Thesis (M.Sc.)--College of William and Mary. / Vita. Includes bibliographical references.

Summer flounder, Paralichthys dentatus, culture is becoming increasingly popular in the United States because of high market prices and consumer demand. In addition, flounder is a marine fish species that can tolerate a wide range of salinities, allowing for inland intensive fish culture. Oxytetracycline (OTC) is one of two available FDA-approved antibiotics for use in foodfish in the United States. Oxytetracycline was chosen for these studies because it is excreted primarily unchanged through the urine and the absorption, distribution and elimination of this drug may be influenced by environmental and physiological conditions. Four experiments were conducted to investigate: 1) pharmacokinetic parameters of oxytetracycline (50 mg/kg) following intravascular (IV), intraperitoneal (IP), intramuscular (IM) and per os (PO) administration in summer flounder maintained at 28 ppt salinity and 20°C; 2) pharmacokinetic parameters of OTC (50 mg/kg) following IM and PO administration in summer flounder maintained at three different salinity levels of 0 ppt, 15 ppt and 32 ppt and the physiological adjustments summer flounder make to acclimate to environmental salinity; 3) OTC retention times in muscle tissue from summer flounder maintained at three different salinity levels (0 ppt, 15 ppt, 32 ppt) and treated with a single 50 mg/kg OTC dose via IM and PO administration; and 4) pharmacokinetic parameters of OTC (50 mg/kg) following IM and PO administration in clinically healthy and clinically diseased summer flounder maintained at 28 ppt and 20°C. Oxytetracycline plasma concentrations were determined using high performance liquid chromatography (HPLC) and analyzed using a non-compartmental pharmacokinetic model for all routes of drug administration. Statistical comparisons were not made between the different routes of OTC exposure, but results from experiment one indicated that IV administration of OTC resulted in the largest area under the curve (AUC) value (8147.9 µg·h/ml) and the highest maximum plasma concentration (Cmax) of 1173.2 µg/ml OTC at 5 min post-injection. Intramuscular injections of OTC resulted in prolonged total body elimination half-life (T ½) of 301.3 h and high fish-to-fish variability (0.6). Per os administration resulted in low Cmax (0.2 µg/ml OTC) and poor systemic bioavailability (0.2 %). Results from experiment two demonstrated that when OTC is administered IM AUC estimates are significantly (p<0.05) lower in summer flounder held at 0 ppt (1684.8 µg·h/ml) than fish maintained at 15 ppt or 32 ppt salinity (2067.8 µg·h/ml and 2241.3 µg·h/ml, respectively). Although not significantly different from other salinity treatments, time to maximum plasma concentration (Tmax) was longer in fish held at 15 ppt and 32 ppt (312 h and 168 h, respectively) compared to cohorts in freshwater (0.5 h) and Cmax values were higher in animals held at 15 ppt and 32 ppt (8.4 µg/ml OTC and 9.2 µg/ml OTC, respectively) than freshwater fish (4.9 µg/ml OTC) when OTC was administered via IM injection. No significant differences were detected in any of the pharmacokinetic parameters following PO dosing of OTC, however, the AUC estimates were lower in the 32 ppt acclimated fish (127.7 µg·h/ml) than in the 0 ppt or 15 ppt acclimated fish (190.2 µg·h/ml and 180.7 µg·h/ml, respectively). In addition, the T ½ was longer in the higher salinity groups (278.1 h and 266.0 h, respectively) than in the freshwater fish group (256.9 h). Physiological adjustments made by summer flounder including plasma and urine osmolality, urine flow rate and urine character, gill chloride cell size and density, and Na+ - K+ ATPase activity demonstrated trends that suggested physiological differences among the salinity groups. Plasma and urine osmolalities were typically significantly (p<0.05) higher in fish maintained at 32 ppt salinity than at the lower salinity treatments. In addition, urine flow rates were generally significantly (p<0.05) greater in freshwater adapted fish (0.13 - 0.21 ml of urine/kg/hour) in comparison to fish in the salinity treatments of 15 ppt and 32 ppt (0.06 - 0.12 ml of urine/kg/hour and 0.09 – 0.11 ml of urine/kg/hour, respectively). Changes in gill chloride cell size and density and enzyme activity of Na+ - K+ ATPase revealed no significant differences between the salinity treatments but summer flounder in saltwater had numerically larger and more chloride cells than summer flounder in freshwater, but enzyme activity was greater in freshwater acclimated summer flounder compared to fish in seawater. Experiment three results revealed similar OTC muscle tissue pharmacokinetic parameters in summer flounder following IM injection. However, there were significant differences (p<0.05) in the AUC parameters of the plasma and muscle OTC concentrations between fish maintained at different salinities following IM OTC treatment. These effects may be the result of a "depot" effect in the muscle tissue or may be related to the reduced solubility of OTC in the muscle tissue of marine fish. A single PO dose administration of OTC at 50 mg/kg did not result in plasma or tissue concentrations higher than the FDA tissue tolerance limit of 2 ppm. Results of the fourth experiment demonstrated that following IM OTC administration healthy fish had significantly (p<0.05) higher AUC (4700.6 µg·h/ml) values than diseased cohorts (2576.2 µg·h/ml). Maximum plasma concentrations were also higher in the healthy fish than in the diseased fish, although values were not significantly different (23.4 µg OTC/ml and 20.2 µg OTC/ml, respectively for healthy and diseased fish). Additionally, in diseased fish, the mean resident time (MRT) (293.7 h) and T ½ (203.5 h) parameters were longer compared to parameters in healthy fish (253.2 h and 175.4 h, respectively), although values were not significantly different. No significant differences were detected in any of the pharmacokinetic parameters following PO OTC administration, however, healthy fish achieved higher maximum plasma OTC concentrations (1.0 µg OTC/ml) than diseased fish (0.7 µg OTC/ml). Fish-to-fish variation was greater in diseased animals than in healthy regardless of route of drug administration. The results of these experiments indicated that OTC pharmacokinetic parameters are influenced by route of drug administration, environmental salinity and fish health status. These factors must be considered by veterinarians and governmental regulators when developing treatment regimens for summer flounder. / Ph. D.

