Dissertations / Theses on the topic 'Acidosis'

Foram utilizados neste experimento quatro garrotes Jersey (J) e quatro Gir (G), providos de cânula ruminal. Dois meses antes da indução da acidose láctica ruminal (ALR), os animais foram alimentados com dieta padronizada a base de feno e concentrado. A ALR foi induzida experimentalmente por meio da administração de sacarose intraruminal, correspondente ao peso metabólico corrigido, segundo técnica descrita por ORTOLANI (l995). Colheitas de sangue, suco de rúmen, urina, fezes e exames clínicos foram realizados nos seguintes momentos após a indução: zero, 14, 16, 18, 20, 22 e 24 horas. O pH e as concentrações de ácido láctico total, D e L e de seus sais foram determinados em todos os materiais biológicos colhidos. No sangue foram avaliados o hematócrito, os exames gasométricos e a concentração de creatinina; esta última substância também foi determinada na urina. Após a última colheita, todo o conteúdo ruminal foi completamente retirado para a determinaçãodo seu volume. Os bovinos de ambas as raças apresentaram marcante e idêntica acidose ruminal, não ocorrendo diferença no pH e na concentração de ácido láctico total, L e D no suco de rúmen. A acidose metabólica sistêmica foi moderada em ambas as raças, porém esta foi mais intensa nos bovinos J, confirmada pelas menores concentrações médias de bicarbonato e TCO2 (P < 0,00001) e pelo menor pH sangüíneo, (p < 0,025). Os garrotes J absorveram maiores quantidades de ácido láctico total e do isômero D; este último apresentou correlação negativa com o pH sangüíneo nesta raça (r = -O,78). Os garrotes G apresentaram maior capacidade homeostática de manutenção de pH sangüíneo no final da indução, provavelmente pela maior metabolização do lactato-L. Entretanto, os mesmos animais tiveram maior grau de desidratação, evidenciado pelas maiores porcentagens de hematócrito e de déficit de volume plasmático (p < 0,00001). Nessa raça ocorreu uma menor filtração glomerular, demonstrada pela maior concentração sérica de creatinina (p < 0,00001), menor depuração deste catabólito (p < 0,003) e menor volume urinário estimado (p < 0,05). Não ocorreram diferenças significativas no pH fecal entre as raças estudadas. Houve correlação negativa entre a concentração de lactato total fecal e o correspondente pH (r = - 0,65).
Four Jersey (J) and four Gir (G) rumen-cannulated steers were used. The steers were fed, for two months before the beginning of the rumen lactic acidosis (RLA) induction, a standard diet of hay and concentrates. The RLA was induced experimentally through the administration of sucrose into the rumen, according to the corrected metabolic weight, after ORTOLANI (1995). Blood, rumen fluid, urine, and fecal samples were collected and clinical examination carried out in the following times after the induction: zero, 14, 16, 18, 20, 22 and 24 hours. The pH, the total lactic acid and its L and D isomers were determined in all samples. The hematocrit, acid-base variables and the creatinine concentration were determined in the blood samples; creatinine was also determined in the urine samples. All the rumen content was evacuated in order to evaluate its volume at the 24th h. A intense rumen acidosis was reached; no differences in the rumen fluid pH and in the concentration of the total lactic acid and its isomers were found in both studied breeds. A moderate level of systemic metabolic acidosis was reached in both breeds, but lower overall mean of bicarbonate and TCO2 (p < 0.0001) as well as blood pH (p < 0.025) were found in the J steers. These steers absorbed higher amounts of total lactic and its D isomer than the G animals; the higher the blood D-lactate concentration, the lower the blood pH (r = - O.78) in the former breed. Better blood pH homeostasis were kept, at the end of induction, by the G steers, probably by their higher efficiency to metabolize L-lactate. However, the G steers exhibited a higher level of dehydration as seen by the greater hematocrit and plasma volume deficit (p < 0.00001). They also presented a lower glomerular filtration as evidenced by the higher creatinine serum levels (p < 0.00001), its lower urinary clearance (p < 0.003) and the lower estimated urinary volume (p < 0.05). There were no differences in the fecal pH values presented by both breeds. There was a negative correlation between the fecal total lactate concentration and the fecal pH (r = - 0.65).

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
O objetivo deste estudo foi avaliar o efeito da restrição alimentar prévia ou exposição anterior a ingredientes concentrados sobre o tempo para adaptação à dietas de alto concentrado, sob os parâmetros da fermentação ruminal e o perfil de microrganismos ruminais de bovinos Nelore canulados confinados. Foram utilizados 6 bovinos da raça Nelore, castrados, com peso vivo inicial aproximado de 236 ± 23 kg, 15 meses de idade e canulados no rúmen, os quais foram divididos em dois quadrados latinos 3 x 3. Os tratamentos diferiram somente sobre o tipo de alimentação estabelecida previamente ao período de adaptação: Controle (forragem ad libitum + suplemento mineral); Restrição (forragem restrita a 1,4% do peso vivo + suplemento mineral) e Concentrado (forragem ad libitum + 0,5% do peso vivo de ingredientes concentrados e suplemento mineral). A duração deste estudo foi de 119 dias, sendo compostos por 3 períodos experimentais (33 dias cada período) e dois intervalos de washout (10 dias) entre os períodos 1 e 2, e 2 e 3. Os períodos foram divididos em: 14 dias de pré- adaptação, 6 dias de adaptação 1 (72% de concentrado), 6 dias de adaptação 2 (79% de concentrado) e 7 dias de dieta de terminação (86% de concentrado). O pH ruminal foi monitorado por meio da utilização de data loggers a cada 10 minutos. Foram avaliados a concentração de ácidos graxos de cadeia curta e amônia ruminal; a quantificação relativa das bactérias celulolíticas e utilizadoras de lactato por meio da técnica de PCR e a contabilidade total e diferencial de protozoários (0,4,8 e 12 horas após o trato). Os animais restritos, na fase de adaptação, tiveram maior concentração de AGCC (P< 0,01), maior duração de pH abaixo de 6,2 (P<0,01) e menor pH máximo nos dias 15 e 16 (P≤ 0,10) em relação ao tratamento controle. Na fase de terminação, esses animais tiveram menor CMS (P=0,02), maior pH médio e menor área de pH abaixo de 6,2 (P≤0,10) em relação ao tratamento controle. Os animais expostos previamente ao concentrado, na fase de adaptação, tiveram menor concentração de butirato e NH3 ruminal (P≤0,10) em relação ao tratamento controle. Apresentaram também menor quantidade relativa de Fibrobacter succinogenes (P=0,10) e maior CMS (P<0,01) em relação ao controle. Já na fase de terminação o CMS não teve diferença significativa em relação ao tratamento controle. Os animais que passaram por restrição alimentar ou por exposição de concentrado na fase de pré-adaptação apresentam características ruminais semelhantes durante a fase a terminação aos animais em forragem ad libitum.
The objective of this study was to evaluate the effect of diet restriction or prior exposure to concentrate ingredientes prior to the adaption period to high concentrate diet on parameters ruminal fermentation patterns and ruminal microorganisms profile of cannulated Nellore cattle. Six Nelore steers was used, with initial body weight about 350 kg, 20 months and cannulated in the rumen, which will be divided into two Latin squares 3 x 3. The treatments differed only with respect to the type of diets prior to the adaptation period: Control (forage ad libitum + mineral supplement); Restriction (forage restricted to 1.4% of body weight + mineral supplement) and concentrate (forage ad libitum + 0.5% of the body weight of concentrated ingredients + mineral supplement). The study was last 119 days, in which animals was submitted to three experimental periods (33 days each one) and two washout intervals (10 days) between the periods 1 and 2, 2 and 3; each period was divided as follows: 14 days of pre-adaptation, 6 days of adaptation 1 (72% concentrate), 6 days of adaptation 2 (79% concentrate) and 7 days of finishing diet (88% concentrate). The rumen pH and temperature was be monitored through the use of data loggers it was evaluated the rumen production of short chain fatty acids and ammonia concentration; the relative quantification of cellulolytic, amylolytic and lactate bacteria utilizing PCR and total and differential quantification of protozoa. Thus, the hypothesis is to test if Nelore cattle exposed to feed restriction or comsuption of concentrate ingredientes prior to the adaptation period present same rumen fermentation patters and microrganism profile when compared to animals of control treatment in the finish diet. Restricted animals, in the adaptation phase, had a higher concentration of SCFA (P<0.01), a higher duration of pH below 6.2 (P <0.01) and lower maximum pH on days 15 and 16 (P≤ 0.10) in relation to the control treatment. In the finish diet, these animals had lower DMI (P = 0.02), higher mean pH and lower pH area below 6.2 (P≤0.10) in relation to the control treatment. The animals previously exposed to the concentrate in the adaptation diet had a lower concentration of butyrate and ruminal NH3 (P≤0.10) in relation to the control treatment. They also presented lower amount of Fibrobacter succinogenes (P = 0.10) and higher DMI (P <0.01) in relation to the control. In the termination diet the DMI did not affect in relation to the control treatment. Cattle previously submitted to either nutricional restriction or intake of concentrate have similar ruminal characteristics during the finishing diet of the animals in control treatment.
2016/04262-8

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
A avaliação da perfusão tecidual com macroparâmetros não permite a detecção precoce de alteração na microvasculatura. A anestesia da gestante requer avaliação da perfusão e a eficácia do lactato na identificação de complicações em crianças após o parto já foi descrita. O presente estudo objetivou validar o lactato sanguíneo e correlacioná-lo a métodos, na avaliação de neonatos de parto normal ou cesariana eletiva sob anestesia geral inalatória. Foram utilizadas oito cadelas para realização de parto normal ou cesariana eletiva, com o protocolo composto de morfina, propofol e sevofluorano. Foram avaliados 24 neonatos de parto normal (GN) e 30 de cesariana (GC), com exames de sangue umbilical no nascimento para dosagem de lactato, hemogasometria, hematócrito, glicose e eletrólitos. No nascimento e aos 10 minutos de vida foram realizadas avaliações Apgar e neurológica. O lactato foi avaliado aos 10 minutos, 4 e 30 dias de vida. Os filhotes apresentaram acidose respiratória, acidemia e hipoxemia ao nascimento, mais elevada no GC. Os animais do GC apresentaram notas de Apgar e resposta neurológica menores ao nascimento, com melhora aos 10 minutos. O lactato sanguíneo foi maior nos animais do GN no nascimento, e foi maior nos animais que morreram. A correlação entre o lactato e as variáveis ocorreu em GN. O lactato sanguíneo associado aos demais parâmetros foi útil na avaliação dos neonatos do GN, porém nos animais do GC não houve correlação com a condição clínica dos animais no momento do nascimento. O procedimento anestésico influencia nos valores de lactato, e a determinação do melhor intervalo para sua avaliação nesses pacientes é necessária.
The assessment of tissue perfusion using macro parameters does not allow early detection of changes in the microvasculature. Anesthesia for pregnant patient requires evaluation of perfusion, and the lactate effectiveness in identifying complications in children after birth has been described. This study aimed to validate the blood lactate and its correlation with other methods, in the evaluation of neonates born from vaginal delivery or elective cesarean section under inhalator anesthesia. Eight dogs were used to perform normal delivery or cesarean section, with the protocol consisting of morphine, propofol and sevoflurane. At birth were evaluated 24 neonates born from vaginal delivery (NG) and 30 born from cesarean section (CG) using umbilical blood to test lactate, blood gas, hematocrit, glucose and electrolytes. Apgar score and neurologic tests were performed at birth and at 10 minutes of life. Lactate levels were evaluated at 10 minutes, 4 and 30 days of life. The puppies showed respiratory acidosis, hypoxemia and acidemia at birth, higher in the CG. The animals of CG presented lower Apgar scores and neurological response at birth, which improved in 10 minutes. Blood lactate was higher in NG animals at birth, and was higher in those who died. The correlation between lactate and the variables occurred on NG. Association of blood lactate with other parameters was useful in the evaluation of neonates on NG, but in the animals of CG there was no correlation with the clinical condition of animals at birth. Anesthesia influences the lactate values, and it is necessary to determine the best interval for evaluation in these patients.

