This thesis is missing pages 87, 172 and 255. No other copies of the thesis have these pages. -Digitization Centre / This thesis examined the effects of exhaustive exercise on acid-base and metabolite status in the intracellular and extracellular compartments of two very different fish species: the active, pelagic rainbow trout, and the sluggish, benthic starry flounder. In both species, exhaustive exercise resulted in an acidosis in the extracellular compartment of mixed respiratory and metabolic origin. Despite the reduction in pHe, red blood cell pHi was well regulated, though more precisely in trout than in flounder. Catecholamines were mobilized into the blood after exercise in trout but not in flounder. Circulating catecholamines may play an important role in regulating red blood cell (RBC) pHi in trout after exercise. In trout, lactate appeared in the blood in excess of H+ ; the reverse pattern was observed in flounder. H+ appearance was similar in both species. Differential release of lactate from the muscle mass was apparently responsible for this discrepancy. After exhaustive exercise, both trout and flounder experienced a severe intracellular acidosis in the white muscle, as measured by 14c-DMO (5,5-dimethyl -2,4-oxazolidinenione) distribution. H+ and lactate were not produced in equimolar quantities, with H+ produced in excess of lactate. Muscle lactate and H+ production was about 3-fold lower in flounder than in trout. The muscle intracellular acid-base disturbance was corrected more rapidly in flounder (4-8h) than in trout (8-12h). In flounder, this occurred prior to , but in trout after , correction of the extracellular acidosis. In flounder, a more rapid correction of muscle metabolite status was associated with the more rapid correction of the intracellular acidosis. After exercise there was a reduction in the whole body extracellular fluid volume and expansion of the intracellular fluid volume, largely reflecting changes within the muscle. This fluid shift resulted in a general hemoconcentration. Exercise led to a transient increase in net H+ excretion in both trout and flounder. Negligible amounts of lactate were transferred to the water. In flounder, about 20% of the total H+ load produced passed through the extracellular space and was transiently stored in the water, which appeared to hasten correction of the intracellular acid-base disturbance. In contrast, in trout, a much smaller portion of the acid load (about 6%), though about the same absolute amount as in flounder, was transferred to the water. This appeared to expedite correction of the extracellular acidosis.The results of this thesis argue against a prominent role for the Cori cycle in the final disposition of the lactate burden produced during exercise. Instead, it is suggested that the bulk of the lactate was metabolized in situ, either by oxidation or glyconeogenesis. In flounder, this was almost the sole fate of lactate, as very little appeared in the blood space. In trout, a significant portion of the lactate was exported to the blood, which was taken up and metabolized by aerobic tissues. / Thesis / Doctor of Philosophy (PhD)

O objetivo deste estudo foi avaliar os efeitos da suplementação de vitamina C nos biomarcadores de estresse oxidativo induzido por exercício físico exaustivo. A amostra foi composta por 13 indivíduos do sexo masculino, fisicamente ativos, com idades entre 18 e 33 anos, IMC médio 23,65 ± 3,5 kg/m² e VO2max de 50,94 ± 5,2 ml.kg-1.min-1. Estes indivíduos foram submetidos a um protocolo de exercício exaustivo (40 minutos de corrida a 70-75% do VO2max, e aumento da velocidade para 90% do VO2max até a exaustão), randomizados duplo cego em duas fases (placebo X vitamina C). Na fase vitamina C eles ingeriram 500 mg de vitamina C por dia, durante 7 dias, antes do protocolo exaustivo, e na fase placebo eles ingeriram por sete dias placebo antes do mesmo protocolo. Foram coletadas amostras de sangue antes do exercício, logo após, 1h, 2h, 4h e 24h após para determinação dos biomarcadores de estresse oxidativo. As duas fases foram homogêneas, os indivíduos apresentaram consumo alimentar similar e não houve diferença na duração e intensidade do exercício. Os resultados mostram que o uso de suplemento de vitamina C favoreceu maiores níveis séricos de ácido ascórbico em relação ao placebo logo depois do exercício até 4 horas após, e por isso poupou a eliminação dos outros