Thesis (Ph.D.)--University of Delaware, 2006. / Principal faculty advisor: Timothy E. Targett, College of Marine and Earth Studies. Includes bibliographical references.

Thesis (Ph.D.)--University of Delaware, 2008. / Principal faculty advisor: Timothy E. Targett, College of Marine and Earth Studies. Includes bibliographical references.

Paralichthys adspersus “lenguado”, especie endémica de las costas de Perú y Chile, es muy valorada comercialmente y los estudios demuestran la factibilidad de su cultivo. En tal sentido, la clasificación del desarrollo gonadal en escalas de madurez, viene a ser una fuente de información para conocer el ciclo reproductivo de esta especie y necesario para el manejo de los recursos en el medio natural así como en cautiverio. Los 162 individuos utilizados en la presente tesis, fueron adquiridos de capturas realizadas en la costa central del Perú. Los peces se evaluaron biométrica y biológicamente. Se analizaron y procesaron histológicamente 96 ovarios y 66 testículos. Se realizó la caracterización del desarrollo de los gametos y de las gónadas microscópica y macroscópicamente; además se evaluó la homogeneidad del ovario, la caracterización morfométrica de las poblaciones ovocitarias en cada estadio de madurez gonadal, la proporción sexual por grupos de longitudes totales, la talla de primera madurez gonadal y otros parámetros indicadores del desarrollo gonadal. Del grupo evaluado, las hembras representaron el 59,3% mientras que los machos el 40,7%. La caracterización de los ovocitos permitió clasificarlos en: Inmaduro, Pre–vitelogénicos, Vitelogénicos, Maduro e Hidratado y de las células espermatogénicas en: Espermatogonia, Espermatocito, Espermátida y Espermatozoide. Además, se observó Folículos Post-ovulatorios (FPO) y Atresia ovocitaria. Se tuvo en cuenta en este estudio, en el caso de las hembras, la no existencia de diferencias significativas en el desarrollo ovocitario en cualquier parte de la gónada; y en el caso de los machos, la diferenciación de una Zona de Crecimiento (ZC) y de Colección (ZCL). Se clasificó microscópicamente y macroscópicamente a los ovarios en los siguientes estadios de madurez gonadal: Inmaduro, En maduración, Maduro, Desovante, Recuperación e Inactivo; y a los testículos en: Inmaduro, En maduración, Maduro, Expulsante, Post-expulsante e Inactivo. En la proporción sexual de los peces se observó un crecimiento favorable de las hembras hacia mayores longitudes. La mayor parte de las capturas estuvo representada por peces maduros, desovantes o expulsantes en los meses que comprenden las estaciones de primavera y verano. El IGS e IHS son buenos indicadores del desarrollo gonadal en el caso de las hembras; contrariamente, el K no es un buen indicador en ninguno de los dos sexos. La prevalencia del parásito nematodo Philometra sp. fue de 19,14% y mayor en hembras, no se observó anormalidades producidas por la infección ni efectos en la maduración gonadal. La comparación de las características macro y microscópicas de las gónadas de peces sometidos al cautiverio no mostraron ninguna diferencia con respecto a las gónadas de los peces silvestres. / Tesis