Orientador: Mário De Beni Arrigoni
Resumo: O presente estudo teve o objetivo de avaliar o impacto da flutuação natural da ingestão de massa seca no desempenho, características de carcaça, comportamento ingestivo, perfil metabólico sanguíneo e aspectos morfofisiológicos ruminais de bovinos alimentados com dietas de alta inclusão de concentrado, por meio de estudo meta-analítico. Os experimentos contidos na meta-análise utilizaram animais com grupamento genético similar ao utilizado em confinamentos comerciais brasileiros. A meta-análise teve como base de dados os resultados de dez experimentos conduzidos no Confinamento Experimental da Faculdade de Medicina Veterinária e Zootecnia da UNESP, em Botucatu, entre os anos de 2006 e 2015. Os dados foram coletados a partir 838 bovinos, divididos em 238 baias, consideradas as unidades experimentais para o estudo. Os valores de flutuação de ingestão de massa seca na baia foram calculados pela diferença da ingestão de massa seca entre dois dias consecutivos e dividido pelo dia anterior para encontrar o valor em porcentagem. Com base na mediana geral de 5,62%, os animais foram divididos em dois grupos: alta e baixa flutuação de massa seca. As análises estatísticas foram feitas com auxílio do programa computacional R (versão 3.2.3). Utilizou-se um modelo de regressão linear misto para analisar os dados, no qual os experimentos e baias foram considerados como variáveis aleatórias e grupos de flutuação como variável fixa. Os grupos de baixa e alta flutuação apresentaram flutuação mé... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: This study aimed to evaluate the inpact of natural dry matter intake fluctuation on performance, carcass characteristics, feeding behavior, blood metabolic profile and morphophysiological ruminal aspects of cattle fed with diets of high inclusion of concentrate. The experiments used for this meta-analysis used cattle with genetic group similar to used in Brazilian commercial feedlots. The meta-analysis was conducted using a database of results of ten experiments conducted in Experimental Feedlot of Faculty of Veterinary Medicine and Animal Science, UNESP, Botucatu, between the years 2006 and 2015. Data were collected from 838 bulls, divided into 238 pens, considered the experimental units for the study. The values of dry matter intake fluctuation were calculated by the difference in dry matter intake between consecutive days and divided by the previous day to find the value in percentage. Based on the overall median of 5.62% cattle were classified into two groups: high- or low-fluctuation. All data analysis was performed in R using a mixed model approach where pens and year were random variables and fluctuation group fixed. The low and high fluctuation groups presented a dry matter intake fluctuation average of 4.79% and 6.74%, respectively. Low fluctuation group showed higher dry matter intake as percent of body weight (P <0.01), higher average daily gain (P = 0.05), higher dry matter intake in kg (P = 0.06), greater total weight gain (P = 0.08), as well as greater depositio... (Complete abstract click electronic access below)
Mestre

In the first section the relative severity of the insult produced by low-flow ischaemia was established through a series of experiments monitoring the change in intracellular pH, high energy phosphates and LVDP during zero-flow, low flow (7.5% of the control flow rate) and moderate flow ischaemia (30% of the control flow rate). This established that in terms of mechanical performance and bioenergetic status reducing the flow to 7.5% of the control flow rate produced a significant ischaemic insult. Separate experiments showed that low-flow ischaemia caused action potential duration (APD) shortening which could be prevented by the K_ATP channel inhibitor glibenclamide, so implicating the K_ATP channel in the shortening process. In the second section it was established that low-flow ischaemia caused an early increase in ⁸⁶Rb efflux which was maximal during the phase of APD shortening. Once again glibenclamide abolished APD shortening but only reduced the degree of ⁸⁶Rb efflux. In the third section the effects of respiratory and metabolic acidosis, both known modulators of channel function, on the APD were investigated to establish whether they caused K_ATP channel activation in isolation in whole heart. In conclusion, this study has demonstrated that in whole heart low-flow ischaemia causes potassium efflux as a result of K_ATP channel activation, with the subsequent APD shortening. Intracellular acidosis and increased intracellular lactate, both known modulators of channel function in isolated patches, cause activation of the channel in isolation in whole heart but their main effect is to cause APD lengthening. Hence, these metabolic changes during ischaemia may serve to reduce overall APD shortening.

Chronic renal failure (CRF) is associated with loss of lean body mass, a high incidence of malnutrition, and with insulin resistance. CRF is often complicated by metabolic acidosis. Metabolic acidosis is known to alter both protein and carbohydrate metabolism. A series of studies have been undertaken to investigate the effect of metabolic acidosis on protein metabolism in both normal and CRF human subjects, and to study whether metabolic acidosis in CRF affects insulin sensitivity. Protein turnover was studied using the technique of primed constant infusions of L-[1-¹³C]leucine. Normal subjects were studied before and after ammonium chloride induced metabolic acidosis. Acidosis was associated with increased protein turnover and amino acid oxidation. In CRF subjects, correction of acidosis with sodium bicarbonate decreased protein turnover and amino acid oxidation. The effect of acidosis in CRF on insulin mediated carbohydrate metabolism was studied using the technique of the hyperinsulinaemic euglycaemic clamp. Insulin sensitivity increased with correction of acidosis. By combining L-[1-¹³C]leucine infusions with hyperinsulinaemic euglycaemic clamps, the response of protein metabolism to hyperinsulinaemia was measured before and after correction of acidosis. The presence of acidosis did not impair the ability of insulin to modulate protein metabolism. There is therefore, dissociation between the effects of acidosis in CRF on insulin mediated carbohydrate metabolism and insulin mediated protein metabolism. In summary, metabolic acidosis increases protein catabolism in both normal and CRF man and may contribute to the loss of lean body mass characteristic of CRF. Insulin resistance in CRF improves with correction of acidosis. However the effects of acidosis on protein metabolism are not mediated via alterations in insulin sensitivity.

Previous work has demonstrated that bone cells are highly sensitive to extracellular acidification. Low pH is a key osteoclast activation factor, and reduces bone formation by inhibiting mineralisation of the matrix. The aim of this thesis was to investigate further the roles played by pH in modulating bone cell function. Transient receptor potential (TRP) channels detect protons within the pathophysiological range, so are potential candidates for mediating the observed effects of acid on bone cells. I found that TRPV1 agonists/antagonists had no effect on osteoclast formation and activation or bone mineralisation by osteoblasts. Additionally, TRPV1-/- osteoclasts demonstrated no functional differences from wildtypes. Furthermore, agonists/antagonists for TRPM8, TRPV4 and TRPV3 were without effect. Pyrophosphate (PPi) a fundamental inhibitor of mineralisation in the bone microenvironment is generated from extracellular nucleotides by ecto-nucleotidases such as E-NPP1. I showed that E-NPP1 mRNA and protein were up-regulated in osteoblasts cultured at pH 6.9; total E-NPP activity was also increased. To determine the role of acidification in tumour-induced osteolysis, I devised an in vitro model to investigate interactions of osteoclasts with breast cancer cells, which metastasise to bone, sometimes causing osteolytic disease. Surprisingly, the overall effect of breast cancer cells on osteoclasts was inhibitory; however, resorption was significantly stimulated relative to buffered controls when breast cancer cells were allowed to acidify the culture medium. These results suggest that local acidification in tumour settings in vivo could be an important factor in determining the degree of osteolysis that occurs. Finally, I investigated whether two key cytokines implicated in the vicious cycle model of tumour-mediated osteolysis, B-cell activating factor (BAFF) and activin A influence osteoclast function. In all experiments only acidosis stimulated osteoclast resorption. These results provide further evidence for the fundamental role of acid as a regulator of bone cell function.

This work aims to investigate metabolic acidosis induced skeletal muscle protein wasting, and the inter-relationships between: metabolic acidosis, glucocorticoids (GC), and the ubiquitin proteasome system in this process.;Experiments were undertaken using three key themes and models: 1. The role of glucocorticoids and apoptosis in the process of protein degradation (PD) in L6G8C5 myoblasts in a cell culture system of metabolic acidosis utilizing dexamethasone and the glucocorticoid antagonist RU38486. Stimulation of PD in these cells by acid and GC does not appear to be an artefact of apoptosis or dedifferentiation, but differentiation state does determine whether PD responds spontaneously to acid or (as in vivo) only does so in the presence of GC. 2. The ability of RU38486 to pharmacologically antagonize the suggested permissive effect of glucocorticoid in an in vivo model of acidosis-induced muscle wasting. RU38486 did not prevent the acidosis-induced muscle wasting in this model despite demonstration of significant GC receptor blockade. 3. The role of the ATP-dependent ubiquitin proteasome system in the malnutrition of patients treated by peritoneal dialysis. When serum bicarbonate increased in these patients weight and body mass index increased significantly as did plasma BCAA. Muscle levels of ubiquitin mRNA decreased significantly; serum tumour necrosis factor-a also decreased. These results indicate that even a small correction of serum bicarbonate improves nutritional status, and provide evidence for down-regulation of BCAA degradation and muscle proteolysis via the ubiquitin proteasome system. Whether acidosis and inflammatory cytokines (such as TNF-alpha) interact to impair nutrition remains to be determined.

ABSTRACT Ruminal acidosis remains an important and prevalent disorder of economic and welfare concern to the dairy industry worldwide. There are inconsistencies in the diagnostic techniques and definitions of ruminal acidosis and a requirement for further information on the pathogenesis of ruminal acidosis, in particular in regard to the role of feed substrates, such as starch, sugar, and protein (Chapter 1). A greater understanding of changes to the microbiome during ruminal acidosis, feed management, and the possible synergistic effects of feed additive control agents is also required (Chapter 1). Consequently, the overall hypothesis of this thesis, which was supported, is that starch-, sugar-, and protein- or amino acid-based feed substrates would produce different ruminal and blood measures and distinct rumen bacterial community composition associated with different risks of ruminal acidosis. Secondly, that partial mixed ration feeding strategies and feed additive control agents would promote favorable ruminal conditions and reduce the risk of ruminal acidosis, which was also supported; however, whether feed additive control agents reduced the risk of ruminal acidosis was equivocal. Heifers exposed to a single feeding of grain and fructose had an increased risk of ruminal acidosis and accumulated ruminal lactate, compared to those fed grain only (Chapter 2). This highlights that diets with high sugar content should be fed with caution and increase the risk of ruminal acidosis when physically effective fiber is inadequate. Different oxidative stress responses were not observed among treatment groups of heifers fed single exposures of different substrates (Chapter 3) or different feed additives over a 20 d period (Chapter 7), but were evident in a heifer with acute clinical ruminal acidosis (Chapter 8). This suggests oxidative stress responses may only occur during acute clinical ruminal acidosis. Distinct ruminal bacterial community composition occurred among heifers fed a single exposure to different substrates (Chapter 4) and also among lactating cows fed different feeding strategies at different supplementary feeding amounts (Chapter 5) and these communities were associated with rumen fermentation characteristics. Cattle appeared to have host specific rumen bacteria and a core microbiome (Chapters 4 and 5). This suggests that host specificity in rumen ecosystems may be associated with the individual susceptibilities of cattle to ruminal acidosis and a need to tailor feed management and control for ruminal acidosis for individual cattle. Supplementary feeding amount and ruminal concentrations of propionate and valerate appeared to have the largest association with ruminal bacterial communities in Chapter 5 and may be good predictors of ruminal acidosis. A partial mixed ration feeding system, compared with component feeding, decreased ruminal acidosis (Chapter 5), suggesting benefits of this feeding system; however, milk production and milk component benefits were not observed for this feeding system. Feed additive control agents perturbed the rumen by different mechanisms but had minimal synergistic effects when combinations of feed additives were fed and ruminal acidosis control was equivocal (Chapters 6 and 7). Feed additives may not be capable of controlling ruminal acidosis in all cattle when large amounts of readily fermentable carbohydrates are fed (Chapter 7). Concentrations of the volatile fatty acids (VFA): butyrate, propionate, valerate, isobutyrate, isovalerate, and caproate were below detectable limits in a heifer with acute clinical ruminal acidosis 24 h after she consumed a ration with 19.1% sugar and 54.1% starch on a DM basis and her acetate concentration was <20 mM. However, concentrations of these VFA were higher 55 h after she consumed the ration. These findings demonstrate that the rumen is extremely dynamic and can rapidly recover from severe perturbation. Throughout this thesis it has been evident that classic models of ruminal acidosis may not be sufficient to describe the pathogenesis of ruminal acidosis when diets with a high sugar content are fed and uncharacterized rumen bacteria may be involved in the pathogenesis of ruminal acidosis. Definitions of ruminal acidosis to describe acidosis when cattle are fed different substrates, in particular diets with a high sugar content are required. The rumen appears to be better adapted to respond to changes in starch intakes, compared with sugar intakes and cattle have individual rumen responses and susceptibilities to ruminal acidosis during shifts in feed substrates. In summary, this thesis has increased our understandings of the pathogenesis of ruminal acidosis and control strategies for ruminal acidosis in cattle.

