Dissertation (Ph.D.)--Auburn University, 2006. / Abstract. Vita. Includes bibliographic references.

Sex steroids are generally considered as natural sex inducers in fish. Aromatase (cytochrome P450 aromatase) catalyses androgens into oestrogens in the steroidogenic pathway. Three different approaches were taken to elucidate the action of aromatase in relation to sex differentiation in the Nile tilapia Oreochromis niloticus. The first was treatment with Fadrozole TM,a non-steroidal aromatase inhibitor (Al), by incorporating it in the diet or by immersing fish in a solution containing Al during the sex differentiation period. The Al treatment masculinised genetic females, indicating the importance of aromatase in sex differentiation. The result revealed that the most sensitive time to Al lies between 11-18 dpf (days post fertilisation). A partial brain type aromatase cDNA (1707bp) was identified from a brain cDNA library of O. niloticus. The amino acid sequence (that corresponds to exon 2-9) derived from this showed 63.7% identity to a previously reported ovarian aromatase gene of this species, and 96.7% identity to the brain type aromatase gene of a closely related species O. mossambicus. A semi-quantitative RT-PCR method was established to investigate expression of brain and ovarian aromatase genes during ontogeny. No sexually dimorphic expression of brain aromatase mRNA was detected. However, expression of ovarian aromatase was down-regulated from 15 to 23 dpf in genetic males but upregulated in genetic females. This period overlaps closely with the most sensitive period to Al. The pattern of temperature-dependent sex determination (TSD) was examined using three different genotypes (XX, XY, YY) at two temperatures (28 and 36°C). The results showed a bidirectional pattern of TSD. YY groups showed a significant percentage of feminisation at the higher temperature, which was suppressed by the Al treatment, implying that aromatisation is mechanistically associated with TSD in this species.All of these data consistently suggest that aromatase plays a crucial role in sex differentiation, and that the decisive aromatisation takes place between 13-25 dpf in this species. Considering the timing (26-30 dpf) of the first appearance of steroid producing cells in the gonadal area, the decisive aromatisation is not likely to take place there. The brain could be the primary aromatisation site in fish sex differentiation.

Thesis (doctoral)--Wageningen Agricultural University, 1998. / Summary and conclusion also in Dutch. Includes bibliographical references.

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.

Orientador: Gilson Luiz Volpato / Resumo: Este estudo investigou o perfil de ansiedade em Juverus de Tilápia-do-Nilo (Ore Niloticus). Num primeiro capitulo, determinamos um perfil comportamental indicativo de níveis de ansiedade. Num segundo capitulo, avaliamos o efeito de diferentes proporções de peixes com ansiedade alta (AA) e pouca (PA) na agressão e na produção de peixes em condições de aquário. No primeiro capitulo, por meio da modulação do comportamento de peixes usando drogas ansiolíticas (Diazepam, DZP) e ansiogênicas (Pentilenotetrazol, PTZ), demonstramos que o tempo gasto na natação foi um indicador confiável do nível de ansiedade (menor a locomoção, maior o nível de ansiedade). Também descobrimos que esse traço comportamental é melhor do que a medida de latência para sair do abrigo e explorar o ambiente novo. No segundo capitulo, outros peixes foram separados pelo critério de locomoção em perfis AA e PA e agrupados (6 peixes/aquário) para observação da agressão, do crescimento, da alimentação e da sobrevivência em 3 proporções: a) 50% de AA + 50% de PA; b) 100% PA e c) 100% AA. Nenhuma das variáveis observadas foi diferente entre essas composições de grupo / Abstract: This study investigated the anxiety profile in Nile tilapia juveniles (Oreochromis niloticus) by determining in a first chapter a behavioral profile to indicate levels of anxiety and, in a second chapter, the effect of different proportion of high (liA) and low (LA) anxiety fish on aggression and fish production in aquarium conditions. ln the first chapter, by modulating fish behavior with ansiolitic (Diazepam DZP) and ansiogenic (pentylenotetrazole PTZ) drugs, we demonstrated that time fish spent in swimming was a reliable indicator of anxiety levei (the lower the swimming, the higher the anxiety levei). We also found that this behavioral trait is better than measure of latency to exit shelter and explore a novel environment. Then, in the second chapter, other fish were separated by the locomotor criterium in HA and LA profiles and grouped (6 fish/aquarium) for aggression, growth, feeding and survival observations at three proportions: a) 50% HA + 50% LA, b) 100% HA, and c) 100% LA. Neither of the observed variables was different among these group compositions / Mestre