antioxidantes, -tocoferol e retinol, que estavam em menores concentrações séricas até 2 horas após o exercício, e do GSH, que se manteve em menores concentrações até 24 horas após. Enquanto com uso de placebo, já que os níveis de ácido ascórbico eram menores, houve uma maior liberação destes outros antioxidantes, e o -tocoferol e retinol começaram a ser consumidos 1 hora após o exercício, para combater os radicais livres formados, até 24 horas após. Apesar da CAT não ser alterada nem pela suplementação nem pelo exercício exaustivo, o FRAP foi maior na fase vitamina C e se manteve estável nas 24h após o exercício. O ácido úrico não apresentou diferença significativa, e seu uso foi poupado por outros antioxidantes séricos nas duas intervenções. Nas duas fases os indivíduos conseguiram combater os radicais livres formados pelo exercício exaustivo, já que os marcadores de peroxidação lipídica (MDA, TBARS e FOX) não aumentaram após o exercício em nenhuma das intervenções, não houve aumento de oxidação protéica (AOPP e PC). Os marcadores de lesão hepática (TGO), lesão muscular (CK) e tecidual (LDH) se comportaram de maneira igual com ou sem uso de vitamina C. As vantagens do uso adicional de vitamina C são: a maior proteção antioxidante desde o ínicio do exercício, já que apresenta maiores concentrações séricas de vitamina C e de FRAP, o menor grau de peroxidação lipídica, avaliado pelo MDA, o menor grau de oxidação protéica, avaliado pelas PC e a menor dependência do GSH como antioxidante endógeno. Sendo assim, uso de uma dose modesta de vitamina C, por um curto período de tempo, foi capaz de auxiliar na proteção antioxidante, sem nenhum efeito danoso aos indivíduos. / The objective of this study was to evaluate the effects of vitamin C supplementation on oxidative stress biomarkers induced by exhaustive exercise. The sample consisted of 13 males, physically active, aged 18 to 33 years, mean BMI 23.65 ± 3.5 kg/m² and 50.94 ± 5.2 VO2max ml.kg-1. min-1. These individuals were subjected to an exhaustive exercise protocol (40 minutes of running at 70-75% of VO2max, and increased speed to 90% VO2max until exhaustion), randomized double-blind in 2 phases (placebo X vitamin C). In vitamin C phase they ingested 500 mg vitamin C per day for 7 days before exhausting protocol and the placebo phase, they ingested placebo for 7 days prior to the same protocol. Blood samples were taken before exercise, immediately after, 1h, 2h, 4h and 24h after for determination of biomarkers of oxidative stress. The two phases were homogeneous, the subjects had similar food consumption and no difference in the duration and intensity of exercise. The results show that the use of vitamin C supplement favored higher serum levels of ascorbic acid relative to placebo soon after exercise to 4 hours after, and therefore spared the elimination of other antioxidants, -tocopherol and retinol, which were lower serum concentrations up to 2 hours after exercise, and GSH, which remained at lower levels within 24 hours. As with the use of placebo, as the ascorbic acid levels were lower, there was a greater release of these other antioxidants, and -tocopherol and retinol began to be consumed in 1 hour of exercise to combat free radicals formed by 24 hours. Despite the CAT not be altered or by supplementation or by exhaustive exercise, the FRAP was higher in vitamin C phase and remained stable in the 24 hours after exercise. Uric acid showed no significant difference, and its use has been spared by other serum antioxidants in 2 interventions. In 2 phases individuals able combat free radicals formed by exhaustive exercise, since the lipid peroxidation markers (MDA, TBARS and FOX) did not increase after exercise in any of the interventions, there was no increase in protein oxidation (AOPP and PC) . The markers of liver injury (AST), muscle damage (CK) and tissue (LDH) behaved equally with or without use of vitamin C. The advantages of the additional use of vitamin C are: the highest antioxidant protection since the beginning of exercise, since it has higher serum concentrations of vitamin C and FRAP, the lowest degree of lipid peroxidation, measured by MDA, the lowest level of protein oxidation, rated by PC and less dependent on GSH as an endogenous antioxidant. Thus, use of a modest dose of vitamin C, for a short period of time, was able to assist in antioxidant protection, with no harmful effect on individuals.

