In contrast to most teleost fish that are ammoniotelic, the gulf toadfish (Opsanus beta) is both facultatively ureogenic and ureotelic. In vivo pharmacological manipulations were used to show that lowering circulating cortisol levels or blocking glucocorticoid receptors (GR) enhanced both urea excretion and urea pulse size. These findings demonstrated that changes in pulsatile urea excretion in the toadfish are mediated by the permissive action of cortisol through GRs. Measurement of urea transport across isolated basolateral gill membranes revealed a cortisol-sensitive carrier mechanism. Cortisol infusion in vivo significantly reduced urea transport capacity, suggesting that cortisol inhibits the recruitment of urea transport proteins (UT) to the basolateral membrane to ultimately decrease the size of the urea pulse in toadfish. A 1.2 kb fragment of the upstream transcription start site for the toadfish urea transporter (tUT) gene was isolated and in silico analysis revealed the presence of several putative glucocorticoid response element (GRE) half sites. Toadfish provided with this regulatory sequence in a reporter gene construct showed increased reporter gene transcription driven by cortisol. The data indicated that cortisol-mediated upregulation of tUT mRNA by GREs may be necessary to maintain tUT activity. Four Rhesus (Rh) glycoproteins (Rhag, Rhbg, Rhcg1, Rhcg2) were isolated from toadfish; these sequences grouped with those of other vertebrates coding for membrane channels that transport ammonia. In vivo increases in circulating cortisol reduced branchial Rh glycoprotein expression and decreased ammonia excretion. These changes were accompanied by cortisol-induced increases in glutamine synthetase activity, an enzyme that captures ammonia for urea synthesis. Taken together, the data indicated that cortisol reduces the loss by branchial excretion of ammonia, instead favouring biochemical pathways that convert ammonia to urea. This thesis confirms that nitrogen excretion in toadfish is controlled and regulated in fashions unlike those in other teleosts. The results demonstrate the importance of the GR signaling pathway in mediating changes in both urea and ammonia transport through molecular mechanisms. As a whole, the data provide a new understanding of branchial nitrogen excretion in the gulf toadfish and enhance our evolutionary perspective of the integrated biological systems involved in nitrogen excretion in fish.

Made available in DSpace on 2016-12-08T16:24:21Z (GMT). No. of bitstreams: 1 PGCA15MA168.pdf: 311594 bytes, checksum: e09666d73e41b74adb26b60363213c88 (MD5) Previous issue date: 2015-04-24 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Condensed tannins can reduces the ruminal degradation of protein, increasing N-duodenal flow and improving animal performance with lower environmental impacts. The aim of this work was to assess if the supplementation with condensed tannins (CT) extracted from black wattle (Acacia mearnsii) could reduce the N urinary excretion and improve milk yield and milk composition of dairy ewes. The treatments fed were two protein levels (16.4 and 22.3% of crude protein) with or without CT extract (0 and 8 g/kg fresh). Eight Texel × Lacaune lactating ewes at mid lactation was assigned in a Latin square 4×4 with four periods of 19 days, 14 for adaptation and a five days measurement period. The N urinary excretion increased in animals receiving high-protein level diet compared with lowprotein level diet and decreased with extract supplementation. However, milk yield and milk solids yield were similar between treatments. The CT extract supplementation was not able to improve animal performance, but reduce the N urinary excretion and environmental impact / Os taninos condensados podem reduzir a degradação proteica no rúmen, aumentando o fluxo duodenal de proteína metabolizável com melhorias no desempenho animal e redução do impacto ambiental. Objetivou-se avaliar o efeito do extrato tanífero de acácia negra (Acacia mearnsii) em ovelhas lactantes recebendo ração totalmente misturada (RTM) com dois níveis de proteína bruta. Os tratamentos experimentais constituíramse de RTM com 16,4 e 22,3% de PB na MS, com ou sem a adição de extrato tanífero de Acacia mearnsii na proporção de 8 g/kg na matéria natural. A quantidade de extrato foi calculada para permitir a ingestão de aproximadamente 15 g/animal/dia. Foram utilizadas oito ovelhas cruza da raça Texel × Lacaune no terço médio de lactação distribuídas num delineamento experimental em Quadrado latino 4 × 4, com quatro períodos de 19 dias, sendo 14 de adaptação e cinco de coleta. A excreção urinária de N aumentou nos animais recebendo a dieta com maior nível de proteína em comparação aos que receberam a dieta com menor nível de proteína. Entretanto, a produção de leite e de sólidos não variou entre tratamentos. A suplementação com extrato tanífero de Acacia mearnsii não foi suficiente para melhorar o desempenho animal, mas reduziu a excreção urinária de N e o impacto ambiental