Made available in DSpace on 2014-06-11T19:30:12Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-02-26Bitstream added on 2014-06-13T19:39:30Z : No. of bitstreams: 1 silva_gv_me_botib_parcial.pdf: 90534 bytes, checksum: e5ae1fd42966c5fcbd0c44e74b7307e3 (MD5) Bitstreams deleted on 2015-03-16T11:30:16Z: silva_gv_me_botib_parcial.pdf,Bitstream added on 2015-03-16T11:31:01Z : No. of bitstreams: 1 000609983.pdf: 275068 bytes, checksum: c5458d6c71bd9cf6f1bf766448d6a3ba (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Nos identificamos percentuais de individuos que se distribuem no continuo hesitayaoousadia na tihipia-do-Nilo, Oreochromis niloticus (Linnaeus, 1758). Avaliamos a reayao de peixes isolados (100 peixes, 22.1 ± 5.9 g) a urn objeto-estimulo introduzido subitamente no aquario. Dividimos a parede frontal do aquario em quatm regioes iguais, atraves de linhas externas verticais. Apos isolamento por 24 h nesse aquario, assim que o peixe estava numa das duas extremidades, introduziamos subitamente 0 objetoestimulo (cilindro de PVC de 10 em diametro) na extremidade oposta (~ 30 em do peixe). Nos 5 min seguintes registravamos: a) tempo para sair da regiao inicial; b) tempo para chegar a regiao do objeto-estimulo; e c) tempo em locomoyao. Identificamos dois perfis extremos de resposta: permanencia na regiao inicial (peixes hesitantes) e aproximayao do objeto-estimulo em ate 40 s (peixes ousados). Este criterio resultou em 20% de individuos hesitantes, 29% de ousados e 51% de intermediarios, sendo os peixes ousados os mais ativos. Esses perfIS nao tiveram associayao com 0 sexo dos animais. Para testar a associayao dos perfis hesitante e ousado com a agressividade, uma nova amostra de peixes foi avaliada (115 peixes) como descrito acima, porem 24h horas depois urn segundo teste foi realizado, mas com outro objeto-estimulo introduzido no aquario. Os peixes considerados hesitantes ou ousados nos dois testes consecutivos foram submetidos a anaIise da agressividade. Esta foi avaliada atraves do teste do espelho (20 peixes de cada perfil) e pareamento entre coespecificos (10 duplas de cada perfil). Os peixes ousados foram mais agressivos que os hesitantes. Em urn terceiro estudo, esses peixes foram submetidos a estresse de confinamento por 30 min e o estresse foi avaliado pe1a mudanya na frequencia ventilatoria e colorayao do olho. Peixes ousados foram mais responsivos ao estresse. Conc1uimos que os perfis hesitante e ousado ocorrem na tilápia-do-Nilo,.... / We identified percentage of fish III the shy-bold continuum III the Nile tilapia, Oreochromis niloticus (Linnaeus, 1758). This was achieved from the reaction of isolated fish (100 fish, 22.1 ± 5.9 g) to a stimulus-object suddenly introduced into the aquarium. The frontal glass of the test aquarium was divided in four regions of same area by vertical lines outside the aquaria. After 24-h isolation in the test aquarium, when the fish was in one of the extremities of the aquarium, the stimulus-object (1O-cm diameter PVC cylinder) was suddenly immersed into the aquarium in the opposite side (- 30 cm from the fish). We registered in the next 5 min: a) latency to the fish exit from the initial area; b) time to reach the stimulus-object area; and c) time spent in locomotion. We could identify two extreme responses: staying in the initial area (fish considered shy) and approaching the stimulus-object in up to 40 s (bold fish). This criterion resulted in 20% shy, 29% bold and 51% intermediate fish, with the bold fish being more active. These profiles were not associated with fish sex. To test the association of shy and bold fish with aggression, these fish were identified in other sample (115 fish) as described above; 24 h later a second test was performed (but now with another object introduced). Fish considered shy or bold in these two consecutive testes were then tested for aggression. Aggression was evaluated by the mirror test (20 fish each profile) and also pairing with conspecific fish (10 pairs in each profile). Bold fish were more aggressive than the shy fish. In a third study, these fish were subjected to stress confinement for 30 min and stress evaluated in terms of changes in ventilation rate and eye color. Bold fish were the most responsive to stress. In conclusion, shy and bold profiles occur in the Nile tilapia, can be identified by reaction to a sudden stimulus and ...