Orientador: Jose Antonio Rocha Gontijo
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
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Resumo: Estudos em animais demonstram que a acidose metabólica promove muitas mudanças morfológicas e bioquímicas nos rins, que estão intimamente associadas com a hipertrofia do órgão Confirmamos que a acidose metabólica crónica causada pela ingestão de cloreto de amónia (NH4Cl), promove a hipertrofia do órgão e também uma diminuição da reabsorção de água e sais pelos rins. Aventamos a hipótese que esses são processos que competem pela energia disponível pelas células tubulares Como a mitocòndria tem sido implicada em uma variedade de processos metabólicos, nós estudamos o comportamento das mitocôndrias isoladas de rins de ratos tratados com NH4Cl As mitocôndrias de rim de ratos (MRR) acidóticos apresentaram uma diminuição de aproximadamente 25% da eficiência da respiração, através do controle respiratório Usando o mesmo substrato (succinato), nós achamos a relação ADP/O significativamente menor nos animais acidóticos quando comparados aos controles Em concordância com esses resultados, o potencial transmembrana mitocondrial, apresentou o tempo de fosforilação aumentado nesses animais, determinado pelo método da safranina. Nenhuma diferença significativa foi observada no potencial em ambas as mitocôndrias. Interessantemente, na determinação do transporte de cálcio, verificamos uma captação inicial mais rápida e maior nas MRR acidóticos, quando comparadas aos controles, mantida equilibrada por mais tempo com a adição do inibidor do poro de transição de permeabilidade mitocondrial, a ciclosporina A (CsA). Não foi observada nenhuma diferença significativa na liberação do cálcio após adição de vermelho de Rutênio (RR) e cloreto de sódio (NaCl). Nós também analisamos todos esses parâmetros em mitocôndrias de fígado de rato, para saber se esse efeito da acidose é particular do rim, ou outros órgãos também apresentam. Não verificamos alterações nas funções mitocondriais, induzidas pela acidose. durante dois dias de tratamento com NH4Cl, nessas mitocôndrias Para sabermos se essas alterações nas mitocôndrias renais estariam relacionadas à proteína desacopladora (UCP), utilizamos em nossos experimentos de controle respiratório inibidores da atividade dessas proteínas CAT, GDP e BSA Observamos indiretamente que a UCP esta ativada nas MRR acidóticos, respondendo o desacoplamento observado na situação de acidose nessas mitocôndrias
Abstract: Studies in animals have shown that metabolic acidosis causes several morphologic and biochemical changes in kidney, which are ultimately associated with organ hypertrophy We have previously shown that chronic metabolic acidosis caused by feeding NH4Cl lead to nephron hypertrophy and to a decreased water-salt reabsortion by the kidneys Since mitochondria have been implicated in a variety of metabolic disorders, we examined energy linked functions in isolated mitochondria from rat kidneys with chronic metabolic acidosis induced by NH4Cl Mitochondria from acidotic rats presented resting respiration supported by succinate oxidation 25% slower when compared to control mitochondria Using the same substrate we found an ADP/O ratio of 1,4 in control and 1,1 in isolated mitochondria from acidotic rats. Accordingly, the transient decrease in ?? that occurs during ADP phosphorylation was much larger in mitochondria from acidotic rat kidneys as evidenced by the safranine method No significant difference in ?? could be detected in both types of mitochondria using this method. Interestingly, determination of Ca2+ transport showed a faster rate of initial Ca2+ up take by mitochondria from acidotics and a lower concentration of extramitochondrial Ca2+ under steady state conditions (Ca2+ set point) by these mitochondria compared to control mitochondria In contrast, no significant differences could be detected in rates of Na+ or ruthenium red induced Ca2+ efflux. The data provided evidence that: succinate oxidation is inhibited in mitochondria isolated from acidotic rats while the Ca2+ influx pathway is activated in these mitochondria
Doutorado
Doutor em Farmacologia

Orientadores: Caroline Dário Capitani, Marcelo Saldanha Aoki
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Aplicadas
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Resumo: O presente estudo teve os seguintes objetivos: caracterizar as demandas fisiológicas de diferentes rotinas de treinamento e, avaliar o efeito da suplementação de citrato de sódio sobre o desempenho de habilidades específicas do Tênis após uma simulação de partida. Dez jogadores de Tênis juvenis, do sexo masculino (17,0 ± 1,2 anos; estatura: 176,7 ± 5,2 cm; peso: 68,4 ± 7,9 kg; gordura corporal: 11,7 ± 1,4%) participaram dos estudos. No primeiro estudo foram realizadas quatro rotinas de treinamento, cada qual com seis pontos. As rotinas se diferiram entre si pelo número de rebatidas por ponto (duas, quatro, sete e dez rebatidas por ponto). Foram avaliados frequência cardíaca (FC), percepção subjetiva de esforço (PSE) e concentração de lactato ([La]) após o primeiro, terceiro e sexto ponto de cada rotina. O segundo estudo consistiu na suplementação de citrato de sódio (0,5 g.kg-1 de peso) ou placebo - NaCl (0,1 g.kg-1 de peso). Após 120 minutos da ingestão do suplemento, foram realizados testes de habilidades específicas no Tênis (Loughborough Tennis Skill Test ¿ LTST e Repeated Sprint Ability Shuttle Test ¿ RSA) antes e após a simulação de uma partida de 60 minutos. Observou-se que todos os parâmetros avaliados no primeiro estudo (FC, PSE e [La]) aumentaram progressivamente durante as rotinas de treinamento, à medida que se aumentava o número de rebatidas por ponto, indicando que rotinas de treinamento com sete e dez rebatidas por ponto induziram respostas maiores associadas ao nível de estresse do que observado nas rotinas mais curtas. No segundo estudo observou-se aumento significante (p<0,05) da [HCO3-] e do excesso de base no grupo suplementado com citrato de sódio comparado ao placebo. Após a simulação de partida houve aumento da [La] e de pH no grupo suplementado com citrato de sódio ([La]: 5,46 mmol.L-1; pH: 7,48) comparado ao grupo placebo ([La]: 2,28 mmol.L-1; pH: 7,34). A capacidade de sprints repetidos avaliada pelo RSA não apresentou diferença (p>0,05) entre os testes pré e pós simulação de partida. Apesar do total de golpes desenvolvidos pelos jogadores não apresentarem diferença significante (p>0,05) antes e após a simulação de partida, observou-se declínio (p<0,05) na consistência dos golpes para o grupo placebo (pré: 53,5%; pós: 40,4%) quando comparado ao grupo suplementado com citrato de sódio (pré: 50,2%; pós: 58,5%). Portanto, sugere-se que a suplementação de citrato de sódio pode prevenir o declínio do desempenho das habilidades específicas do Tênis, após uma simulação de partida
Abstract: This study had the following objectives: To characterize the physiological demands of different training routines and evaluate the effect of sodium citrate supplementation on the performance of specific tennis skills after a simulated match. Ten young male tennis players (17,0 ± 1,2 years old; height: 176,7 ± 5,2 cm; weight: 68,4 ± 7,9 kg; body fat: 11,7 ± 1,4%) participated in the studies. In the first study, four training routines were performed, each with six points. The routines differed according to the number of hits per point (two, four, seven and ten hits per point). Heart rate (HR), rating of perceived exertion (RPE) and blood lactate concentration ([La]) were assessed after the first, third and sixth points of each routine. The second study consisted of supplementing with sodium citrate (0,5 g.kg-1 body weight) or placebo - NaCl (0,1 g.kg-1 wt). One-hundred and twenty minutes after taking the supplement, tests were performed for specific tennis skills (Loughborough Tennis Skill Test - LTST and Repeated Sprint Ability Test Shuttle - RSA) before and after simulating a match of 60 minutes. All parameters evaluated in the first study (HR, RPE and [La]) were found to increase progressively during training routines, as the number of hits per point increased, indicating that training routines with seven and ten hits per point were associated with higher levels of stress than for the shorter routines. The second study demonstrated a significant increase (p <0,05) in [HCO3-] and base excess in the group supplemented with sodium citrate, compared with placebo. After the match simulation, increases were observed in [La] and pH in the group supplemented with sodium citrate ([La]: 5,46 mmol.L-1; pH: 7,4), compared to the placebo group ([La]: 2,28 mmol.L-1; pH: 7,34). The ability to repeat sprints, as evaluated by RSA, did not differ (p > 0,05) between the pre and post match simulation. Although the total number of hits performed by the players was not significantly different (p>0,05) before and after starting the simulation, we observed a decrease (p<0,05) in the consistency of the hits in the placebo group (pre: 53,5%; post: 40,4%), when compared to the group supplemented with sodium citrate (pre: 50,2%; post: 58,5%). Therefore, we suggest that supplementation of sodium citrate may prevent the decline in performance of specific tennis skills after a simulated match
Mestrado
Nutrição
Mestra em Ciências da Nutrição e do Esporte e Metabolismo

Bibliography: leaves 122-138. Investigates the use of microbial inoculants to prevent the onset of acidosis in acutely grain fed animals; and, the most effective combination of virginiamycin and lactic acid utilising bacteria (selenomonas ruminantium subsp. lactilytica and Megasphaera elsdenii) in controlling lactic acid accumulations in vitro.

The human ether-a-go-go-related gene (hERG) encodes channels mediating the rapid delayed rectifier K+ current (IKr). IKr participates in cardiac action potential (AP) repolarisation and may also protect the ventricles against premature stimulation. The heart is exposed to acidosis (low pH) in a number of pathological conditions including myocardial ischemia. Extracellular acidosis is known to modulate hERG current (IhERG) function, although a number of aspects of the modulation remain incompletely understood. The aims of this investigation were to establish the effects of acidosis on: (i) IhERG amplitude, kinetics and the response to premature stimulation at mammalian physiological temperature; (ii) the hERG blocking potency of selected anti arrhythmic drugs. Whole-cell patch-clamp recordings of IhERG were made from mammalian cells (CHO or HEK 293) at 37 QC. Lowering external pH from 7.4 to 6.3 reduced the magnitude of IhERG by reducing macroscopic hERG conductance and modulating IhERG kinetics, with positively shifted activation and accelerated deactivation. Results from experiments using an acidic pipette solution showed that the actions of protons occurred from the external surface and not from secondary intracellular acidosis. Experimental and computer simulation work demonstrated that acidosis impairs the protective role of IhERG against premature stimulation. The effects of extracellular acidosis on IhERG kinetics were preserved when the shortened hERG I b isoform was studied, indicating that a full-length N-terminus is not necessary for acidic modulation of hERG channel function. Interestingly, the inhibitory effect of acidosis on IhERG was greater for hERG 1 band hERG lall b than for hERG la. Extracellular acidosis decreased the hERG blocking potency of flecainide, dofetilide and ranolazine, whilst the potency of amiodarone was unaffected. IhERG inactivation was found to be important for ranolazine's inhibitory action and a series of S6 and inner helix residues (Y652, F656, T623, S624 and V625) were identified as contributing to ranolazine binding. I.

Increased cancer risk is associated with select dietary factors. Dietary lifestyles can alter systemic acid-base balance over time. Acidogenic diets, which are typically high in animal protein and salt and low in fruits and vegetables, can lead to a sub-clinical or low-grade state of metabolic acidosis. The relationship between diet and cancer risk prompts questions about the role of acidosis in the initiation and progression of cancer. Cancer is triggered by genetic and epigenetic perturbations in the normal cell, but it has become clear that microenvironmental and systemic factors exert modifying effects on cancer cell development. While there are no studies showing a direct link between diet-induced acidosis and cancer, acid-base disequilibrium has been shown to modulate molecular activity including adrenal glucocorticoid, insulin growth factor (IGF-1), and adipocyte cytokine signaling, dysregulated cellular metabolism, and osteoclast activation, which may serve as intermediary or downstream effectors of carcinogenesis or tumor promotion. In short, diet-induced acidosis may influence molecular activities at the cellular level that promote carcinogenesis or tumor progression. This review defines the relationship between dietary lifestyle and acid-base balance and discusses the potential consequences of diet-induced acidosis and cancer occurrence or progression.

Orientador: Danilo Domingues Millen
Resumo: O objetivo deste estudo foi avaliar a redução do tempo de adaptação à rações com alto teores de energia para bovinos Nelore canulados em confinamento com a utilização de virginiamicina (VM) como aditivo alimentar, em que foram avaliadas as seguintes variáveis: pH, temperatura, e potencial de óxido-redução do rúmen, taxa de desaparecimento, digestibilidade total e degradabilidade ruminal de nutrientes, seletividade da ração e comportamento ingestivo, produtos da fermentação ruminal, assim como dados de ingestão de matéria seca. O delineamento experimental foi em quadrado latino 5x5, e cada período durou 21 dias, com 7 dias de washout entre o final e o início de um novo período. Foram utilizados 5 bovinos inteiros, com peso vivo aproximado de 414.86 ± 21.71 kg e com 17 meses de idade. Neste estudo os animais foram divididos em cinco tratamentos: 1) Monensina (MON; 30 mg/kg) com adaptação por 14 dias; 2) MON (30 mg/kg) + VM (25 mg/kg) com adaptação por 14 dias; 3) VM (25 mg/kg) com adaptação por 14 dias; 4) VM (25 mg/kg) com adaptação por 9 dias; e 5) VM (25 mg/kg) com adaptação por 6 dias. Bovinos que consumiram apenas MON tiveram menor concentração de butirato (P=0,02) e maior concentração de propionato (P=0,04), quando comparados aos que consumiram VM e foram adaptados por 14 dias. E animais que consumiram VM e foram adaptados por 14 dias tiveram maiores proporções AGCC quando comparados com animais que consumiram VMMON (P= 0,05) e quando comparados com animais que consumiram... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The objective of this study was to evaluate the reduction of adaptation length to the high-energy diet offered to cannulated Nellore cattle using Virginiamycin (VM) as sole feed additive, in which the following variables were assessed: pH, temperature,and ox-redox potential of the rumen, rate of disappearance, total tract digestibility and rumen nutrient degradability, feed particles sorting and feeding behavior, ruminal fermentation end-products, as well as dry matter intake. It was adopted the experimental design in a 5 x 5 Latin square, where each period lasted 21 days, with 7-day washout between the end and the beginning of a new period. Were used 5 Nellore yearling bulls,with approximately 414.86 ± 21.71 kg of body weight and 17 mo-old. In this study, the animals were divided into five treatments: 1) MON (30 mg/kg) adapted by 14 days; 2) MON (30 mg/kg) + VM (25 mg/kg) adapted by 14 days; 3) VM (25 mg/kg) adapted by 14 days; 4) VM (25 mg/kg) adapted by 9 days and 5) VM (25 mg/kg) adapted by 6 days.Bovine animals that consumed only MON had lower concentration of butyrate (P = 0.02)and higher concentration of propionate (P = 0.04) when compared to those that consumed VM and were adapted for 14 days. Animals that consumed VM and were adapted for 14 days had higher AGCC ratios when compared to animals that consumed VMMON (P = 0.05) and when compared to animals that consumed only MON (P = 0.04). As the adaptation time increased for animals that consumed VM, the dry matter inta... (Complete abstract click electronic access below)
Mestre