Glutamine synthetase (GS) is a key enzyme in nitrogen (N) assimilation, especially during seed development. This thesis has identified three cytosolic GS isoforms (HvGS1) in barley (Hordeum vulgare L. cv Golden Promise). The quantitation of gene expression, isoform localisation and response to N supply has revealed that each gene plays a non-redundant role in different tissues throughout seedling development. The localisation of HvGS1_1 in vascular cells of different tissues, combined with its abundance in the stem and its response to changes in N supply, indicate that HvGS1_1 is important in N transport and remobilisation. HvGS1_1 is located on chromosome 6H at 72.54 cM, close to the marker HVM074 which is associated with a major quantitative trait locus (QTL) for grain protein content (GPC). HvGS1_1 may be a potential candidate gene to manipulate barley GPC. HvGS1_2 mRNA was localised to the leaf mesophyll cells, in both the cortex and the pericycle of roots and was the dominant HvGS1 isoform in these tissues. HvGS1_2 expression increased in the leaves with an increasing supply of N, suggesting that its role is in the primary assimilation of N. HvGS1_3 was specifically and predominantly localised in the grain, being highly expressed throughout grain development. HvGS1_3 expression increased specifically in the roots of plants grown on high NH₄⁺ suggesting that it has a primary role in grain N assimilation and also in the protection from ammonium toxicity in roots. The expression of the HvGS1 genes is directly correlated with both protein and enzymatic activity, indicating that transcriptional regulation is of prime importance in the control of GS activity in barley. Analysis of 15 different barley cultivars found no correlation between HvGS expression and various desirable attributes. Transgenics which over-express and silence individual HvGS1 isoforms have been produced and confirmed, to analyse for changes in beneficial traits.

In contrast to most teleost fish that are ammoniotelic, the gulf toadfish (Opsanus beta) is both facultatively ureogenic and ureotelic. In vivo pharmacological manipulations were used to show that lowering circulating cortisol levels or blocking glucocorticoid receptors (GR) enhanced both urea excretion and urea pulse size. These findings demonstrated that changes in pulsatile urea excretion in the toadfish are mediated by the permissive action of cortisol through GRs. Measurement of urea transport across isolated basolateral gill membranes revealed a cortisol-sensitive carrier mechanism. Cortisol infusion in vivo significantly reduced urea transport capacity, suggesting that cortisol inhibits the recruitment of urea transport proteins (UT) to the basolateral membrane to ultimately decrease the size of the urea pulse in toadfish. A 1.2 kb fragment of the upstream transcription start site for the toadfish urea transporter (tUT) gene was isolated and in silico analysis revealed the presence of several putative glucocorticoid response element (GRE) half sites. Toadfish provided with this regulatory sequence in a reporter gene construct showed increased reporter gene transcription driven by cortisol. The data indicated that cortisol-mediated upregulation of tUT mRNA by GREs may be necessary to maintain tUT activity. Four Rhesus (Rh) glycoproteins (Rhag, Rhbg, Rhcg1, Rhcg2) were isolated from toadfish; these sequences grouped with those of other vertebrates coding for membrane channels that transport ammonia. In vivo increases in circulating cortisol reduced branchial Rh glycoprotein expression and decreased ammonia excretion. These changes were accompanied by cortisol-induced increases in glutamine synthetase activity, an enzyme that captures ammonia for urea synthesis. Taken together, the data indicated that cortisol reduces the loss by branchial excretion of ammonia, instead favouring biochemical pathways that convert ammonia to urea. This thesis confirms that nitrogen excretion in toadfish is controlled and regulated in fashions unlike those in other teleosts. The results demonstrate the importance of the GR signaling pathway in mediating changes in both urea and ammonia transport through molecular mechanisms. As a whole, the data provide a new understanding of branchial nitrogen excretion in the gulf toadfish and enhance our evolutionary perspective of the integrated biological systems involved in nitrogen excretion in fish.