Made available in DSpace on 2015-07-22T20:49:55Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-04-28 / Objetivo: Verificar o efeito inflamatório do exercício agudo exaustivo sobre diferentes depósitos de tecido adiposo branco (mesentérico e retroperitoneal), assim como, sobre músculos esqueléticos (sóleo: fibra muscular tipo I; EDL: fibra muscular tipo II). Métodos: os animais (ratos Wistars) foram submetidos a uma sessão exaustiva de exercício, após período de adaptação. Os depósitos de tecido adiposo branco, retroperitoneal e mesentérico, como, os músculos EDL e sóleo, foram retirados logo após o sacrifício e analisados para a expressão gênica (PCR-real time) e protéica (ELISA) da citocinas IL-6, IL-10, TNF-α, IL-2 e IL-4. O sacrifício foi realizado logo após a sessão de exercício, duas e seis horas após, e o grupo controle não foi submetido à sessão exaustiva de exercício. Por Western Blotting foi analisado o conteúdo de TLR-4, e sua via de sinalização TRAF6 e MYD88. Resultados: As citocinas IL-6, IL-10 e TNF-α foram encontradas elevadas em todos os tecidos e grupos dos ratos exercitados. No entanto, a razão IL-10/TNF-α foi maior no músculo e encontrou-se diminuída no tecido adiposo. O TLR-4, assim como sua via (TRAF6 e MYD88), foi aumentada nos depósitos de tecido adiposo branco de ratos submetidos ao exercício exaustivo, sem alteração no EDL e sóleo. Já as citocinas IL-2 e IL-4 tiveram suas concentrações alteradas no sóleo e EDL, mas não foram moduladas no tecido adiposo pelo protocolo de exercício exaustivo. Conclusão: O exercício exaustivo apresentou um aumento da resposta antiinflamatória no músculo esquelético, pelo aumento da razão IL-10/TNF-α. Já o tecido adiposo observado (mesentérico e retroperitoneal) apresentou um aumento da resposta inflamatória, evidenciada pela diminuição da razão IL-10/TNF-α e pelo aumento da expressão protéica do TLR-4. Além disso, a IL-4 foi aumentada 6 horas após o protocolo de exercício no EDL, concomitante com o aumento do MyoD. Esse aumento pode levar a um aumento da hiperplasia muscular. / Aim: to verify the inflammatory effects of exhaustive exercise on adipose tissue (mesenteric and retroperitoneal), and skeletal muscle (sóleus: fiber type I, EDL: Fyber type II). Methods: Wistar rats were randomly assigned to four groups: group control and group exercised until exhaustion, these rats were sacrificed in three different times, after the exhaustion, two hours after and six hours after exhaustive exercise. Cytokines IL-2, IL-4, TNF-α, IL-6 and IL-10 were analyzed by mRNA (PCR Real Time) and protein (ELISA) expression. TLR-4 and your inflammatory via (TRAF6, MYD88), were analyzed by Western Blotting. Results: IL-6, IL-10 and TNF-α were elevated in all tissues and groups compared with control group. However, IL-10/TNF-α ratio was higher in skeletal muscle, and opposite response was found in adipose tissue. TLR-4 and your via was elevated in adipose tissue, but not altered in skeletal muscle after exhaustive exercise. The concentrations of IL-2 and IL-4 were altered in skeletal muscle, but were not modulated in adipose tissue, after exercise protocol. Conclusion: exhaustive exercise showed one increased in anti-inflammatory response in skeletal muscle, by increased of IL-10/TNF-α ratio. On the other hand, adipose tissue showed increased in inflammatory response, evidenced by decreased in IL-10/TNF-α ratio and increased in TLR-4 content. Moreover, the IL-4 was increased in EDL, concomitant with raised in Myod. This results could showed one increased in hyperplasia in skeletal muscle. / TEDE / BV UNIFESP: Teses e dissertações

Orientadores: Denise Vaz de Macedo, Dagmar Ruth Stach-Machado / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-15T21:15:50Z (GMT). No. of bitstreams: 1 Ferrucci_DaniloLopes_M.pdf: 1810214 bytes, checksum: 4728c1ea4c33f76583148c1588199a23 (MD5) Previous issue date: 2018-08-15T18:15:37Z / Resumo: Os músculos esqueléticos são constituídos por fibras musculares e pela matriz extracelular. Em resposta ao exercício, o músculo apresenta a habilidade de transformar o estresse mecânico da contração muscular em adaptações de cunho biológico, a esse processo deu-se o nome de mecanotransdução. Embora pouco se saiba sobre a importância da matriz extracelular como mecanoreceptor, trabalhos anteriores mostram que o exercício pode causar alterações na expressão e atividade das metaloproteinases, que podem resultar em modificações na matriz extracelular. Nesse contexto, as MMPs são vistas como pivôs centrais e, através da degradação da matriz extracelular podem gerar fragmentos protéicos com funções distintas das proteínas integras, liberando ainda citocinas e fatores de crescimento associados à matriz, que em interação com receptores celulares fornecem informações à célula sobre o microambiente extracelular, regulando o comportamento e adaptação do tecido. O objetivo do presente estudo foi avaliar o efeito de três sessões diárias de exercício exaustivo, realizados com diferentes pausas recuperativas entre as sessões, grupo E1(1h) e grupo E2 (3h), durante 6 dias consecutivos, na expressão das MMPs 2 e 9 e dos TIMPs 1 e 2; e nas concentrações e atividade biológica das MMPs nas porções vermelha (GV) e branca (GB) do músculo Gastrocnêmio de ratos Wistar. Os animais foram sacrificados após 1°, 3° e 6° dias de exercício e os fragmentos coletados do músculo gastrocnêmio foram analisados via ELISA, zimografia e qRT-PCR. Como controle (C) do protocolo experimental