Several flatfish species including southern flounder (Paralichthys lethostigma) recruit to estuaries during early life. Therefore, the evaluation of estuarine sites and habitats that serve as nurseries is critical to conservation and management efforts. I used biochemical condition and growth measurements in conjunction with catch-density data to evaluate settlement sites used by southern flounder in the Galveston Bay Estuary (GBE). In 2005, beam-trawl collections were made in three major sections of the GBE (East Bay, West Bay, Galveston Bay), and three sites were sampled in each bay. Within each sampling site, replicate collections were taken from 1) the marsh edge, 2) an intermediate zone, and 3) the open bay. The average size of southern flounder collected was between 12 and 19 mm standard length, and peak densities occurred in January and February. Catch data indicated that numeric densities of southern flounder were significantly greater in East Bay (2.75 per 100 m2) than in West Bay (0.45 per 100 m2) or in Galveston Bay (0.91 per 100 m2). Habitat-specific variation in density was not found. Otolith-based estimates of age indicated that the majority of southern flounder collected were 35-45 days old and derived from early December to early January hatch-dates. Growth rate differences were negligible across bays and habitats, with the average growth rate being 0.40 mm/day (range 0.21-0.76 mm/day). RNA:DNA ratios indicated that newly settled southern flounder in the GBE were in relatively high condition. Habitat-specific differences in RNA:DNA ratios were not observed; however, ratios were significantly lower in West Bay (average 8.0) than in East Bay (average 9.5) or in Galveston Bay (average 9.8), suggesting the condition of new recruits may vary across the GBE. Findings from this study indicate that southern flounder use a variety of habitats within the GBE during early life, and survival and recruitment success appear favorable regardless of settlement site. As a result, recruitment success of southern flounder may be less a function of the quality of nursery sites/habitats within the GBE than of other factors (e.g., larval supply to the estuary).

Thesis (M.S.)--University of Delaware, 2008. / Principal faculty advisor: Timothy E. Targett, College of Marine & Earth Studies. Includes bibliographical references.

