytes increased from 0-15% (at day 1) to 65-72% by day 10 after spawning, which coincided with the high levels of Ca2+ and T whereas E2 levels peaked at day 5 and then decreased at day 10 after spawning. Females at day 10 post-spawning had, therefore, completed vitellogenesis and spawning occurred at the median time of 13 days. In addition, average hormonal levels, egg quality and quantity over 2 to 3 spawning cycles were monitored in eight individual females. Females were bled twice a week after their first spawning. The median of spawning cycles of these females for the first and second cycles were 13 (short cycle) and 28 days (long cycle), respectively, and their overall median spawning cycle was 15 days (short cycle). Levels of E2 were significantly (P<0.05; r2=0.79) correlated to the volume fractions of stage 6 oocytes and their peak levels were significantly correlated (P<0.05; r2=0.49) to fertilisation rates of eggs in subsequent spawns. Fecundity and fertilisation rates of eggs from those females in the second and third spawning were higher than the first spawning which indicated that the females that had spawned previously tend to ovulate more eggs than those that had spawned for the first time. The spawning history showed no effect on their fertilisation rates. The females which were selected by their external characteristics were either injected (10 to 300μg D-Ala6-Gly10-LHRH + 0.1mg pimozide/kg body weight) or implanted (fast or slow release pellets containing LHRH; 100μg/kg) with the hormones. Neither the injections nor LHRH pellets were effective in inducing the females to spawn. At day 10 after each spawning, a mixture of 100μg LHRH + 0.1mg pimozide/kg body weight was injected into the females kept under two spawning conditions. Females were held in either separated compartments (limited contact) or under normal communal spawning conditions (unlimited contact). Spawning environment affected the success of induced spawning. The females which were held in the separated compartments spawned within 2 to 6 days post-injection whereas the sham controls spawned in 7 to 8 days postinjection. In contrast, the females in the communal spawning environment did not respond to hormone induction. The timing at day ten post-spawning and the conditions of spawning were found to be the important factors affecting exogenous hormonal administration in this fish species.

Typescript. / Thesis (Ph. D.)--University of Hawaii at Manoa, 1984. / Bibliography: leaves 103-110. / Microfiche. / x, 110 leaves, bound ill., map 29 cm

Thesis (Ph.D.)--Memorial University of Newfoundland, 2004. / Bibliography: leaves 172-204.

Orientador: Joaquim Gonçalves Machado Neto / Banca: Júlio Vicente Lombardi / Banca: Robinson Antônio Pitelli / Resumo: Os objetivos deste trabalho foram avaliar a retenção do cátion do sulfato de cobre, usado para o controle de bloom algal, nos compartimentos biótico e abiótico de mesocosmos; determinar a influência das variáveis limnológicas no processo de retenção e a eficiência das concentrações de sulfato de cobre na redução do bloom algal (clorofila a). Foi avaliada a retenção de Cu+2 nos seguintes compartimentos dos mesocosmos: peixes: guarus - Poecilia reticulata; tilápias do Nilo - Oreochromis niloticus; gastrópodes - Lymnaea columella; sedimento e a concentração de Cu+2 na água. Os mesocosmos foram compostos por caixas de cimento amianto de 500 L contendo 0,08 m3 de sedimento, 420 L de água 80 guarus, 20 tilápias e 40 caramujos. A água dos mesocosmos foi mantida em sistema de fluxo contínuo de água com ciclo de renovação de 4 dias. O delineamento experimental utilizado foi o inteiramente ao acaso, com cinco tratamentos (0; 0,5; 1,0; 2,0 e 4,0 ppm de sulfato de cobre) e cinco repetições. Os compartimentos avaliados foram amostrados nos 0, 1, 4, 8, 12, 16, 20, 24, 28 dias após a aplicação do produto na água. O Cu+2 nas amostras foi quantificado em espectrofotômetro de absorção atômica. Também foram analisadas algumas variáveis limnológicas nas águas dos mesocosmos. A concentração de Cu+2 na água foi inversamente proporcional ao tempo de exposição, dependente do tempo de renovação da água e maior no 1º dia após a aplicação do sulfato de cobre para todos os tratamentos. A acumulação do Cu+2 no sedimento ocorreu até o 4º dia, para as doses 0,5 e 1,0 ppm (23,11 e 22,38 mCu. g-1 m.s.) e até o 8º dia, para 2,0 e 4,0 ppm (25,53 e 26,27 mCu.g-1 m.s.). Contudo, foi erodido nesses tratamentos, inclusive o Cu+2 existente no sedimento... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The aims of this paper were to evaluate the retention of the cation from the copper sulfate used to control algal bloom, over mesocosms ligands; to determine the influence of the limnological variables over this retention and the efficiency of the copper sulfate in reducing the algal bloom (chlorophyll a). The retention was evaluated on the following mesocosm ligands: fish: - guppy (Poecilia reticulata); - Nile tilapia (Oreochromis niloticus); gastropods - Lymnaea collumella; sediment and the Cu2+ concentration at the water. Mesocosms were composed by 500 L capacity cement asbestos boxes with 0.08 m3 of sediment, 420 L of water, 80 guppy, 20 tilapia and 40 gastropods. The mesocosm water was handled by a continuous water flux system with four days renewal cycle. Complete randomized experimental design was used and consisted of five treatments (0; 0.5; 1.0; 2.0 and 4.0 ppm of copper sulfate) and five repetitions. The evaluated ligands were sampled at 0, 1, 4, 8, 12, 16, 20, 24 and 28 days past the copper sulfate was applied on the water. The Cu2+ in the samples was quantified through atomic absorption spectrophotometer. Some limnological variables were also accessed on the water. The concentration of Cu2+ on water was inversely proportional to the exposition period, it depended on the water renewal cycle and was greater on the 1st day past the product application for all treatments. The accumulation of the Cu2+ occurred on sediment until the 4th day past the treatment for the 0.5 and 1.0 ppm doses (23.11 and 22.38 mCu.g-1 dry matter) and until the 8th day for 2.0 and 4.0 ppm (25.53 and 26.27 mCu.g-1 dry matter). Still the cation was eroded from the system, including the preexisting Cu2+. The guppy did not present bioaccumulation of the Cu2+ during the 28 days, except for the 16th day, when copper concentration was enhanced, mainly on the 4.0 ppm treatment (27.67 mCu.g-1 dry matter)... (Complete abstract, access undermentioned eletronic address) / Mestre