utilizou-se ratos sedentários. Os animais exercitados (E) mostraram aumento de desempenho a partir do 4° dia. Não houve diferença significativa na perfomance dos grupos E1 e E2. Os genes analisados mostraram-se igualmente expressos em resposta ao exercício em ambas as porções do músculo gastrocnêmio, contudo, o grupo E1 apresentou alterações mais acentuadas do que E2 para todos os biomarcadores analisados. O grupo E1 mostrou aumento na expressão das MMPs 2 e 9 no 3° dia de exercício, em ambas as regiões do músculo gastrocnêmio. Com relação aos TIMPs 1 e 2, os dados obtidos demonstram o aumento na expressão no GV e GB em resposta ao exercício (E1 e E2), em todos os tempos experimentais analisados. Todavia esse aumento foi mais acentuado para o grupo E1. A concentração total de MMP-2 nos grupos e E1 e E2 mostrou-se diminuída em todos os períodos analisados, para GV e GB. A atividade das isoformas latente e intermediária da MMP-2 apresentou-se diminuída em ambos os grupos exercitados no GB e apenas no grupo E1 para o GV. A atividade e concentração da MMP-9 não foram detectadas neste estudo. O protocolo agudo utilizado nesse estudo induziu um aumento significativo no desempenho dos animais, independente do tempo de pausa entre as sessões. O efeito adaptativo observado nas MMPs 2 e 9 em GV e GB foi de diminuição quantitativa e qualitativa. Possivelmente o aumento significativo na expressão gênica tanto das MMPs quanto TIMPs no grupo E1, permitiu um remodelamento acelerado na matriz extracelular do tecido, que possibilitou a melhora significativa no desempenho a partir do 3° dia. Considerando que ambos os tempo de pausa foram igualmente eficientes para aumentar a performance, seria melhor utilizar uma pausa de 3h entre as sessões, pois o processo de sinalização de síntese protéica demanda grande quantidade de energia, e depende da oferta de nutrientes em quantidade e qualidade adequada para a resposta / Abstract: Skeletal muscles are composed by muscle fibers and extracellular matrix. In response to exercise, muscles have the ability to transform mechanical stress from their contraction in biological adaptations, and, this process was called echanotransduction. Although little is known about the importance of the extracellular matrix acting as mechanoreceptors in this process, previous studies have shown that exercise can cause changes in the MMPs genetic expression and activity on the extracellular matrix. In this context, MMPs play the central role, once that the extracellular matrix degradation can generate fragments that are bioactive compounds and can interact with cell receptors to provide information regarding to extracellular microenvironment to the cells. Therefore, MMPs can regulate the behavior and adaptation of the muscle. The aims of this study were to evaluate the effect of three daily sessions of exhaustive exercise (performed with different recuperative breaks between sessions for six consecutive days) on the MMP-2, MMP-9 and tissue inhibitors of metalloproteinases 1 and 2 (TIMPs) gene expression, as well as, MMP's concentration and biological activities on the rat gastrocnemius muscle red (RG) and white (WG) portions. The animals were sacrificed on exercise-days 1, 3 and 6 and muscle fragments were collected and stored for later analysis through zymography, ELISA and qRT-PCR. Sedentary rats were used as controls. The exercised animals (E) showed an increased performance from the 4th day (p <0.05). There was no statistical difference between the performance of E1 and E2 groups. The genes examined were similarly expressed in response to exercise on the analyzed muscle regions, however, the group E1 have more pronounced changes than E2 when these biomarkers was analyzed. The E1 showed an increased expression of MMP-2 and -9 on the 3rd day of exercise comparing to the control group for RG e WG. Regarding TIMPs 1 and 2, our data showed an increase on their expression in RG and WG responsive to exercise (E1 and E2) during all time experimental points, but this increase was more pronounced in group E1. Furthermore, total concentration of MMP-2 in both groups was significantly reduced when compared to sedentary animals for all time points and muscle regions. The activity of latent and intermediate MMP-2 isoforms were significantly reduced in both exercised groups in the WG and, only, in the E1 to the RG. MMP-9 concentration and activities were not detected in this study. The acute protocol designed for this study induced an increase in the animal performances, regardless of the pauses employed between sessions. The adaptive effect observed in MMP-2 and 9 in the RG and WG was reduced quantitatively and qualitatively, due to the increased expression of both MMPs and TIMPs in group E1 leading to an accelerated remodeling the gastrocnemius extracellular matrix, allowing a significant improvement on performance since the third day onwards. Considering that both rest periods were equally effective to improve physical performance, rest periods of 3 hours between sessions are more adequate due to processes of protein synthesis, once that demands a great amount of energy and depends on the supply of nutrients in quantity and quality suitable for the positive response / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular

O objetivo deste estudo foi avaliar os efeitos da suplementação de vitamina C nos biomarcadores de estresse oxidativo induzido por exercício físico exaustivo. A amostra foi composta por 13 indivíduos do sexo masculino, fisicamente ativos, com idades entre 18 e 33 anos, IMC médio 23,65 ± 3,5 kg/m² e VO2max de 50,94 ± 5,2 ml.kg-1.min-1. Estes indivíduos foram submetidos a um protocolo de exercício exaustivo (40 minutos de corrida a 70-75% do VO2max, e aumento da velocidade para 90% do VO2max até a exaustão), randomizados duplo cego em duas fases (placebo X vitamina C). Na fase vitamina C eles ingeriram 500 mg de vitamina C por dia, durante 7 dias, antes do protocolo exaustivo, e na fase placebo eles ingeriram por sete dias placebo antes do mesmo protocolo. Foram coletadas amostras de sangue antes do exercício, logo após, 1h, 2h, 4h e 24h após para determinação dos biomarcadores de estresse oxidativo. As duas fases foram homogêneas, os indivíduos apresentaram consumo alimentar similar e não houve diferença na duração e intensidade do exercício. Os resultados mostram que o uso de suplemento de vitamina C favoreceu maiores níveis séricos de ácido ascórbico em relação ao placebo logo depois do exercício até 4 horas após, e por isso poupou a eliminação dos outros antioxidantes, -tocoferol e retinol, que estavam em menores concentrações séricas até 2 horas após o exercício, e do GSH, que se manteve em menores concentrações até 24 horas após. Enquanto com uso de placebo, já que os níveis de ácido ascórbico eram menores, houve uma maior liberação destes outros antioxidantes, e o -tocoferol e retinol começaram a ser consumidos 1 hora após o exercício, para combater os radicais livres formados, até 24 horas após. Apesar da CAT não ser alterada nem pela suplementação nem pelo exercício exaustivo, o FRAP foi maior na fase vitamina C e se manteve estável nas 24h após o exercício. O ácido úrico não apresentou diferença significativa, e seu uso foi poupado por outros antioxidantes séricos nas duas intervenções. Nas duas fases os indivíduos conseguiram combater os radicais livres formados pelo exercício exaustivo, já que os marcadores de peroxidação lipídica (MDA, TBARS e FOX) não aumentaram após o exercício em nenhuma das intervenções, não houve aumento de oxidação protéica (AOPP e PC). Os marcadores de lesão hepática (TGO), lesão muscular (CK) e tecidual (LDH) se comportaram de maneira igual com ou sem uso de vitamina C. As vantagens do uso adicional de vitamina C são: a maior proteção antioxidante desde o ínicio do exercício, já que apresenta maiores concentrações séricas de vitamina C e de FRAP, o menor grau de peroxidação lipídica, avaliado pelo MDA, o menor grau de oxidação protéica, avaliado pelas PC e a menor dependência do GSH como antioxidante endógeno. Sendo assim, uso de uma dose modesta de vitamina C, por um curto período de tempo, foi capaz de auxiliar na proteção antioxidante, sem nenhum efeito danoso aos indivíduos. / The objective of this study was to evaluate the effects of vitamin C supplementation on oxidative stress biomarkers induced by exhaustive exercise. The sample consisted of 13 males, physically active, aged 18 to 33 years, mean BMI 23.65 ± 3.5 kg/m² and 50.94 ± 5.2 VO2max ml.kg-1. min-1. These individuals were subjected to an exhaustive exercise protocol (40 minutes of running at 70-75% of VO2max, and increased speed to 90% VO2max until exhaustion), randomized double-blind in 2 phases (placebo X vitamin C). In vitamin C phase they ingested 500 mg vitamin C per day for 7 days before exhausting protocol and the placebo phase, they ingested placebo for 7 days prior to the same protocol. Blood samples were taken before exercise, immediately after, 1h, 2h, 4h and 24h after for determination of biomarkers of oxidative stress. The two phases were homogeneous, the subjects had similar food consumption and no difference in the duration and intensity of exercise. The results show that the use of vitamin C supplement favored higher serum levels of ascorbic acid relative to placebo soon after exercise to 4 hours after, and therefore spared the elimination of other antioxidants, -tocopherol and retinol, which were lower serum concentrations up to 2 hours after exercise, and GSH, which remained at lower levels within 24 hours. As with the use of placebo, as the ascorbic acid levels were lower, there was a greater release of these other antioxidants, and -tocopherol and retinol began to be consumed in 1 hour of exercise to combat free radicals formed by 24 hours. Despite the CAT not be altered or by supplementation or by exhaustive exercise, the FRAP was higher in vitamin C phase and remained stable in the 24 hours after exercise. Uric acid showed no significant difference, and its use has been spared by other serum antioxidants in 2 interventions. In 2 phases individuals able combat free radicals formed by exhaustive exercise, since the lipid peroxidation markers (MDA, TBARS and FOX) did not increase after exercise in any of the interventions, there was no increase in protein oxidation (AOPP and PC) . The markers of liver injury (AST), muscle damage (CK) and tissue (LDH) behaved equally with or without use of vitamin C. The advantages of the additional use of vitamin C are: the highest antioxidant protection since the beginning of exercise, since it has higher serum concentrations of vitamin C and FRAP, the lowest degree of lipid peroxidation, measured by MDA, the lowest level of protein oxidation, rated by PC and less dependent on GSH as an endogenous antioxidant. Thus, use of a modest dose of vitamin C, for a short period of time, was able to assist in antioxidant protection, with no harmful effect on individuals.