Dissertaçao(mestrado)- Universidade Federal do Rio Grande, Programa de Pós-Graduação em Aqüicultura, Instituto de Oceanografia, 2008. / Submitted by Cristiane Silva (cristiane_gomides@hotmail.com) on 2012-07-10T00:50:00Z No. of bitstreams: 1 CarlosFrederico.pdf: 3216882 bytes, checksum: 2323f88ad7d58d3bbb5164aa5335dd23 (MD5) / Approved for entry into archive by Bruna Vieira(bruninha_vieira@ibest.com.br) on 2012-11-06T16:46:35Z (GMT) No. of bitstreams: 1 CarlosFrederico.pdf: 3216882 bytes, checksum: 2323f88ad7d58d3bbb5164aa5335dd23 (MD5) / Made available in DSpace on 2012-11-06T16:46:35Z (GMT). No. of bitstreams: 1 CarlosFrederico.pdf: 3216882 bytes, checksum: 2323f88ad7d58d3bbb5164aa5335dd23 (MD5) Previous issue date: 2008 / O linguado Paralichthys orbignyanus é um importante recurso pesqueiro do oceano Atlântico Sul e é considerado uma espécie com grande potencial para a aqüicultura. A qualidade do sêmen é uma importante variável no manejo reprodutivo de espécies cultivadas, influenciando diretamente na produção de ovos viáveis. Além disso, o conhecimento das características físicas e químicas do sêmen é um importante fator no desenvolvimento de protocolos de resfriamento e criopreservação do sêmen. O objetivo deste trabalho foi caracterizar o sêmen de P. orbignyanus ao longo da estação reprodutiva e utilizar essas informações para auxiliar no processo de criopreservação do sêmen e no desenvolvimento de um protocolo de transferência de genes mediada por espermatozóides (TGME). Na primeira etapa deste estudo, amostras de sêmen de linguados selvagens foram coletadas ao longo da estação reprodutiva. Através dos resultados obtidos foi verificado que a produção de espermatozóides e a motilidade progressiva aumentam significativamente à medida que a estação reprodutiva progride para o seu final (P<0.05). A motilidade progressiva dos espermatozóides durou em média 10 min, porém o tempo total de motilidade alcançou cerca de 100 min. Com relação ao pH, a osmolalidade e a concentração dos íons K+, Cl- e Mg+ dosados no plasma seminal, assim como, para o percentual de células móveis, nenhuma diferença foi verificada ao longo da estação reprodutiva. No meio da estação foi observada uma diminuição na concentração do íon Ca2+, a qual coincidiu com o menor tempo de motilidade dos espermatozóides. A concentração de Na+ foi aumentando ao longo dos períodos monitorados, atingindo a maior concentração no fim da estação reprodutiva (P<0,05). Na segunda parte deste estudo, a criopreservação do sêmen do linguado P.orbignyanus foi realizada. Para isso, foram testadas duas soluções crioprotetoras: uma contendo glicerol diluído numa solução a base de sais e outra contendo DMSO (dimetilsulfóxido) diluído numa solução à base de sacarose. Os dados de fertilização, eclosão e viabilidade larval demonstraram que as duas soluções utilizadas são eficientes na criopreservação do sêmen do linguado. Na última etapa deste estudo, alguns fatores limitantes na TGME em peixes foram avaliados e um protocolo de incorporação de DNA exógeno pelos espermatozóides foi desenvolvido. Os resultados obtidos nesta parte do trabalho demonstraram que o plasma seminal do P.orbignyanus apresenta uma forte atividade da enzima DNase. Entretanto, a atividade dessa enzima pode ser eliminada ou diminuída lavando o sêmen com soluções contendo EDTA. Além disso,foi verificado que uma quantidade de DNA exógeno similar ou inferior a 50 ng/106 espermatozóides deve ser usada na TGME em peixes. Por último, foi demonstrado que os espermatozóides do linguado P. orbignyanus são capazes de incorporar o DNA exógeno espontaneamente após a eliminação da atividade das DNases. Isto ficou evidente através da amplificação do DNA exógeno através de PCR. Desta forma, os resultados obtidos neste estudo podem auxiliar no manejo reprodutivo dessa espécie e,conseqüentemente, na implementação de programas de melhoramento genético tanto da forma clássica através da criopreservação do sêmen dos reprodutores que apresentam as melhores características fenotípicas, como através de técnicas modernas como a TGME. / The Brazilian flounder Paralichthys orbignyanus is an important fishing resource from southern Atlantic coast of America and it has being considered for aquaculture. The sperm quality is an important variable in reproductive management of cultured species, influencing directly on the production of viable eggs. Moreover, the adequate knowledge of physical and chemical characteristics of the sperm is an important factor for developing short-term storage and cryopreservation protocols of sperm. The aim of this study was to characterize the P. orbignyanus sperm throughout the reproductive season to facilitate the sperm cryopreservation and to help in development of a sperm mediated gene transfer (SMGT) protocol. In the first part of this study, sperm samples were collected from wild flounders throughout the reproductive season. It was verified that the sperm production and the progressive motility of spermatozoa increased significantly towards the end of reproductive season (P<0.05). The mean time of progressive motility of spermatozoa was 10 min, however the total time of motility reached about 100 min. No difference was observed during the reproductive season in pH, osmolality and concentrations of K+, Cl- e Mg+ measured in seminal plasma as well as in percentage of cells motility. In the middle of the breeding season was observed a reduction in Ca+2 concentration which has coincided with the lesser motility time of spermatozoa. The Na+ concentration increased throughout the season, reaching the highest concentration at the end of reproductive period (P<0.05). In the second part of this study, the cryopreservation of the flounder sperm was carried out. Two different cryosolutions were tested: glycerol saline and DMSO - sucrose. The results of fertilization, hatching rate and larval viability demonstrate that both solutions are efficient in the cryopreservation of the flounder sperm. In the last part of this study, some limitant factors for exogenous DNA uptake by spermatozoa were evaluated and a SMGT protocol was developed. It was observed a strong DNase activity in P.orbignyanus seminal plasma. However, it was verified that DNase activity can be eliminated or decreased washing the sperm with EDTA-containing solutions. Moreover, it was verified that an amount of exogenous DNA similar or inferior to 50 ng/106 cells should be used in fish SMGT. At last, it was demonstrated that fish spermatozoa are capable to uptake exogenous DNA spontaneously after DNase activity elimination. This was evidenced through exogenous DNA amplification by PCR. The results of the present study should help in reproductive management of this species and consequently in implementation of genetic improvement programs based not only in the sperm cryopreservation, but also using modern techniques such as SMGT.