No description available.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / This study aimed to evaluate the effect of tannin extract from Acacia mearnsii in the reduction of total and urinary nitrogen (N) excretion, in optimizing nutritional and productive performance of lactating cows grazing on temperate or tropical pastures, and also aimed to evaluate its effects on the digestive process, N excretion as well as the flow of metabolites through the portal system and liver of sheep fed temperate grass or tropical grass based diets. Four assays were carried out, two with sheep and two with lactating cows. In assay 1, six sheep were used in a crossover design for two 21-day periods to evaluate the intake, digestibility and N excretion. The diets were composed of Tifton hay 85 and concentrate either without the inclusion of tannin extract (control) or with the addition of 1% tannin extract (dry matter (DM) basis). In assay 3, five sheep were used in a crossover design for two 21-day periods to evaluate the intake, digestibility, N excretion and the flow of blood metabolites through the portal system and liver. The diets were a mixture of oats and ryegrass hay and concentrate either without the addition of tannin extract (control) or with the addition of 2% tannin extract. In assays 2 and 4 were used 14 lactating cows in each assay through a random block design. The experiments lasted for 49 days, and were divided into two periods. In the first 21 days all cows received, for standardization, the same diet and were later randomly assigned, within each block, one of the treatments. In experiment 2, cows were grazing on Tifton 85 pasture and supplemented with concentrate containing 1%, or not (control), of tannin extract. In experiment 4, the cows were kept in mixed oats and ryegrass pasture, and supplemented with concentrate either with the addition of 2% tannin extract or without (control) the addition of tannin extract. For the sheep assays were collected food, scraps, feces, urine and samples as well as arterial, liver and portal blood samples, for assay 3. For assays with cows were collected food scraps, feces, urine and milk samples as well as blood samples from the coccygeal vein. The data were submitted to variance analysis and the effect of treatment compared by F test using the SAS statistical program. P≤0.05 values were considered significant and 0.05<P≤0.10 were considered trend. In assay 1 the inclusion of tannin in the diet promoted a reduction in the true digestibility of N and endogenous N excretion (P≤0.05), without affecting consumption, fecal and urinary excretion of N and digestibility of other dietary fractions (P>0.05). In assay 2 the tannin extract did not cause any changes (P>0.05) in consumption, and milk yield or composition, or the N use efficiency for synthesis of milk proteins, as well as in plasma urea concentrations. There was, however, an increase in N excretion via urine and hence reduction in the overall N use efficiency. In assay 3 there was a reduction (P≤0.05) of the true and apparent N digestibility and urea excretion by urine without affecting the parameters of intake, digestibility of organic matter, neutral detergent fiber and synthesis of microbial rumen nitrogenous compounds. The N-urea concentrations in arterial, portal and hepatic blood samples reduced (P≤0.05) and hepatic glucose concentration tended to decrease (P=0.065) due to the intake of the tannin extract. However, there was a reduction in N-urea flow only in total visceral tissue (P=0.053), and neither concentrations nor the N-NH3 flow changed (P>0.05). In assay 4 the intake of tannins did not affect any milk yield and composition parameters, neither plasma concentrations of urea. It did, however, promote increase (P≤0.05) pasture intake and fecal N excretion and tended to decrease (P≤0.10) N excretion through urine without altering the N use efficiency. The inclusion of 2% tannin extract of Acacia mearnsii in the concentrate fed to dairy cows in the proportion of approximately 25% of the diet based on temperate grasses promotes increased fodder consumption and has the potential to reduce the environmental impact due to the reduction of urinary N excretion, without short term interference in milk yield and composition. Furthermore, based on the study with sheep, offer of up to 40% of concentrate in the diet promotes a more marked reduction in urinary N excretion and may have positive effects on the concentrations of urea in milk and plasma. In assays with tropical grass the inclusion of tannin in the diet did not show results that justify the use of this additive in the assessed dietary conditions. / O objetivo deste estudo foi avaliar o potencial do uso dietético do extrato tanífero de Acacia mearnsii em reduzir a excreção de nitrogênio (N), melhorar a eficiência nutricional e o desempenho produtivo de vacas em lactação recebendo dietas à base de gramínea temperada ou gramínea tropical, e avaliar o seu efeito sobre os processos de digestão, excreção de N, e o fluxo de metabólitos através do sistema portal e fígado de ovinos recebendo dietas à base de feno de gramínea temperada ou gramínea tropical. Foram conduzidos dois ensaios com ovinos e dois ensaios com vacas em lactação. No ensaio 1, foram utilizados seis ovinos em um delineamento inteiramente casualizado (DIC) em esquema de reversão simples, em dois períodos de 21 dias, para avaliar o consumo, a digestibilidade e a excreção de N. As dietas foram constituídas de feno de Tifton 85 e concentrado sem inclusão de extrato tanífero ou adição de 1% de extrato tanífero (base MS). No ensaio 3, cinco ovinos foram utilizados em um DIC em esquema de reversão simples, em dois períodos de 21 dias, para avaliar o consumo, a digestibilidade, a excreção de N e o fluxo de metabólitos sanguíneos através do sistema portal e fígado. As dietas foram constituídas de feno misto de aveia e azevém e concentrado sem inclusão de extrato tanífero ou adição de 2% de extrato tanífero. Nos ensaios 2 e 4 foram utilizadas 14 vacas em lactação em cada um dos ensaios através de um delineamento em blocos ao acaso. Os experimentos tiveram duração de 49 dias, divididos em dois períodos. Nos primeiros 21 dias todas as vacas receberam a mesma dieta para padronização e, posteriormente, foi atribuído aleatoriamente às vacas, dentro de cada bloco, um dos tratamentos. No experimento 2, as vacas foram mantidas em pastagem de Tifton 85 e suplementadas com concentrado contendo 1% ou não (controle) de extrato tanífero. No experimento 4, as vacas foram mantidas em pastagem mista de aveia e azevém, e suplementadas com concentrado contendo 2% ou não (controle) de extrato tanífero. Nos ensaios com ovinos foram coletadas amostras de alimentos, sobras, fezes, urina, e também amostras de sangue arterial, portal e hepático no ensaio 3. Nos ensaios com vacas foram coletadas amostras de alimentos, sobras, fezes, urina, leite e sangue através da veia coccígea. Os dados foram submetidos à análise de variância e o efeito de tratamento comparado pelo teste F através do programa estatístico SAS. Valores de P≤0,05 foram considerados significativos e 0,05<P≤0,10 foram considerados tendência. No ensaio 1 a inclusão de tanino na dieta promoveu redução na digestibilidade verdadeira do N e na excreção de N endógeno (P≤0,05), sem influenciar no consumo, excreção fecal e urinária de N e digestibilidade das demais frações da dieta (P>0,05). No ensaio 2 o extrato tanífero não promoveu nenhuma alteração (P>0,05) no consumo, produção e composição do leite, eficiência de utilização do N para síntese das proteínas do leite, bem como nas concentrações de ureia plasmática. No entanto, houve aumento na excreção de N através da urina e, consequentemente, redução na eficiência de uso geral do N. No ensaio 3 houve redução (P≤0,05) das digestibilidades aparente e verdadeira do N e da excreção de ureia através da urina, sem afetar os parâmetros de consumo, digestibilidade da matéria orgânica, fibra em detergente neutro e síntese de compostos nitrogenados microbianos ruminais. As concentrações de N-ureia nas amostras de sangue arterial, portal e hepático reduziram (P≤0,05) e a concentração de glicose hepática tendeu a reduzir (P=0,065) devido à ingestão do extrato tanífero. Entretanto, houve redução no fluxo de N-ureia apenas nos tecidos viscerais totais (P=0,053) e, nem as concentrações, nem os fluxos de N-NH3 foram alterados (P>0,05). No ensaio 4 a ingestão de taninos pelos animais não afetou nenhum dos parâmetros de produção e composição do leite, assim como as concentrações plasmáticas de ureia. Entretanto, promoveu aumento (P≤0,05) no consumo de pasto e na excreção de N fecal pelos animais e tendeu a reduzir (P≤0,10) a excreção de N através da urina, sem alterar a eficiência de uso deste nutriente. A inclusão de 2% de extrato tanífero de Acacia mearnsii no concentrado fornecido a vacas em lactação na proporção de aproximadamente 25% da dieta à base de gramíneas temperadas promove aumento do consumo de forragem e tem o potencial de reduzir o impacto ambiental pela menor proporção de N excretado através da urina, sem interferir na produção e composição do leite em curto prazo. Além disso, com base no estudo com ovinos, o fornecimento de até aproximadamente 40% deste concentrado na dieta promove uma redução mais acentuada na excreção de N urinário e pode apresentar reflexos positivos sobre as concentrações de ureia no leite e no plasma. Nos ensaios com gramínea tropical a inclusão de tanino na dieta não apresentou resultados que justificam o uso deste aditivo nas condições dietéticas avaliadas.