O presente trabalho avaliou a possibilidade de duas diferentes quantidades de melão, com alto teor de açúcares, em causar acidose ruminal em ovinos não adaptados. Foram utilizados 12 ovinos mestiços Santa Inês, machos, providos de cânula ruminal, com 25 kg de P.V. e 8 m de idade, que nunca receberam rações concentradas, frutas ou raízes, anteriormente. Os animais foram mantidos em baias coletivas com dieta basal composta de volumoso (feno de capim Cynodon dactylon - Coast cross) na base de 2,3% de seus pesos vivos, providos de água e sal mineralizado à vontade. Após 30 dias de adaptação, os ovinos foram distribuídos aleatoriamente em dois grupos iguais, assim constituídos: G1: 25% da M.S. de melão e G2: 75% de melão. O melão inteiro, sem as sementes, com 12º Bx de grau Brix (120 g sacarose/kg polpa e 7,8 % M.S.) foi triturado administrando-se pela cânula ruminal 130 g e 389,4 g de sacarose no G1 e G2, respectivamente, após os animais permanecerem em jejum por 12 horas. Foram realizados exames físicos, coletadas amostras de fluido ruminal e sangue nos seguintes tempos após a administração do substrato: zero, 3, 6, 12, 18 e 24 h. Os animais do G1 apresentaram durante a 3ª a 6ª h quadro de acidose ruminal por AGVs (subaguda) caracterizado por pH ruminal ligeiramente inferior a 5,6; discreto aumento no teor de ácido láctico-L, no potencial de oxirredução (POR) e no tempo de redução do azul de metileno (RAM) ruminais, sem que os animais manifestassem quaisquer sintomatologia clínica. Os animais do G2 desenvolveram quadro de acidose láctica ruminal, evidenciado pelo baixo pH (< 5,0), altos teores de lactato-L, valores de POR e da RAM, aumento temporário de osmolaridade ruminal, porém os animais não se tornaram desidratados, e apresentaram uma intensa diurese enquanto perdurou marcada hiperglicemia. Os animais evoluíram para uma moderada acidose metabólica sistêmica. A taquicardia e a taquipnéia foram provocadas pelo aumento da circunferência abdominal, devido ao grande volume de conteúdo administrado pela cânula, não sendo no caso da taquicardia gerada pelo aumento do volume globular, oriunda da desidratação e encontrada classicamente nesse tipo de acidose. A diminuição dos movimentos do rúmen foi correspondente a queda no pH ruminal e elevação dos teores de lactato-L no rúmen. O baixo pH e os altos valores de POR interferiram no aumento do tempo da RAM. Não se recomenda o oferecimento de altas quantidades de melão (75% da M.S.), porém acredita-se que os ovinos não terão problema de ingerir a quantia de 25% da M.S., desde que se tomem cuidados para a adaptação gradual dos animais ao substrato.
The present study evaluated the possibility of ruminal acidosis being caused by two distinct amounts of melon with high sugar content on non-adapted sheep. Twelve crossbreed Santa- Inês sheep with rumen cannula were used. Animals were male, 8 months old, 25 kg B. W., and had never eaten concentrated feed, fruits or roots of any kind before. They were kept in collective pens and their basal diet was composed on 2.3% of their B. W. of Coast cross (Cynodon dactylon) hay, water, and mineral salt Ad libitum. After 30 days of adaption, animals were divided in two groups: G1: 25% of D. M. of melon; and G2: 75% of D. M. of melon. The animals fasted for 12 hours. Then the whole melon, without seeds, 12º Bx (120 g sucrose/kg pulp and 7.8% D. M.) was crushed and administered through the rumen cannula, it represented 130 g and 389.4 g of sucrose on G1 and G2, respectively. Physical examinations along with the collection of ruminal fluid and blood were done after the administration of the substrate at: 0, 3, 6, 12, 18, and 24 h. G1 sheep presented VFAs ruminal acidosis (sub-acute) between the 3ª and 6ª h, characterized by rumen pH slightly inferior to 5.6, discrete lactic-L acid content increase, increased redox potential (RP) and methylene blue redox (MBR) time of the ruminal fluid, and lack of clinical signs. G2 presented lactic ruminal acidosis, ruminal fluid pH < 5.0, high lactate-L content, increased RP and MBR time, increased ruminal fluid osmolarity, no dehydration, and increased diuresis during hypoglycemic period. Animals had moderate systemic acidosis. Tachycardia and tachypnea were caused by an increase on abdominal circumference, resultant of the large amount of melon administrated through the cannula. Therefore these conditions were not caused by the increased globular volume and dehydration typically found on this type of acidosis. Reduced ruminal movements frequency corresponded to decreased ruminal pH and increased Lactate-L ruminal content. Reduced rumen pH and increased RP caused the increase on MBR time. Therefore, it is not recommended offering of large amounts of melon (75% da D.M.) on the diet, though it is believed that sheep will have no problems ingesting 25% of D.M. of melon, whenever the proper gradual adaptation is performed previously.

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
O objetivo deste estudo foi avaliar os efeitos da duração do período de adaptação à dieta de alto teor de concentrado sobre o desempenho, comportamento ingestivo, características de carcaça, saúde do rúmen e digestibilidade do amido, FDN e MS em bovinos Nelore e ½ Angus/Nelore terminados em confinamento. Setenta e dois bovinos machos (com 313,5 kg ± 24,5 em média), não castrados, 36 da raça Nelore e 36 ½ Angus/Nelore, provenientes de sistema de recria em pasto, foram aleatorizados e mantidos em 24 baias (3 animais por baia) de chão batido (24 m² e 2,0 m de cocho por animal), de acordo com os tratamentos: bovinos Nelore adaptados por 9 dias, bovinos Nelore adaptados por 14 dias, bovinos ½ Angus/Nelore adaptados por 9 dias, e bovinos ½ Angus/Nelore adaptados por 14 dias, constituindo-se assim delineamento experimental em blocos casualizados em arranjo fatorial 2 × 2 (2 grupos genéticos e 2 durações do período de adaptação) com medidas repetidas no tempo, as quais foram tomadas de acordo com a variável analisada. Cada tratamento foi composto por 6 baias, as quais foram consideradas as unidades experimentais neste estudo. Após 9 ou 14 dias de adaptação, os animais receberam a ração final com 86% de concentrado. No início do período experimental e a cada 28 dias todos os animais foram pesados. A IMS foi medida todos os dias, para então calcular-se ganho de peso diário e conversão alimentar. Da mesma forma, os animais foram submetidos a avaliações de ultrassonografia no início e no final do período de confinamento para avaliação da deposição de músculos e gordura na carcaça, sendo abatidos em frigorífico comercial após 89 dias de alimentação, alcançando peso vivo final médio de 446,2 kg. Amostras de epitélio ruminal e ração, sobra e fezes foram coletadas para avaliações de saúde ruminal e digestibilidade dos nutrientes, respectivamente. Animais ½ Angus/Nelore apresentaram melhor (P<0,05) desempenho produtivo, eficiência alimentar e características de carcaça que os da raça Nelore. Foi observado (P<0,05) interação para escore de rumenites, onde animais Nelore adaptados por 14 dias apresentaram o maior escore. Bovinos adaptados em 14 dias apresentaram (P<0,05) seleção contra às partículas maiores que 19,0 mm e seleção a favor das partículas menores que 1,18 mm. Animais adaptados por 9 dias apresentaram flutuação da IMS maior (P<0,05) que os adaptados em 14 dias durante o período de adaptação. Conclui-se que o tempo de adaptação mais seguro para adaptar ambos genótipos foi de 14 dias e que o genótipo de melhor desempenho para confinamento foi o ½ Angus/Nelore.
The objective of this study was to evaluate the length of adaptation period to high concentrate diets on performance, feeding behavior, carcass traits, rumen health and starch, NDF and DM digestibility of Nellore and ½ Angus/Nellore cattle finished in feedlot. Seventy-two yearling bulls (313.5 kg ± 24.5 on the average), 36 Nellore and 36 ½ Angus/Nellore, backgrounded on pasture were randomly allocated and kept in 24 pens (3 animals/pen) with concrete floor (24 m² and 2,0 m bunk per animal) according to the treatments: Nellore adapted for 9 days, Nellore adapted for 14 days, ½ Angus/Nellore adapted for 9 days, and ½ Angus/Nellore adapted for 14 days, characterizing a randomized block design with a 2 x 2 factorial arrangement of treatments (2 genotypes and 2 lengths of adaptation period) with repeated measures over time according to the dependent variable measured. Each treatment was composed by 6 pens which was considered the experimental unit to this study. After 9 or 14 days of adaptation, yearling bulls received the finishing diet containing 86% concentrate. At the beginning of experimental period and every 28 days, yearling bulls were weighted. Dry matter intake was measure every day, and average daily gain and feed efficiency were calculate. Likewise, the deposition of muscle and fat were evaluate by ultrasound at the beginning and at the end of the study where the yearling bulls were slaughter in a commercial abattoir after 89 days of feed reaching average live body weight of 446.2 kg. Samples of rumen epithelium, diet, refusals and feces were collected for analysis of rumen health, and nutrients digestibility, respectively. Animals ½ Angus/Nellore presented better (P<0.05) performance, feed efficiency and carcass traits than the Nellore. Interaction (P<0.05) to rumenitis was observed, where Nellore adapted in 14 days presented the higher score. Yearling bulls adapted in 14 days presented (P<0.05) selection against particles higher than 19.0 mm and selection in favor of the particles lower than 1,18 mm. Animals adapted in 9 days showed higher DMI variation (P<0.05) than the adapted in 14 days during the adaptation period. Thus, it is possible to concludes that the most security adaptation length to both genotypes is the 14 days and the better genotype to feedlot performance is the ½ Angus/Nellore.
FAPESP: 2013/21666-7

Orientador: Valéria Nobre Leal de Souza Oliva
Banca: Paulo Sérgio Patto dos Santos.
Banca: Rodrigo Cardoso Rabelo.
Resumo: A avaliação da perfusão tecidual com macroparâmetros não permite a detecção precoce de alteração na microvasculatura. A anestesia da gestante requer avaliação da perfusão e a eficácia do lactato na identificação de complicações em crianças após o parto já foi descrita. O presente estudo objetivou validar o lactato sanguíneo e correlacioná-lo a métodos, na avaliação de neonatos de parto normal ou cesariana eletiva sob anestesia geral inalatória. Foram utilizadas oito cadelas para realização de parto normal ou cesariana eletiva, com o protocolo composto de morfina, propofol e sevofluorano. Foram avaliados 24 neonatos de parto normal (GN) e 30 de cesariana (GC), com exames de sangue umbilical no nascimento para dosagem de lactato, hemogasometria, hematócrito, glicose e eletrólitos. No nascimento e aos 10 minutos de vida foram realizadas avaliações Apgar e neurológica. O lactato foi avaliado aos 10 minutos, 4 e 30 dias de vida. Os filhotes apresentaram acidose respiratória, acidemia e hipoxemia ao nascimento, mais elevada no GC. Os animais do GC apresentaram notas de Apgar e resposta neurológica menores ao nascimento, com melhora aos 10 minutos. O lactato sanguíneo foi maior nos animais do GN no nascimento, e foi maior nos animais que morreram. A correlação entre o lactato e as variáveis ocorreu em GN. O lactato sanguíneo associado aos demais parâmetros foi útil na avaliação dos neonatos do GN, porém nos animais do GC não houve correlação com a condição clínica dos animais no momento do nascimento. O procedimento anestésico influencia nos valores de lactato, e a determinação do melhor intervalo para sua avaliação nesses pacientes é necessária.
Abstract: The assessment of tissue perfusion using macro parameters does not allow early detection of changes in the microvasculature. Anesthesia for pregnant patient requires evaluation of perfusion, and the lactate effectiveness in identifying complications in children after birth has been described. This study aimed to validate the blood lactate and its correlation with other methods, in the evaluation of neonates born from vaginal delivery or elective cesarean section under inhalator anesthesia. Eight dogs were used to perform normal delivery or cesarean section, with the protocol consisting of morphine, propofol and sevoflurane. At birth were evaluated 24 neonates born from vaginal delivery (NG) and 30 born from cesarean section (CG) using umbilical blood to test lactate, blood gas, hematocrit, glucose and electrolytes. Apgar score and neurologic tests were performed at birth and at 10 minutes of life. Lactate levels were evaluated at 10 minutes, 4 and 30 days of life. The puppies showed respiratory acidosis, hypoxemia and acidemia at birth, higher in the CG. The animals of CG presented lower Apgar scores and neurological response at birth, which improved in 10 minutes. Blood lactate was higher in NG animals at birth, and was higher in those who died. The correlation between lactate and the variables occurred on NG. Association of blood lactate with other parameters was useful in the evaluation of neonates on NG, but in the animals of CG there was no correlation with the clinical condition of animals at birth. Anesthesia influences the lactate values, and it is necessary to determine the best interval for evaluation in these patients.
Mestre