Made available in DSpace on 2014-08-20T13:32:53Z (GMT). No. of bitstreams: 1 dissertacao_vinicius_campos.pdf: 483644 bytes, checksum: 9caf292597102dea88bfa2da68dc9158 (MD5) Previous issue date: 2009-03-03 / Food intake in vertebrates is a complex process involving several endocrine and neural pathways. Among the orexigenic factors, the most notably are the neuropetides as the neuropeptide Y (NPY) that is a 36 amino acid peptide which plays a key role in food intake. Studies evaluating fish NPY expression showed that this peptide is involved in appetite stimulation. However, despite the recent advances, our present knowledge of the regulation of feeding behavior in fish is limited and based in a few fish species, and there is increasing evidence of speciesspecific differences. The Brazilian flounder Paralichthys orbignyanus is being considered for aquaculture, and it is important to understand the mechanisms regulating feeding in order to improve its performance in captivity. The objectives of this study were to clone NPY cDNA, evaluate the mRNA levels in different tissues of flounder, and also evaluate brain NPY expression during 24 h by real-time RT-PCR. A 597 bp NPY cDNA was cloned from Brazilian flounder brain by 3´RACE method. NPY expression was detected in all peripheral tissues analyzed, but with predominant expression in the brain. No significant differences were observed in brain NPY gene expression over a 24 h evaluation period. No correlation was observed among plasma glucose, total protein, cholesterol, triglycerides and NPY expression levels during 24 hours. These results indicated that NPY may play roles in flounder peripheral tissues and may be not involved short-term regulation of food intake at low-temperatures in Brazilian flounder. / O apetite e a ingestão de alimentos em vertebrados é um processo complexo que envolve várias rotas neurais e endócrinas. Dentre os fatores orexigênicos, destacamse os neuropeptídeos, como o neuropeptídeo Y (NPY), o qual é constituído de 36 aminoácidos e desempenha papel chave na regulação do apetite. Estudos avaliando a expressão do NPY em peixes demonstraram que este peptídeo está envolvido no estímulo da ingestão. Entretanto, apesar dos avanços recentes, o atual conhecimento sobre a regulação fisiológica do apetite é limitado e baseado em poucas espécies de peixes com evidencias de diferenças interespecíficas. O linguado Paralichthys orbignyanus tem sido considerado para a aqüicultura e com isso, torna-se importante compreender os mecanismos de regulação do apetite para incrementar o seu desempenho em cativeiro. Os objetivos deste trabalho compreenderam clonar o gene do NPY, avaliar os níveis de RNA mensageiro nos diferentes tecidos do linguado e também avaliar a expressão do NPY no cérebro durante 24 h por RT-PCR em tempo real. Um fragmento de 597 pb do cDNA do NPY foi clonado a partir do RNAm do cérebro do linguado através do método RACE 3 . A expressão do NPY foi detectada em todos os tecidos analisados (cérebro, baço, coração, intestino, estômago, brânquia, fígado, rim, músculo e testículo), mas com predominante expressão no cérebro. A expressão do NPY não cérebro demonstrou variações durante 24 horas de avaliação. Nenhuma correlação foi observada entre glicose, proteínas totais, colesterol e triglicerídeos plasmáticos. Estes resultados indicam que o NPY pode desenvolver funções em outros tecidos além do cérebro e também pode não estar associado na regulação da alimentação em curto prazo em baixas temperaturas.

Essential fatty acids are necessary for growth, survival, and development of larval fishes, but there is limited information on the essential fatty acid requirements of larval southern flounder (Paralichthys lethostigma). The objectives of this study were to elucidate connections between dietary supply of docosahexaenoic acid (DHA) and arachidonic acid (ARA) and deposited fatty acids in the head or body, and then link diet and stored fatty acids in the head or body with larval quality traits. From 4-15 days posthatch (dph), southern flounder larvae were fed rotifers enriched with four different combinations of DHA-rich Algamac 3050 and ARA-rich Algamac ARA. Fatty acid concentrations in the head and body were measured at 15 dph, and relationships between fatty acids in head or body and in the diet were determined. Larval quality traits, including specific growth rate (SGR), survival, and eight behavioral performance variables were measured. Results showed that concentrations of DHA and ARA in the head and in the body were correlated with concentrations of DHA and ARA in the diet. Growth rate did not vary among the four diets, but survival was positively correlated with the amount of lipid in the diet. Responsiveness to a visual stimulus was positively correlated with the concentration of DHA in the diet, the ratio DHA:EPA in the head, and total energy content of the diet. Turning rate during routine swimming was correlated with body DHA. This study demonstrates the influence of DHA content, total lipid content, and energy levels in the diet of southern flounder and provides a foundation for future studies examining causal factors of recruitment variability or larviculture production success. / text