Freshwater sport fisheries contribute substantially to the economies of England and Wales. However, many trout fisheries rely partly or entirely on stocking farmed trout to maintain catches within freshwater fisheries. Farmed trout often differ genetically from their wild counterparts and wild trout could be at risk of reduced fitness due to interbreeding or competition with farmed fish. Therefore, to protect remaining wild brown trout (Salmo trutta L) populations and as a conservation measure, stocking policy has changed. Legislation introduced by the Environment Agency (EA, 2009) will now only give consent to stocking of rivers and some stillwaters with sterile, all-female triploid brown trout. There are reliable triploidy induction protocols for some other commercially important salmonid species however; there is limited knowledge on triploid induction in brown trout. Previously, triploid brown trout have been produced by heat shocks although reduced survivals were obtained suggesting that an optimised heat shock had not been identified, or that heat shock gives less consistent success than hydrostatic pressure shock (HP), which is now recognised as a more reliable technique to produce triploid fish. Thus the overall aim of this thesis was to conduct novel research to support the aquaculture and freshwater fisheries sector within the United Kingdom by optimising the production and furthering the knowledge of triploid brown trout. Firstly, this PhD project investigated an optimised triploidy induction protocol using hydrostatic pressure (Chapter 2). In order to produce an optimised hydrostatic pressure induction protocol three experiments were conducted to (1) determine the optimal timing of HP shock application post-fertilisation, (2) define optimal pressure intensity and duration of the HP shock and (3) study the effect of temperature (6-12 °C) on triploid yields. Results indicated high survival to yolk sac absorption stage (69.2 - 93.6 %) and high triploid yields (82.5 - 100 %) from the range of treatments applied. Furthermore, no significant differences in triploid rates were shown when shock timings and durations were adjusted according to the temperature used. In all treatments deformity prevalence remained low during incubation (<1.8 %) up to yolk sac absorption (~550 degree days post hatch). Overall, this study indicated that the optimised pressure shock for the induction of triploidy in brown trout delivering high survival and 100 % triploid rate (a prerequisite to brown trout restocking) is a shock with a magnitude of 689 Bar applied at 300 Centigrade Temperature Minutes (CTM) for 50 CTM duration. Regarding the assessment of triploid status, the second experimental chapter tested the accuracy and efficacy of three ploidy verification techniques (Chapter 3). Techniques studied were erythrocyte nuclei measurements (Image analysis), flow cytometry (Becton Dickinson Facscalibur flow cytometer) and DNA profiling (22 polymorphic microsatellite loci) to assess the effectiveness of triploidy induction in brown trout. Results indicated the validity of using erythrocyte indices major nuclear axis measurements, flow cytometric DNA distributions expressed as relative fluorescence (FL2-Area), and polymorphic microsatellite loci (Ssa410UOS, SSa197, Str2 and SsaD48) for assessing ploidy status in brown trout. Accuracy of each technique was assessed and indicated that all techniques correctly identified ploidy level indicating 100 % triploid rate for that commercial batch of brown trout. These techniques may be utilised within aquaculture and freshwater fisheries to ensure compliance with the legislation introduced by the EA. As a result of the legislation introduced by the Environment Agency triploid brown trout will freely interact with diploid trout therefore there is a need to assess feeding response and behavioural differences between diploid and triploid trout prior to release. Therefore, in the third experimental chapter (Chapter 4) diploid and triploid brown trout were acclimated for six weeks on two feeding regimes (floating/sinking pellet). Thereafter, aggression and surface feeding response was compared between pairs of all diploid, diploid and triploid and all triploid brown trout in a semi natural stream (flume). In each pairwise matching, fish of similar size were placed in allopatry and rank determined by the total number of aggressive interactions initiated. Dominant individuals initiated more aggression than subordinates, spent more time defending a territory and positioned themselves closer to the food source (Gammarus pulex) whereas subordinates occupied the peripheries. When ploidy was considered, diploid trout were more aggressive than triploid, and dominated their siblings when placed in pairwise matchings. However, surface feeding did not differ statistically between ploidy irrespective of feeding regime. Triploids adopted a sneak feeding strategy while diploids expended more time defending a territory. In addition, an assessment of whether triploids exhibited a similar social dominance to diploids when placed in allopatry was conducted. Although aggression was lower in triploid pairs than in the diploid/triploid pairs, a dominance hierarchy was observed between individuals of the same ploidy. Dominant triploid fish were more aggressive and consumed more feed items than subordinate individuals. Subordinate fish displayed a darker colour index than dominant fish suggesting increased stress levels. However, dominant triploid fish seemed more tolerant of subordinate individuals and did not display the same degree of invasive aggression as observed in the diploid/diploid or diploid/triploid matchings. These novel findings suggest that sterile triploid brown trout feed similarly but are less aggressive than diploid trout and therefore may provide freshwater fishery managers an alternative to stocking diploid brown trout. In addition to research at the applied level in triploid brown trout, this thesis also examined the fundamental physiological effects of ploidy in response to temperature regime. Triploid salmonids have been shown to differ in their tolerance to environmental temperature. Therefore the fourth experimental chapter (Chapter 5) investigated whether temperature tolerance affected feed intake and