A series of experiments were conducted to determine nutritional strategies to improve the efficiency of N utilization in dairy cows when feeding co-products including wheat-based (W-DDGS) and corn-wheat blend distillers grains with solubles (B-DDGS), and dried whey permeate (DWP). In Experiment 1, the objective was to determine the effects of replacing canola meal (CM) as the major protein source with W-DDGS on ruminal fermentation, microbial protein production, omasal nutrient flow, and animal performance. Cows were fed either a standard barley silage-based total mixed ration containing CM as the major protein supplement (0% W-DDGS, control) or diets formulated to contain 10, 15 and 20% W-DDGS (dry matter [DM] basis), with W-DDGS replacing primarily CM. Diets were isonitrogenous (18.9% crude protein [CP]). Inclusion of W-DDGS to the diet did not negatively affect ruminal fermentation, microbial protein production, and omasal nutrient flow. However, there was a 0.7- to 2.4-kg increase in DM intake, and a 1.2- to 1.8-kg increase in milk yield after the addition of W-DDGS in place of CM. In Experiment 2, the objective was to delineate the effects of including either W-DDGS or B-DDGS dried distillers grains with solubles as the major protein source in low or high CP diets fed to dairy cows on ruminal function, microbial protein synthesis, omasal nutrient flows, urea-N recycling, and milk production. The treatment factors were type of distillers co-product (W-DDGS vs. B-DDGS) and dietary CP content (15.2 vs. 17.3%; DM basis). The B-DDGS was produced from a mixture of 15% wheat and 85% corn grain. All diets were formulated to contain 10% W-DDGS or B-DDGS on a DM basis. Feeding up to 10% of dietary DM as B-DDGS or W-DDGS as the major source of protein did not have negative effects on metabolizable protein (MP) supply and milk production in dairy cows. However, reducing dietary CP content from 17.3 to 15.2% decreased milk production. This response was attributed to an insufficient supply of ruminally degradable protein (RDP) that suppressed microbial nonammonia N (NAN) synthesis in the rumen, thus decreasing intestinal MP supply. In Experiment 3, the objective was to determine the effects of replacing barley or corn starch with lactose (as DWP) in diets containing 10% W-DDGS on ruminal function, omasal nutrient flow, and lactation performance. The treatment factors were source of starch (barley vs. corn) and dietary inclusion level of DWP (0 vs. 6%; DM basis) as a partial replacement for starch. Diets were isonitrogenous (18% CP) and contained 3 or 8% total sugar. The starch content of the low sugar diet was 24% compared to 20% for the high sugar diet. Dry matter intake, and milk and milk component yields did not differ with diet. However, partially replacing dietary corn or barley starch with sugar up-regulated ruminal acetate and propionate absorption, and reduced ruminal NH3-N concentration, but had no effect on ruminal pH, microbial protein synthesis, omasal nutrient flow and production in dairy cows. In summary, data presented in this thesis indicate that W-DDGS and B-DDGS can be included as the major source of protein in dairy cow diets without compromising ruminal function, nutrient supply and milk production in dairy cows. Feeding medium to low CP diets, and partial replacement of starch with sugar in diets containing W-DDGS and B-DDGS can improve N utilization efficiency in dairy cows. Additionally, an upregulation of facilitated transport of acetate and propionate across epithelial cells possibly prevents the occurrence of ruminal acidosis when lactose partially replaces starch in cow diets.