Orientador: Danilo Domingues Millen
Resumo: O objetivo deste estudo foi avaliar os efeitos da duração do período de adaptação à dieta de alto teor de concentrado sobre o desempenho, comportamento ingestivo, características de carcaça, saúde do rúmen e digestibilidade do amido, FDN e MS em bovinos Nelore e ½ Angus/Nelore terminados em confinamento. Setenta e dois bovinos machos (com 313,5 kg ± 24,5 em média), não castrados, 36 da raça Nelore e 36 ½ Angus/Nelore, provenientes de sistema de recria em pasto, foram aleatorizados e mantidos em 24 baias (3 animais por baia) de chão batido (24 m² e 2,0 m de cocho por animal), de acordo com os tratamentos: bovinos Nelore adaptados por 9 dias, bovinos Nelore adaptados por 14 dias, bovinos ½ Angus/Nelore adaptados por 9 dias, e bovinos ½ Angus/Nelore adaptados por 14 dias, constituindo-se assim delineamento experimental em blocos casualizados em arranjo fatorial 2 × 2 (2 grupos genéticos e 2 durações do período de adaptação) com medidas repetidas no tempo, as quais foram tomadas de acordo com a variável analisada. Cada tratamento foi composto por 6 baias, as quais foram consideradas as unidades experimentais neste estudo. Após 9 ou 14 dias de adaptação, os animais receberam a ração final com 86% de concentrado. No início do período experimental e a cada 28 dias todos os animais foram pesados. A IMS foi medida todos os dias, para então calcular-se ganho de peso diário e conversão alimentar. Da mesma forma, os animais foram submetidos a avaliações de ultrassonografia no início e no... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The objective of this study was to evaluate the length of adaptation period to high concentrate diets on performance, feeding behavior, carcass traits, rumen health and starch, NDF and DM digestibility of Nellore and ½ Angus/Nellore cattle finished in feedlot. Seventy-two yearling bulls (313.5 kg ± 24.5 on the average), 36 Nellore and 36 ½ Angus/Nellore, backgrounded on pasture were randomly allocated and kept in 24 pens (3 animals/pen) with concrete floor (24 m² and 2,0 m bunk per animal) according to the treatments: Nellore adapted for 9 days, Nellore adapted for 14 days, ½ Angus/Nellore adapted for 9 days, and ½ Angus/Nellore adapted for 14 days, characterizing a randomized block design with a 2 x 2 factorial arrangement of treatments (2 genotypes and 2 lengths of adaptation period) with repeated measures over time according to the dependent variable measured. Each treatment was composed by 6 pens which was considered the experimental unit to this study. After 9 or 14 days of adaptation, yearling bulls received the finishing diet containing 86% concentrate. At the beginning of experimental period and every 28 days, yearling bulls were weighted. Dry matter intake was measure every day, and average daily gain and feed efficiency were calculate. Likewise, the deposition of muscle and fat were evaluate by ultrasound at the beginning and at the end of the study where the yearling bulls were slaughter in a commercial abattoir after 89 days of feed reaching average live body weig... (Complete abstract click electronic access below)
Mestre

Esta tesis doctoral se planteó con el fin de profundizar en el estudio de la acidosis ruminal y evaluar nuevas estrategias de prevención.
En el experimento 1, se desarrolló la PCR cuantitativa para detectar y cuantificar Streptococcus bovis y Megasphaera elsdenii, bacterias relacionadas con la producción y utilización del lactato, respectivamente. La técnica se desarrolló con unas eficiencias del 88% (S. bovis) y 99% (M. elsdenii). Posteriormente se empleó esta técnica en los experimentos siguientes, permitiendo progresar en comprender el rol que tienen estas bacterias en el proceso de la acidosis.
El experimento 2 se realizó con el objetivo de determinar los efectos de administrar altos niveles de carbohidratos no fibrosos (CNF) en la dieta sobre la fermentación ruminal y el metabolismo de vacas lecheras. Para ello se utilizaron 62 vacas Holstein en un diseño cross-over 2x2. Los tratamientos fueron: una dieta tradicional de vacas de alta producción (TR, CNF = 43%) y una dieta alta en CNF (HC, CNF = 47%). El grupo HC incrementó la producción de leche y su contenido en proteína, pero afectando negativamente a la salud de los animales (síndrome de baja grasa, mayor riesgo de cojeras, más horas a pH ruminal subóptimo).
El experimento 3 consistió en describir los cambios fisiológicos en la fermentación ruminal durante la inducción a la acidosis en terneras de engorde y su control mediante una preparación comercial de anticuerpos policlonales (mayoritariamente contra S. bovis). Se utilizaron 12 terneras cruzadas que se distribuyeron completamente al azar en 2 grupos de 6 animales cada uno: el grupo control (CTR) y el grupo que recibía el tratamiento de anticuerpos (PAP). El 83,3% y el 50,0% de los animales entraron en acidosis en el grupo CTR y PAP, respectivamente, y tardaron de media 5,25 ± 0,17 d. Asimismo, el perfil de fermentación de los animales acidóticos fue similar entre tratamientos. El grupo PAP tuvo mayores valores de pH a las 0h post-alimentación en los días 16, 18 y 19 de experimento comparado con el grupo CTR. Estos resultados indican que PAP puede ser efectivo para controlar la acidosis en terneras durante una transición rápida a concentrado.
El objetivo del experimento 4 fue evaluar los efectos de un inhibidor de la actividad α-amilasa (acarbosa) en el control de la acidosis ruminal sobre la fermentación ruminal y el metabolismo en vacas lecheras. Se usaron 8 vacas Holstein en un diseño cross-over 2x2. Los tratamientos fueron: control (sin aditivo, CTR) y acarbosa (0,75 g/animal/d, AMI). Se observó que el grupo AMI estuvo menos horas a pH ruminal subóptimo y tuvo un pH ruminal medio superior comparado con el grupo CTR. Microbiológicamente, los animales AMI tendieron a tener una menor ratio S. bovis:M. elsdenii comparado con los CTR. No se observaron diferencias en el resto de parámetros. Estos resultados indican que el suplemento de dietas con acarbosa en vacas lecheras alimentadas con raciones de alta producción puede ser efectivo en reducir la acidosis ruminal sin repercusiones en la fermentación ruminal del animal ni en su metabolismo.
This doctoral thesis was designed to increase our knowledge of ruminal acidosis and to valuate new strategies
In experiment 1, quantitative PCR probes were developed to detect and quantify S. bovis and M. elsdenii, lactic acid-producing and lactic acid-utilizing bacteria, respectively. The two PCR probes had efficiencies of 88% (S. bovis) and 99% (M. elsdenii). In the following experiments of this thesis, these techniques were used to progress in the understanding of the role that these bacteria have in the development of acidosis.
The experiment 2 was designed to study the effects of administrating high levels of NFC in the diet on ruminal fermentation and animal metabolism of lactating dairy cows. Sixty-two lactating Holstein cows were used in a cross-over 2x2. Treatments were: a traditional ration of high-producing dairy cows (TR, NFC = 43%) and a high-NFC ration (HC, NFC = 47%). Milk yield and milk protein content were greater in HC group, but had negative effects on animal health (milk fat depression, greater lameness risk, more hours at suboptimal pH).
In experiment 3, physiological changes in rumen fermentation during acidosis induction and its control using a multivalent polyclonal antibody preparation against S. bovis (PAP) in heifers were studied. A completely randomized experiment was designed using 12 crossbred heifers. Treatments were control (CTR) and PAP. After the feeding challenge, acidosis was declared 5.25 ± 0.17 d after the start of transition, during which 83.3 and 50.0% of the animals entered in acidosis in CTR and PAP groups, respectively. However, the fermentation profile of animals with acidosis was similar between treatments. Heifers fed PAP had greater pH before feeding on days 16, 18 and 19 of the experiment compared with CTR. These results indicate that PAP may be effective in controlling acidosis of heifers during a rapid transition to a high-concentrate diet.
The objective of experiment 4 was to evaluate the effects of an amylase inhibitor (acarbose) in dairy cows to control ruminal acidosis in a 2x2 cross-over design. Eight Holstein cows were used to study animal metabolism and ruminal fermentation. Treatments were: control (no additive, CTR) and acarbose (0.75 g/cow/d, AMI). AMI group stayed less hours below pH = 5.6 and had greater daily average pH compared with CTR group. AMI animals tended to have lower S. bovis to M. elsdenii ratio than CTR. These results indicate that supplementing diets with acarbose to dairy cattle fed high-NFC rations may be effective in reducing acidosis in lactating cows with no negative effects on ruminal fermentation and animal metabolism.

Hereditary myopathy with lactic acidosis (HML, OMIM#255125) is an autosomal recessive disorder which originates from Västerbotten and Ångermanland in the Northern part of Sweden. HML is characterized by severe exercise intolerance which manifests with tachycardia, dyspnea, muscle pain, cramps, elevated lactate and pyruvate levels, weakness and myoglobinuria. The symptoms arise from malfunction of the energy metabolism in skeletal muscles with defects in several important enzymes involved in the TCA cycle and the electron transport chain. All affected proteins contain iron-sulfur (Fe-S) clusters, which led to the suggestion that the disease was caused by malfunctions in either the transportation, assembly or processing of Fe-S clusters. The aim of my thesis was to identify the disease causing gene of HML and to investigate the underlying disease-mechanisms. In paper I we identified a disease-critical region on chromosome 12; a region containing 16 genes. One of the genes coded for the Fe-S cluster assembly protein ISCU and an intronic base pair substitution (g.7044G>C) was identified in the last intron of this gene. The mutation gave rise to the insertion of intron sequence into the mRNA, leading to a protein containing 15 abberant amino acids and a premature stop. In paper II we investigated why a mutation in an evolutionary well conserved protein with a very important cellular role, which in addition is expressed in almost all tissues, gives rise to a muscle-restricted phenotype. Semi-quantitative RT-PCR analysis showed that the mutant transcript constituted almost 80% of total ISCU mRNA in muscle, while in both heart and liver the normal splice form was dominant. We could also show that, in mice, complete absence of Iscu protein was coupled with early embryonic death, further emphasizing the importance of the protein in all tissues. These data strongly suggested that tissue-specific splicing was the main mechanism responsible for the muscle-specific phenotype of HML. In paper III the splicing mechanisms that give rise to the mutant ISCU transcript was further investigated. We identified three proteins; PTBP1, IGF2BP1 and RBM39, that could bind to the region containing the mutation and could affect the splicing pattern of ISCU in an in vitro system. PTBP1 repressed the inclusion of the intronic sequence, while IGF2BP1 and RBM39 repressed the total ISCU mRNA level though the effect was more pronounced for the normal transcript. Moreover, IGF2BP1 and RBM39 were also able to reverse the effect of PTBP1. IGF2BP1, though not a splicing factor, had higher affinity for the mutant sequence. This suggested that the mutation enables IGF2BP1 binding, thereby preventing the PTBP1 induced repression seen in the normal case. In conclusion, we have determined the genetic cause of HML, identifying a base pair substitution in the last intron of the ISCU gene that gives rise to abnormally spliced transcript. The muscle-specific phenotype was also analyzed and tissue-specific splicing was identified as the main disease-mechanism. Furthermore, nuclear factors with ability to affect the splicing pattern of the mutant ISCU gene were identified. This work has thoroughly investigated the fundamental disease mechanisms, thus providing deeper understanding for this hereditary myopathy.

This thesis describes two groups of experiments, both relating to the condition of distal renal tubular acidosis (dRTA). In the first, an alternative diagnostic test of dRTA to the ‘gold standard’ short ammonium chloride (NH4Cl) test was assessed. This was achieved by the simultaneous oral administration of the diuretic furosemide and the mineralocorticoid fludrocortisone to increase distal sodium delivery and a-intercalated cell proton secretion. I evaluated 11 control subjects and 10 patients with known dRTA by giving oral NH4Cl or furosemide/fludrocortisone in random order on separate days. 3 subjects were unable to complete the study due to vomiting after the NH4Cl, however there were no adverse effects with furosemide/fludrocortisone administration. The urine pH decreased to less than 5.3 in the controls with both tests, whereas no patients were able to lower their urine pH below 5.3 with either test. The simultaneous administration of furosemide/fludrocortisone proved to be an easy, effective and well-tolerated alternative to the standard NH4Cl test for the diagnosis of dRTA. The second group were laboratory-based molecular physiology experiments. Anion exchanger 1 (AE1) mediates electroneutral anion exchange across cell membranes. It is the most abundant protein in the red cell membrane, but is also found in the basolateral membrane of renal a-intercalated cells, where it is required for urinary acidification. Point mutations have been described that convert the red cell AE1 into a cation conductance. AE1 mutations can also cause hereditary dRTA. I investigated the properties of four dRTA associated AE1 mutations (R586H, G609R, S613F and G701D) by heterologous expression in Xenopus Laevis oocytes. These mutants proved to be functional anion exchangers, unlike the red cell mutants, but also demonstrated a cation ‘leak’. I found a very large leak property in the G701D mutant, which is prevalent in SE Asia. I hypothesised that this property might confer a survival advantage. I characterised three other AR dRTA-associated AE1 mutants found in SE Asia, S773P, \Delta850 and A858D via similar transport experiments in AE1-expressing Xenopus oocytes. These three SE Asian mutants also had cation leaks of similar magnitude to that seen in G701D, a property that distinguishes them as a discrete group. The clustering of these cation-leaky AE1 mutations to malarious areas of SE Asia suggests that they may confer malaria resistance.