exercise recovery. Diploid and triploid brown trout were exposed to an incremental temperature challenge (10 and 19 °C) and subsequent survival and feed intake rates were monitored. Triploids took longer to acclimate to the increase in temperature however feed intake were significantly greater in triploids at high temperature. In a follow on study, we investigated post-exercise recovery processes under each temperature regime (10 and 19 °C). Exhaustion was induced by 10 minutes of forced swimming, with subsequent haematological responses measured to determine the magnitude of recovery from exercise. Plasma parameters (alkaline phosphatase, aspartate aminotransferase, calcium, cholesterol, triglycerides, phosphorous, total protein, lactate, glucose, pH, magnesium, osmolality, potassium, sodium, chloride, lactate dehydrogenase) were measured for each ploidy. Basal samples were taken prior to exercise and then at: 1; 4, and 24 hours post-exercise. Contrary to previous studies, there was no triploid mortality during or after the exercise at either temperature. Although diploid and triploid brown trout responded metabolically to the exercise, the magnitude of the response was affected by ploidy and temperature. In particular, triploids had higher levels of plasma lactate, osmolality, and lower pH than diploids at 1 hour post exhaustive exercise. By 4 hours post-exercise plasma parameters analysed had returned to near basal levels. It was evident that the magnitude of the physiological disturbance post-exercise was greater in triploids than diploids at 19 °C. This may have implications where catch and release is practiced on freshwater fisheries. Overall, this work aimed to develop and/or refine current industry induction and assessment protocols while better understand the behaviour and physiology of diploid and triploid brown trout. The knowledge gained from this work provides aquaculture and freshwater fisheries with an optimised protocol, which delivers 100 % triploid rates and profitability without compromising farmed trout welfare, thus ultimately leading towards a more sustainable brown trout industry within the United Kingdom.

Estudos epidemiológicos vêm demonstrando que quantidades apropriadas de atividade física regular estão associadas a significativa redução do risco para o desenvolvimento de câncer de cólon em até 40% entre os indivíduos mais fisicamente ativos. Por outro lado, tem sido observado que o exercício físico exaustivo aumenta a produção sistêmica de radicais livres, com concomitante aumento dos danos oxidativos ao DNA, além de deprimir a função imune global, eventos relacionados ao aumento do risco para o desenvolvimento de câncer. Relativamente, poucos estudos experimentais exploram essas relações, principalmente no que concerne ao exercício exaustivo e o câncer. A taxa de proliferação celular epitelial, os focos de criptas aberrantes (FCAs) e as criptas com acúmulo de beta-catenina (CABC) têm sido marcadores biológicos (biomarcadores) usados para a identificação precoce de fatores que poderiam influenciar a carcinogênese colônica em ratos. Através da aplicação do carcinógeno químico 1,2dimetilhidrazina (DMH), logo após os protocolos de exercício, foram estudados quatro grupos principais de ratos previamente sedentários (grupo-controle, e três grupos submetidos, respectivamente, ao treinamento crônico de natação, à natação até a exaustão, e à natação até a exaustão após treinamento crônico), sendo sacrificados quinze dias após a injeção da DMH. Após análise histológica e imunohistoquímica, os biomarcadores estudados mostraram resultados coerentes nas diferentes situações experimentais aferidas. Foi observada uma diminuição estatisticamente significante da resposta hiperproliferativa, do número de FCAs e de CABC no grupo que realizou o treinamento crônico, enquanto o grupo submetido ao exercício exaustivo apresentou aumento estatisticamente significante dos parâmetros citados, quando comparados ao grupo-controle. No grupo submetido à associação dos dois protocolos de exercício, quando comparado ao grupo-controle, não houve alterações significantes nos biomarcadores de transformação neoplásica estudados. Nesse estudo experimental, o treinamento físico crônico teve efeito protetor contra o desenvolvimento dos biomarcadores precoces de transformação neoplásica do cólon, enquanto o exercício físico exaustivo aumentou a prevalência destes em ratos sedentários. O treinamento físico crônico, quando associado ao exercício físico exaustivo, não alterou a prevalência dos biomarcadores estudados. Desse achado, acrescido do que se pode encontrar na literatura científica atual, levanta-se a hipótese de que, de maneira similar à relação entre exercício físico e infecções, também o exercício poderia ou proteger contra o desenvolvimento, ou aumentar o risco para o câncer, dependendo do tipo, intensidade e duração da atividade desenvolvida. / Ephitelial cell proliferation, aberrant crypt foci (ACF) and â-catenin-accumulated crypts (BCAC) have been used for early detection of factors that influence colorectal carcinogenesis in rats. It has been observed that moderate and regular physical activity may prevent colon cancer up to 40% in humans. However, exhaustive exercise increases free radical DNA oxidative damage and depresses immune function, events also related to the increased risk for cancer development. Fifteen days after either a single exhaustive swimming bout or a swimming physical training for 8 weeks, or both, in untrained rats treated with a colon carcinogen, we observed a statistically significant decreased number of those biomarkers in rats under training protocol, and a statistically significant increased number of them in rats under exhaustive protocol, when compared to the non-exercised group. For the rats under exhaustive protocol after being trained, data was not evident. Thus, we concluded that training protected against the biomakers development and exhaustive exercise improved colorectal carcinogenesis biomarkers development in sedentary rats. From our finding and literature data, we hypothesize that, similarly to the suggested relationship between exercise and infections, exercise could be protective against cancer or it could increase the risk for this disease depending on its type, dose and duration.