Ruminal acidosis is a digestive disorder in dairy and beef cattle and the current definitions are based on the pH of rumen fluid and may occur as an acute, chronic or subclinical (sub-acute) condition. With the aim to achieve the increase of production of milk, highly productive dairy cows are fed cereal-based and energy-dense diets and the rapid ruminal fermentation of starch and sugars leads to an accumulation of volatile fatty acid (VFA) and/or lactate and reduced rumen buffering which cause a drop of pH. Subacute ruminal acidosis (SARA) is characterized by a spontaneous recovery of repeated bouts of low ruminal pH, transient or no accumulation of ruminal lactate, and subtle clinical signs during low ruminal pH bouts. SARA causes consistent direct and indirect economic losses in dairy farming due to the decrease of quality and quantity of milk, increased veterinary costs, premature and involuntary culling. The clinical signs of SARA in the individual cow are subtle, delayed for weeks or months after acidotic insult, not-pathognomonic, and they can be seen in a number of the other diseases. Moreover, ruminal pH varies considerably by different factors like: time after feeding, time of the day, site of sampling within the rumen, techniques of sampling, and individual characteristics. These reasons and the problems in obtaining representative rumen fluid samples make that SARA is under-diagnosticated. Currently, the measurement of rumen pH is used to diagnose SARA, but some diagnostic techniques are invasive (rumen cannulation), or mild invasive (rumenocentesis), give spot sampling of rumen fluid pH decreasing the sensibility on diagnosis of rumen acidosis (rumenocentesis, oral-stomach tube) or cause saliva contamination that alters the values of ruminal pH (oral-stomach tube). So the aim of thesis was the study of easy, rapid and no invasive and economic direct and indirect diagnostic methods in order to diagnose SARA and indicate the severity of the ruminal acidosis (chapters 3 and 4). Moreover, the effectiveness of different methods of prevention of ruminal acidosis was tested (chapters 5, 6, 7 and 8). The first experimental contribution of the thesis (Effect of induced ruminal acidosis on blood variables in heifers; chapter 3) was carried out according to a 3 × 3 Latin square arrangement (3 dietary treatments × 3 periods). Six crossbred Valdostana x Belgian Blue non–pregnant heifers were randomly assigned to one of three dietary treatments with different starch levels (% DM): control (CT) as low starch (17.3%), medium starch (MS) for SARA (33.4%), and high starch (HS) to induce acute ruminal acidosis (42.8%). The animals were fed three times a day at 0800, 1200 and 1800 h. Ruminal pH was continuously measured every 10 minutes using wireless sensors featured to fluctuate into the rumen and by rumenocentesis performed at 4 hours after total mixed ration distribution on the 4th day of each experimental period. The regression coefficient between the two methods (wireless sensors vs. rumenocentesis) was 0.56 (P = 0.040). Feeding the CT, MS and HS led to differences in the time spent below the 5.8, 5.5 and 5.0 pH thresholds and in several variables, including dry matter intake (7.7 vs. 6.9 vs. 5.1 kg/d for CT, MS, and HS, respectively; P = 0.002), ruminal nadir pH (5.69 vs. 5.47 vs. 5.44; P = 0.042), mean ruminal pH (6.50 vs. 6.34 vs. 6.31; P = 0.012), haemoglobin level (11.1 vs. 10.9 vs. 11.4 g/dL; P = 0.010), platelet count (506 vs. 481 vs. 601 K/μL; P = 0.008), HCO3- (31.8 vs. 31.3 vs. 30.6 mmol/L; P = 0.071) and lipopolysaccharide-binding proteins (5.9 vs. 9.5 vs. 10.5 μg/mL; P < 0.001). Applying a canonical discriminant analysis (CDA), five plasma variables (hemoglobin, mean platelet volume, β-hydroxybutyrate, glucose, and reduced hemoglobin) were able to discriminate among physiological ruminal status: SARA or acute ruminal acidosis. The CDA was characterized by two significant (Wilks’ λ = 0.282, F approx = 3.76, df1 =15, df2 = 97, P < 0.0001) axes, which accounted for 60% and 38% of the existing variation. The second experimental contribution (Blood parameters modification at different ruminal acidosis conditions; chapter 4) showed that the metabolic condition related to the ruminal acidosis can be evaluated with one daily blood sample because no differences were observed on blood sampling time (8:00 and 12:00 h). The third experimental contribution (Effect of feeding fine maize particles on the reticular pH, milk yield and composition of dairy cows; chapter 5) evaluated whether feeding finely ground maize (Fg) could cause an increased risk of SARA in high-producing dairy cows. The pH measures were performed through the use of wireless sensors featured to remain in the reticulum, continuously recording reticular pH and temperature. Twelve Holstein-Friesian cows were assigned to one of two experimental groups and were exposed to one of two dietary treatments (maize meal ground to 1.0 mm, control group (Ct) and maize meal ground to 0.5 mm, Fg group) in a 2 × 2 cross-over design (2 diets × 2 periods) over a period of 21 days (14 days of an adjustment phase followed by 7 days of data collection). The pH and temperature of the reticulum were continuously measured in eight cows using indwelling sensors throughout the trial. Data were submitted to statistical analyses according to a mixed model procedure. Reducing the maize particle size, greater dry matter intake (19.0 vs. 20.3 kg/d, for Ct and Fg, respectively; P = 0.067), an increase in milk protein (3.18 vs. 3.31%; P = 0.021), milk casein (2.48 vs. 2.57%; P = 0.035), a reduction in feeding efficiency (1.63 vs. 1.52; P = 0.008), and an increase in starch digestibility (0.94 vs. 0.98; P = 0.078) were observed. The cows fed Fg diet spent a significantly higher time below the 5.5 pH threshold (15 vs. 61 min/day; P = 0.047), had an average daily variation in reticular pH characterized by a lower nadir pH (5.95 vs. 5.72; P < 0.001) and a higher pH range (0.79 vs. 0.94; P = 0.003). Neither reticular temperature parameters nor the daily time spent below the temperature thresholds were affected by dietary treatments. This study demonstrated that the grain particle size should be carefully considered during the ration formulation to maximize the production and quality without excessively increasing the risk of ruminal acidosis, since reducing the maize particle size resulted in a drop in the reticular pH, an increase in milk protein content and a slight reduction in feeding efficiency. The second part of the thesis was to identify prevention strategies against SARA in dairy cows using some feed supplements which could help to prevent SARA and identify the mechanisms of action through which they act on ruminal fermentation (chapters 6, 7, and 8). In the fourth experimental contribution (Effects of carbohydrase inhibiting compounds on in vitro rumen fermentation; chapter 6) an in vitro rumen fermentation study used plant-derived supplements (bilberry, phaseolamin, white mulberry, and common flax) at two different doses (15 mg and 150 mg) that were added to 0.5 g of maize meal (maize grain ground through a 2-mm screen). The supplements showed to modify fermentation pattern and suggested having effect on the microbial composition in vitro given the differences in ammonia N concentration and VFA proportion. In particular, compared to positive control (acarbose), bilberry and mulberry caused the highest drop in pH due to the rapid fermentation of their sugar content. In addition, bilberry resulted in an increase in propionate and in an apparently lower ammonia N concentration and showed an activity against starch degradation, although this effect was concealed by the fermentation of sugars present in the supplement. In the last two experimental contributions (Use of dicarboxylic acids and polyphenols to attenuate reticular pH drop and acute phase response in dairy heifers fed a high grain diet; chapter 7 and Effect of dicarboxylic acids and polyphenols on rumen microbial population in dairy heifers fed a high grain diet though metagenomic analysis; chapter 8) the dicarboxylic acids (fumarate-malate) and natural plant extracts (polyphenol-essential oil mixture) were used in an in vivo study with the aim to determine their effects in attenuating the drop of rumen pH and the changes in metabolites and inflammatory markers in blood and rumen fluid in dairy heifers fed high-grain diet (chapter 7) and evaluate the changes on the rumen bacterial population (chapter 8). In the fifth and sixth experimental contributions according to a 3 × 3 Latin square experiment (3 dietary treatments × 3 periods) six Holstein-Friesian non-pregnant heifers were fed a low starch (LS) diet for 14 d (NDF 39.8%, starch 24.0% DM), followed by a high starch (HS) diet for 8 d (NDF 33.6%, starch 30.0% DM). During HS feeding, all animals were randomly assigned to one of the following three dietary treatments: no supplement/control (CT), a daily dose of 60 g/d of a fumarate-malate mixture (FM), or 100 g/d of polyphenol-essential oil mixture (PM). Reticular pH was continuously measured using the reticular wireless boluses and on rumen fluid collected by rumenocentesis (1400 h) on d 21 of each period together with blood (0800 h) and fecal samples (0800, 1400, and 2100 h). An aliquot of rumen fluid was collected to study the effects of the supplements (FM and PM) on the rumen bacterial populations using quantitative real-time PCR (qPCR) and Illumina sequencing. The correlation coefficient (r) between the pH values obtained using the reticular boluses and rumenocentesis was 0.83 (P < 0.001). Nadir pH was lowest during CT treatment (5.40, 5.69, and 5.62 for CT, FM, and PM, respectively; P = 0.037), confirming the effectiveness of both supplements in reducing the pH drop caused by high maize feeding. Moreover, the PM treatment demonstrated to be more effective than FM treatment in reducing the inflammatory response compared to CT treatment with a decrease of the concentrations of neutrophils (2.9, 3.2, and 2.8 109/L; P = 0.084), acute phase proteins as serum amyloid A (37.1, 28.6, and 20.1 µg/mL; P = 0.036), lipopolysaccharide-binding proteins (4.1, 3.8, and 2.9 µg/mL; P = 0.048), and haptoglobin (675, 695, and 601 µg/mL; P = 0.084). The pH and lipopolysaccharide concentration of feces were not affected by dietary treatment, whereas fecal pH values were affected by time (P = 0.042) and were 6.38, 6.71, and 6.69 at 0800, 1400, and 2100 h, respectively (chapter 7). Both supplements showed to modify the rumen microbial population in dairy heifers fed a high grain diet (chapter 8). In conclusion, direct and indirect diagnostic methods such as blood variables and indwelling boluses resulted a promising diagnostic approach to monitor the ruminal pH changes that may be easily used in intensive dairy farms. Regarding the prevention, some feed supplements such as fumarate-malate and polyphenols attenuate the effects of diets with high concentration of cereals acting on ruminal microbial population that may lead to positive effects on rumen fermentations with effects on animal health and welfare, productivity and profitability in intensive dairy farms
L’acidosi ruminale è un disturbo metabolico-fermentativo presente nell’allevamento della vacca da latte e nel bovino da carne. Attualmente la definizione di acidosi si basa sul pH ruminale e può manifestarsi in forma acuta, cronica e subclinica (sub-acuta). Al fine di ottenere un aumento delle produzioni di latte, le vacche ad alta produzione sono alimentate con diete altamente energetiche e a base di cereali che a seguito della rapida fermentazione ruminale dell’amido e degli zuccheri inducono un accumulo di acidi grassi volatili e/o lattato, che associato a una ridotta capacità tampone a livello ruminale causa una caduta del pH. L’acidosi ruminale subacuta (SARA) è caratterizzata da un recupero spontaneo di ripetuti picchi di basso pH ruminale, assente o transitorio accumulo di lattato ruminale e lievi sintomi clinici durante i cali di pH ruminale. SARA causa ingenti danni economici sia diretti che indiretti nell’allevamento della vacca da latte a causa della diminuita qualità e quantità di latte prodotto, delle maggiori spese veterinarie, della prematura macellazione dell’animale. I sintomi clinici di SARA nel singolo animale sono subdoli, ritardati di settimane o mesi dopo l’evento acidotico, non patognomonici e comuni ad altre numerose patologie. Il pH ruminale inoltre varia considerevolmente a seconda di vari fattori quali: tempo intercorso dopo il pasto, momento del giorno, punto di campionamento all’interno del rumine, tecniche di campionamento utilizzate per raccogliere il liquido ruminale. Per queste ragioni e per i problemi nell’ottenere campioni di liquido ruminale rappresentativi, SARA risulta essere molto spesso sottovalutata. Allo stato attuale, per diagnosticare SARA si misura il pH ruminale utilizzando varie tecniche: alcune di esse sono considerate invasive (fistolizzazione ruminale), o moderatamente invasive (ruminocentesi), forniscono rilievi puntiformi del pH ruminale, perdendo quindi sensibilità nella diagnosi di acidosi ruminale (ruminocentesi, sonda orogastrica) o possono portare a contaminazioni con la saliva che altera il pH del campione (sonda orogastrica). Per ovviare a tali inconvenienti con questa tesi si è cercato di individuare metodi diagnostici (diretti ed indiretti) poco invasivi, rapidi, economici e di facile esecuzione per rilevare la presenza e definire la gravità dell’acidosi ruminale (capitoli 3 e 4). Si è inoltre indagata l’efficacia di diversi metodi di prevenzione (capitoli 5, 6, 7 e 8). Il primo contributo sperimentale (Effect of induced ruminal acidosis on blood variables in heifers; capitolo 3) è stato eseguito utilizzando un quadrato latino 3 × 3 (3 diete × 3 periodi). A sei manze non gravide (incrocio Valdostana x Blu Belga) è stata somministrata una delle tre diete contenenti differenti livelli di amido (% sostanza secca): basso (17.3%) come controllo (CT), medio (MS) per indurre SARA (33.4%) e alto (HS) per indurre l’acidosi ruminale acuta (42.8%). Gli animali sono stati alimentati tre volte al giorno (ore 08:00, 12:00 e 18:00). Il pH ruminale è stato misurato di continuo ogni 10 minuti tramite boli, che hanno la caratteristica di fluttuare all’interno del liquido ruminale, e tramite ruminocentesi eseguita 4 ore dopo la distribuzione della razione al 4° giorno di ogni periodo sperimentale. Il coefficiente di regressione tra i due metodi (boli vs. ruminocentesi) è risultato pari a 0.56 (P = 0.040). La somministrazione di CT, MS e HS ha determinato delle differenze nel tempo speso sotto le soglie di pH 5.8, 5.5 e 5.0 e in diverse variabili compresa l’ingestione (7.7 vs. 6.9 vs. 5.1 kg/d per CT, MS e HS, rispettivamente; P = 0.002), pH ruminale nadir (5.69 vs. 5.47 vs. 5.44; P = 0.042), pH ruminale medio (6.50 vs. 6.34 vs. 6.31; P = 0.012), livello di emoglobina (11.1 vs. 10.9 vs. 11.4 g/dL; P = 0.010), conta piastrinica (506 vs. 481 vs. 601 K/μL; P = 0.008), HCO3- (31.8 vs. 31.3 vs. 30.6 mmol/L; P = 0.071) e proteina legante i lipopolisaccaridi (LBP) (5.9 vs. 9.5 vs. 10.5 μg/mL; P < 0.001). Applicando un’analisi discriminante canonica multifattoriale (CDA), cinque variabili plasmatiche (emoglobina, volume piastrinico medio, β-idrossibutirrato, glucosio ed emoglobina ridotta) sono state in grado di discriminare significativamente lo stato ruminale fisiologico: SARA o acidosi ruminale acuta (Wilks’ λ = 0.282, F approx = 3.76, df1 =15, df2 = 97, P < 0.0001). A seguito della CDA, si sono ottenute due variabili latenti (CAN 1 e CAN 2) che spiegano il 60% e il 38% della varianza. Il secondo contributo sperimentale (Blood parameters modification at different ruminal acidosis conditions; capitolo 4) ha dimostrato che per la valutazione dello stato di acidosi ruminale può essere sufficiente un solo rilievo di sangue al giorno, stante la mancanza di differenze statisticamente significative per tutte le variabili ematologiche determinate nel confronto tra i due tempi di campionamento (08:00 vs. 12:00). Il terzo contributo (Effect of feeding fine maize particles on the reticular pH, milk yield and composition of dairy cows; capitolo 5) ha previsto l’utilizzo di una differente tipologia di boli che hanno la caratteristica di rimanere sul fondo del reticolo. Questi boli hanno registrato in continuo il pH e la temperatura reticolare ed è stato possibile valutare se la somministrazione di mais finemente macinato (Fg) potesse causare un maggiore rischio di SARA in vacche da latte ad alta produzione. Dodici vacche Frisone sono state assegnate a uno dei due gruppi sperimentali: Ct (mais macinato a 1.0 mm) o Fg (mais macinato a 0.5 mm) utilizzando un disegno sperimentale a cross over (2 diete × 2 periodi) basato su una fase di adattamento di 14 giorni e una di rilievo dati di 7 (periodo di 21 giorni). Il pH e la temperatura reticolare sono stati misurati in continuo in 8 vacche utilizzando i boli durante tutta la prova sperimentale. I dati raccolti sono stati sottoposti ad analisi statistica secondo un modello MIXED. Riducendo le dimensioni delle particelle di mais si è osservato un appena significativo aumento dell’ingestione (19.0 vs. 20.3 kg/d per Ct e Fg, rispettivamente; P = 0.067), un significativo incremento del livello di proteina grezza (3.18 vs. 3.31%; P = 0.021) e caseina nel latte (2.48 vs. 2.57%; P = 0.035); per contro, si è osservata una significativa riduzione dell’efficienza alimentare (1.63 vs. 1.52; P = 0.008) benché sia aumentata significativamente la digeribilità dell’amido (0.94 vs. 0.98; P = 0.078). Le vacche alimentate con Fg hanno significativamente speso maggior tempo sotto la soglia di pH 5.5 (15 vs. 61 min/giorno; P = 0.047), hanno avuto una più alta variazione giornaliera media del pH reticolare caratterizzata da un più basso pH nadir (5.95 vs. 5.72; P < 0.001) e un più alto range di pH (0.79 vs. 0.94; P = 0.003). I trattamenti non hanno influenzato i parametri della temperatura reticolare e neppure il tempo speso giornalmente sotto le soglie di temperatura. Questo studio ha dimostrato che anche le dimensioni delle particelle di mais devono essere prese in considerazione durante la formulazione di una razione volta a massimizzare produzione e qualità del latte, stante il fatto che una loro riduzione sembra favorire un aumento del tenore proteico del latte a scapito di una seppur modesta diminuzione dell’efficienza alimentare; tuttavia, si deve porre attenzione a non aumentare il rischio di acidosi ruminale. La seconda parte della tesi ha avuto lo scopo di individuare alcune strategie per prevenire SARA nelle vacche da latte tramite l’utilizzo di alcuni supplementi alimentari e di cercare di identificare i meccanismi d’azione attraverso i quali questi agiscono sulla fermentazione ruminale (capitoli 6, 7 e 8). Il quarto contributo sperimentale (Effects of carbohydrase inhibiting compounds on in vitro rumen fermentation; capitolo 6) è stato uno studio in vitro sulla fermentazione ruminale che ha previsto l’utilizzo di derivati vegetali (mirtillo, faseolamina, gelso bianco e lino comune) a due dosi (15 o 150 mg) aggiunti a 0.5 g di farina di mais (mais macinato attraverso un setaccio di 2 mm). Gli additivi hanno dimostrato sia di poter modificare il pattern fermentativo sia di poter influenzare la composizione batterica in vitro stante le differenze nella concentrazione di azoto ammoniacale e nelle proporzioni di acidi grassi volatili. In particolare, rispetto a un controllo positivo (acarbosio), mirtillo e gelso hanno determinato una maggiore caduta di pH probabilmente a causa della rapida fermentazione del loro contenuto in zuccheri. Il mirtillo inoltre ha aumentato il propionato e comportato un apparente calo della concentrazione di azoto ammoniacale; il mirtillo sembra avere la capacità di limitare la degradazione dell’amido mascherato, sebbene questo effetto sia stato limitato dalla fermentazione degli zuccheri presenti nel supplemento. Negli ultimi due contributi (Use of dicarboxylic acids and polyphenols to attenuate reticular pH drop and acute phase response in dairy heifers fed a high grain diet; capitolo 7. Effect of dicarboxylic acids and polyphenols on rumen microbial population in dairy heifers fed a high grain diet though metagenomic analysis; capitolo 8) acidi dicarbossilici (fumarato-malato) ed estratti di piante (miscela di olii essenziali e di polifenoli) sono stati utilizzati per una prova in vivo allo scopo di determinare i loro effetti sulla caduta del pH ruminale, sui metaboliti e gli indicatori infiammatori del sangue e del liquido ruminale in manze alimentate con diete ad alto contenuto di mais (capitolo 7) e di valutare i loro effetti sulla popolazione batterica ruminale (capitolo 8). Secondo un protocollo sperimentale a quadrato latino 3 × 3 (3 diete × 3 periodi) sei manze Frisone non gravide sono state alimentate con una dieta a basso contenuto d’amido (LS) per 14 giorni (NDF 39.8%, amido 24.0% SS), seguita da una dieta ad alto contenuto d’amido (HS) per 8 giorni (NDF 33.6%, amido 30.0% SS). Durante la somministrazione della dieta HS gli animali sono stati assegnati a uno dei tre trattamenti: nessun supplemento/controllo (CT), una dose giornaliera di 60 g al giorno di una miscela di fumarato-malato (FM), o 100 g al giorno di una miscela di olii essenziali e polifenoli (PM). Il pH ruminale è stato misurato in continuo mediante boli fissi all’interno del reticolo e confrontato con quello rilevato tramite un pH-metro portatile sul liquido ruminale raccolto mediante ruminocentesi il 21° giorno di ogni periodo sperimentale alle 14:00. Un’aliquota di liquido ruminale è stata raccolta per studiare gli effetti dei supplementi sulla produzione di acidi grassi volatili (AGV) e lipopolisaccaridi (LPS) e sulla popolazione microbica ruminale utilizzando la real-time PCR (qPCR) e il sequenziamento Illumina. Lo stesso giorno sono stati inoltre raccolti campioni di sangue (08:00) e feci (08:00, 14:00 e 21:00). Il coefficiente di correlazione tra i valori di pH acquisiti tramite i boli reticolari o la ruminocentesi è stato pari a 0.83 (P < 0.001). Il pH nadir è risultato più basso nella dieta controllo (5.40, 5.69 e 5.62 per CT, FM e PM, rispettivamente; P = 0.037), confermando l’efficacia di entrambi i supplementi nel ridurre la caduta del pH ruminale causata da alimenti ricchi di cereali. Inoltre, PM ha dimostrato di essere più efficace rispetto a FM nel ridurre la risposta infiammatoria con una diminuzione delle concentrazioni di neutrofili (2.9, 3.2 e 2.8 109/L; P = 0.084), proteine della fase acuta come siero amiloide A (37.1, 28.6 e 20.1 µg/mL; P = 0.036), LBP (4.1, 3.8 e 2.9 µg/mL; P = 0.048) e aptoglobina (675, 695 e 601 µg/mL; P = 0.084). Il pH e la concentrazione di lipolisaccaridi nelle feci non sono state influenzate dai trattamenti, mentre i valori di pH nelle feci sono risultati influenzati dal tempo di raccolta (6.38, 6.71 e 6.69 alle ore 08:00, 14:00 e 21:00, rispettivamente; P = 0.042) (capitolo 7). Entrambi i supplementi hanno dimostrato di modificare la popolazione microbica ruminale in manze alimentate con una dieta ad alto contenuto d’amido (capitolo 8). Concludendo, strumenti diagnostici (diretti e indiretti) come boli e variabili del sangue sono risultati essere promettenti per monitorare variazioni di pH ruminale. Tali strumenti diagnostici possono essere facilmente utilizzati negli allevamenti di vacche da latte ad alta produzione. Per quanto riguarda inoltre la prevenzione di SARA, tra gli additivi alimentari testati, gli acidi dicarbossilici (fumarato-malato) e soprattutto i polifenoli si sono dimostrati efficaci nell’attenuare la caduta del pH ruminale dovuta all’uso di diete ad alto contenuto di cereali, agendo sulla popolazione microbica del rumine. Tali risultati suggeriscono il loro utilizzo negli allevamenti per aiutare a garantire la salute e il benessere delle bovine da latte, favorendo di conseguenza produttività e redditività per gli allevatori

Multiple Sclerosis (MS) is an inflammatory demyelinating disease of the Central Nervous System (CNS) featuring severe neuronal degenerative processes. Emerging findings indicate that several injurious molecular and cellular cascades are contributing to neurodegeneration. Among them, CNS acidosis has been recently demonstrated to have a detrimental role in Experimental Autoimmune Encephalomyelitis (EAE). Good candidates to trigger acidosis-mediated neurodegeneration are represented by Acid Sensing Ion Channels (ASICs) which are H+-gated cation channels of the CNS. We measured CNS acidosis in EAE mice by a non-invasive magnetic resonance spectroscopy (MRS), establishing the presence of a substantial extracellular acidification in the Cerebellum (CB). We next examined the ability of Diminazene Aceturate (DA), a new specific ASICs inhibitor, to promote neuroprotective effects in chronic EAE mice. Behavioral and histological evaluations indicated that DA significantly improved walking performances in EAE mice, ameliorated their neurological deficits, as well as, reduced myelin and axonal loss in both CB and Spinal Cord. Additionally, we model in vitro the harmful electrophysiological alterations elicited by acidosis using primary neuronal cultures coupled with Micro Electrode Array (MEA) devices. While an acute acidic treatment caused a rapid and transient reduction of firing activity, long term acidosis caused the chronic impairment of synchronized neuronal electrophysiological activity, and a significant loss of pre-synaptic boutons. DA efficiently compensated the loss of firing activity derived from acute acidosis, as well as protected neurons from injurious effects elicited by chronic acidosis. In conclusion our data suggest that ASICs activation is involved in mediating neuronal derangement during acute neuroinflammation and that the early intervention with specific ASICs antagonists may attenuate these detrimental effects.