Estudos epidemiológicos vêm demonstrando que quantidades apropriadas de atividade física regular estão associadas a significativa redução do risco para o desenvolvimento de câncer de cólon em até 40% entre os indivíduos mais fisicamente ativos. Por outro lado, tem sido observado que o exercício físico exaustivo aumenta a produção sistêmica de radicais livres, com concomitante aumento dos danos oxidativos ao DNA, além de deprimir a função imune global, eventos relacionados ao aumento do risco para o desenvolvimento de câncer. Relativamente, poucos estudos experimentais exploram essas relações, principalmente no que concerne ao exercício exaustivo e o câncer. A taxa de proliferação celular epitelial, os focos de criptas aberrantes (FCAs) e as criptas com acúmulo de beta-catenina (CABC) têm sido marcadores biológicos (biomarcadores) usados para a identificação precoce de fatores que poderiam influenciar a carcinogênese colônica em ratos. Através da aplicação do carcinógeno químico 1,2dimetilhidrazina (DMH), logo após os protocolos de exercício, foram estudados quatro grupos principais de ratos previamente sedentários (grupo-controle, e três grupos submetidos, respectivamente, ao treinamento crônico de natação, à natação até a exaustão, e à natação até a exaustão após treinamento crônico), sendo sacrificados quinze dias após a injeção da DMH. Após análise histológica e imunohistoquímica, os biomarcadores estudados mostraram resultados coerentes nas diferentes situações experimentais aferidas. Foi observada uma diminuição estatisticamente significante da resposta hiperproliferativa, do número de FCAs e de CABC no grupo que realizou o treinamento crônico, enquanto o grupo submetido ao exercício exaustivo apresentou aumento estatisticamente significante dos parâmetros citados, quando comparados ao grupo-controle. No grupo submetido à associação dos dois protocolos de exercício, quando comparado ao grupo-controle, não houve alterações significantes nos biomarcadores de transformação neoplásica estudados. Nesse estudo experimental, o treinamento físico crônico teve efeito protetor contra o desenvolvimento dos biomarcadores precoces de transformação neoplásica do cólon, enquanto o exercício físico exaustivo aumentou a prevalência destes em ratos sedentários. O treinamento físico crônico, quando associado ao exercício físico exaustivo, não alterou a prevalência dos biomarcadores estudados. Desse achado, acrescido do que se pode encontrar na literatura científica atual, levanta-se a hipótese de que, de maneira similar à relação entre exercício físico e infecções, também o exercício poderia ou proteger contra o desenvolvimento, ou aumentar o risco para o câncer, dependendo do tipo, intensidade e duração da atividade desenvolvida. / Ephitelial cell proliferation, aberrant crypt foci (ACF) and â-catenin-accumulated crypts (BCAC) have been used for early detection of factors that influence colorectal carcinogenesis in rats. It has been observed that moderate and regular physical activity may prevent colon cancer up to 40% in humans. However, exhaustive exercise increases free radical DNA oxidative damage and depresses immune function, events also related to the increased risk for cancer development. Fifteen days after either a single exhaustive swimming bout or a swimming physical training for 8 weeks, or both, in untrained rats treated with a colon carcinogen, we observed a statistically significant decreased number of those biomarkers in rats under training protocol, and a statistically significant increased number of them in rats under exhaustive protocol, when compared to the non-exercised group. For the rats under exhaustive protocol after being trained, data was not evident. Thus, we concluded that training protected against the biomakers development and exhaustive exercise improved colorectal carcinogenesis biomarkers development in sedentary rats. From our finding and literature data, we hypothesize that, similarly to the suggested relationship between exercise and infections, exercise could be protective against cancer or it could increase the risk for this disease depending on its type, dose and duration.