Dissertação de Mestrado Integrado em Medicina Veterinária
A acidose metabólica é um desequilíbrio electrolítico muito frequente. O seu controlo e o restabelecimento do equilíbrio electrolítico são parte constante nas urgências veterinárias. Este estudo inclui uma amostra de 31 animais, dos quais 18 cães e 13 gatos. O estudo tem como objectivos a avaliação e reconhecimento das alterações electrolíticas e ácido-básicas em pacientes críticos, classificação do tipo de desequilíbrio e avaliação da resposta compensatória, relacionamento do tipo de patologia com os desequilíbrios ácido-básicos e avaliação da necessidade de instituir terapêutica alcalinizante. A detecção e avaliação de factores de risco em relação à morbilidade (dias de internamento) e fatalidade foram também tentados. As causas de acidose metabólica mais representadas nesta amostra foram a insuficiência renal (45%) e a diabetes mellitus, complicada ou não (19%). A restante patologia subjacente à acidose metabólica foi variada: patologia digestiva, intoxicações, hipoadrenocorticismo, neoplasia, obstrução urinária e golpe de calor. Apenas 14 (45%) dos animais tinham um diagnóstico primário de insuficiência renal. Porém, pode verificar-se que todos tinham compromisso da função renal, em estádios IRIS desde o grau 2 ao 4. Foi encontrada uma associação estatisticamente significativa (p=0.037) entre os valores de ureia plasmática e a duração do internamento, mas não em relação ao desfecho do caso. Os resultados obtidos sugerem que o sistema i-STAT®, com utilização dos cartuchos CG4+, permite uma caracterização do tipo de acidose metabólica presente em animais em cuidados intensivos, bem como a avaliação da necessidade de instituição de terapêutica alcalinizante, o que pode ser crítico em termos de sobrevivência de pacientes cujas causas primárias de acidose metabólica não sejam reversíveis. A dimensão relativamente pequena da amostra e a variabilidade das causas subjacentes à acidose metabólica acentuaram a dificuldade de interpretação das variáveis clínicas registadas e comprometeram a sua expressão estatística. O mesmo aconteceu em relação ao reconhecimento de factores de risco clínicos e laboratoriais de morbilidade e evolução dos casos.
ABSTRACT - Metabolic acidosis is a very common electrolyte imbalance. Its control and electrolyte correction are a constant part in veterinary emergency medicine. The present study includes a sample of 31 animals; 18 dogs and 13 cats. The goals were the evaluation and detection of electrolyte and acid-base imbalances in critical patients, classification of the type of acid-base imbalance as well as the compensatory response, relationship of the type of pathology present and the acid-base imbalance and the need to institute alkalinizing therapy. Furthermore, the detection and evaluation of risk factors for morbility and fatality were also pursued. The main causes of metabolic acidosis in our sample were renal failure (45%) and diabetes mellitus, simple or complicated (19%). The remainder primary causes of metabolic acidosis were varied: digestive conditions, poisoning, hypoadrenocorticism, neoplasia, urinary obstruction and heat stroke. Only 14 (45%) of the animals had a primary diagnosis of renal failure. However, all animals were found to have renal function compromise, in IRIS stages from 2 to 4. A statiscally significant association (p=0.037) between BUN and duration of internment was found, but not regarding the outcome. Our results suggest that the i-STAT® system, with the use of CG4+ cartridges, allows the characterization of the type of metabolic acidosis present in critical care animals, as well as the evaluation of the need to institute alkalinizing therapy, which may be essential in terms of survival in those patients whose primary causes of metabolic acidosis are not reversible. The small size of the sample used in this study and the variability of the underlying causes of metabolic acidosis hindered the interpretation of the clinical variables recorded and compromised its statistical expression. The same happened regarding the detection of clinical and laboratory risk factors towards morbility and outcome of the cases.

Subacute rumen acidosis (SARA) represent one of the most important metabolic disorders in intensive dairy farms that affects rumen fermentations, animal welfare, productivity and profitability. Many are the factors that can increase the possible occurrence of SARA in dairy farms and in particular in intensive dairy farms: food type, food administration modality, rumen ability in absorbing short chain fatty acids (SCFA), year period, ingestion frequency, dry-lactation transition period and others conditions able to contribute to the SARA onset. SARA is possibly under diagnosed because of lack of pathognomonic signs, diurnal fluctuations in rumen metabolism, and problems in obtaining representative rumen fluid samples. Clinical signs of SARA include decreased dry matter intake (DMI), laminitis, rumenitis, liver abscesses, pulmonary bacterial emboli and, furthermore, displacement of the abomasums, mastitis and metritis, low fertility. These problems are linked to rumen pathology because of the exceptional osmotic capacity shown by the rumen and the increase in the adsorption rate of ruminal products, which may have toxic and vasomotor effects. Considering the above statement it is impractical to base the diagnosis of SARA only on the outcome of a clinical sign, but the only way to obtain an early and correct diagnosis is to observe and correlate multiple aspects of the condition: a thorough collection of the history in the farm; evaluation of the diet; evaluation of the ruminal fluid. The determination of ruminal pH is a key factor for the diagnosis of SARA. Different methods are available for the collection of rumen fluid for pH analysis. According several researcher rumenocentesis may be useful for the collection of rumen fluid for pH determination. A group of at least 12 cows (early or middle lactation) is defined as having SARA when more than 30% of them show a ruminal pH lower than 5.5. Anyway rumenocentesis remains a mildly invasive diagnostic method so it results hardly used by freelancer veterinarian and poorly accepted by farmer. A practical need for Veterinarians and Food practitioner, is to have a simple and economic method of diagnosis of SARA, in order to implement in a timely manner the appropriate corrective diet, eliminate the causes of arrival of this disorder and avert the danger of progress in the same very high levels. In response to these requests, the aim of this PhD thesis, was to lead three different studies related to each other but, in order to evaluate the occurrence of subacute rumen acidosis in intensive Italian dairy herds to determine the relationship among diet composition, ruminal pH and short chain fatty acids (SCFA) concentration, to evaluate the effect of the subacute ruminal acidosis on some blood parameters in lactating dairy cows and finally to evaluate the effects of rumenocentesis on the health and welfare status of lactating dairy cows, evaluated by physical examination, blood analysis, milk production and superficial temperature on the area of rumenocentesis using infrared thermography. Trial 1: The aim of the present study was to study the occurrence of subacute rumen acidosis in intensive Italian dairy herds and to determine the relationship among diet composition, ruminal pH and SCFA concentration. Ten commercial dairy herds were investigated; twelve cows in good body condition, between 5 and 60 DIM and without clinical signs of disease were selected randomly from each herd, to perform rumenocentesis and obtain rumen fluid. Ruminal pH was determined immediately after sampling and concentration of SCFA in ruminal fluid was determined on samples after storage. We further studied the effects of rumenocentesis on animal health. Our data confirm the extreme validity of this technique as ruminal sampling. Results were subject to ANOVA and correlation analysis using SIGMA STAT 2.03. The results indicated the presence of SARA in 3 herds (more than 33% cows with rumen pH < 5.5), a critical situation (more than 33% cows with rumen pH < 5.8) in 5 farms and a normal rumen pH condition in 2 herds. In particular dairy herds show an average SCFA concentration of 150, 145, 123 mmol/L for low pH, critical pH and normal pH herds, respectively. The differences among diet composition were not significant even if herds with SARA showed a light discordance between initial composition and residual feed. In the affected herds it was not possible to understand the exact causes of SARA. Animal management seems to be one of the most important factor in developing SARA, including total mixed ration preparation. Trial 2: The aim of this study was to evaluate the effect of subacute ruminal acidosis on some blood parameters in lactating dairy cows. The study was carried out on ten highly productive farms, stationed in different zones throughout northern Italy. In all farms, ruminal fluid was collected through rumenocentesis from 12 dairy cows in early lactation, and ruminal pH was measured with a pH meter. Blood samples for hematochemical and hematological profiles were obtained from the same cows that had rumenocentesis performed by jugular venipuncture. The herds were divided into three groups depending on the mean rumen pH: group A counted farms with average ruminal pH > 5.8 (normal), group B included farms with average ruminal pH between 5.6 and 5.8 (risk), and in group C, dairy farms presented an average ruminal pH < 5.6 (acidosis). Data were statistically analyzed by analysis of variance to verify the effect of the group. The Bonferroni test was applied to determine statistical significances between the three groups. Statistically significant differences (p < 0.05) were observed for various parameters. It was concluded that during subacute ruminal acidosis in dairy cows, modifications of the concentrations of some blood parameters can be observed. Trial 3: The aim of this study was to evaluate the effects of rumenocentesis on the health and welfare status of lactating dairy cows, evaluated by physical examination, blood analysis, milk production and superficial temperature on the area of rumenocentesis using infrared thermography. Two groups of 6 cows either underwent ruminocentesis (GA) or sham (GB) procedures. Data were statistically analyzed by analysis of variance to verify the effect of the group. No effect was observed on white blood cell count, haptoglobin, total protein, and electrophoretic fractions between GA and GB groups. Milk yield was not affected by rumenocentesis. On the region of rumenocentesis in group GA, skin temperature increased 1.0°C immediately following rumen fluid collection, returned to the baseline after 48 h, and remained constant till the end of the study. This study suggested that ruminocentesis used to diagnose subacute ruminal acidosis has no negative impact on animal health and welfare of lactating cows.

Acidose metabólica é um fenômeno comum e clinicamente significativo em pacientes com sepse grave ou choque séptico. Entretanto, sua composição não é bem estabelecida. Neste estudo, descrevemos a composição da acidose metabólica em pacientes com sepse grave ou choque séptico desde sua internação em unidade de terapia intensiva (UTI) até os quinto dia de internação em unidade de terapia intensiva (UTI). Na admissão à UTI, a acidose metabólica foi um fenômeno muito freqüente. Ela era composta principalmente pelo componente derivado dos íons inorgânicos (dado principalmente pela diferença sódio cloro), seguido em magnitude pelo componentes decorrentes de ânions não mensuráveis e lactato e atenuada por hipoalbuminemia. A magnitude da acidose metabólica e hipercloremia foram maiores entre os pacientes não-sobreviventes (considerando a mortalidade hospitalar). Em análise multivariada o grau de acidose por íons inorgânicos, além do escore de gravidade APACHE II e nível inicial de creatinina sérica, esteve associada a mortalidade hospitalar. Ao longo do período de estudo, os pacientes sobreviventes apresentaram melhora da acidose metabólica por diminuição dos níveis de ânions não-mensuráveis e lactato. Os não sobreviventes mantiveram a mesma magnitude de acidose metabólica e apresentaram queda do pH por aumento da PCO2
Metabolic acidosis is frequently found in patients with severe sepsis and septic shock. Several studies have shown that the amount of metabolic acidosis measured by the standard base excess (SBE) at hospital admission and its evolution throughout the first days of intensive care unit (ICU) stay are correlated with clinical outcome. However, the precise composition of the metabolic acidosis in patients with severe sepsis and septic shock is not well known. In this study, we have described the composition of metabolic acidosis in patients with severe sepsis or septic shock at ICU admission and throughout the first five days of ICU stay, by applying the quantitative physicochemical methodology. Metabolic acidosis was extremely frequent at admission to the ICU. Its main component was attributable to the inorganic ion difference disturbance (mainly determined by the Na Cl difference), followed in magnitude by unmeasured anions and lactate´s components. Hypoalbuminemia represented the most frequent and important alkalinizing component. The degree of metabolic acidosis and hyperchloremia was more pronounced in the non-survivors group (according to hospital mortality). In a multivariate analysis the degree of metabolic acidosis due to disturbances in innorganic ion difference was associated to hospital mortality. Acidosis in survivors was corrected during the study period due to a decrease in lactate and SIG levels, whereas non-survivors did not correct their metabolic acidosis and suffered a decrease in the pH due to an increase in PCO2 levels

A heterogeneidade intratumoral é um fenômeno extremamente importante para entender a progressão tumoral e a resposta à intervenção terapêutica. A galectina-3 pertence à família das lectinas, possuem a função de reconhecimento e ligação à ?-galactosídeos ramificados de glicolipídeos e glicoproteínas, e está envolvida em processos fisiológicos e patológicos como o câncer. Nesse trabalho, a heterogeneidade intratumoral em relação à expressão de galectina-3 foi observada em amostras de diferentes lesões melanocíticas de pacientes. Além disso, o inóculo de células de melanoma murino negativas para galectina-3 em animais gal3-/- gerou tumores constituídos por uma fração de células tumorais que passaram a expressar de novo galectina-3, sugerindo que pressões do microambiente tumoral modulam a expressão dessa lectina em melanomas. A acidose extracelular atuou como regulador negativo de galectina-3 in vitro, diminuindo a expressão dessa lectina tanto em células de melanoma murino e humano quanto em melanócito murino. Entretanto, a hipóxia, seja pela exposição aguda ou intermitente, não alterou a expressão in vitro de galectina-3 em células de melanoma humano. Por fim, tumores originados pelo inóculo de células tumorais positivas e negativas para galectina-3 (mimetizando tumores heterogêneos) obtiveram a maior taxa de crescimento tumoral comparados aos tumores constituídos por uma única população de células, seja positiva ou negativa para galectina-3. Portanto, foram apresentadas evidências de que a heterogeneidade intratumoral em relação à galectina-3 parece estar envolvida com o sucesso evolutivo do melanoma e que a acidose é indicada como uma das pressões microambientais que contribuem para o estabelecimento e manutenção da fração de células tumorais negativas para galectina-3 dentro da massa tumoral
The intratumoral heterogeneity observed in human tumors is extremely important to understand tumor progression and its therapeutic response. Galectin-3 belongs to animal lectin family and it is a ?-galactosidase binding protein which is involved in physiological and pathological processes, including cancer. In this work, an intratumor heterogeneous galectin-3 expression was observed in tissue sessions containing different human melanocytic lesions. Moreover, negative galectin-3 murine cells injected into gal3-/- mice were able to generate tumors composed of a positive galectin-3 cell fraction, suggesting that selective forces in tumor microenvironment modulate galectin-3 expression in melanoma. Extracellular acidosis acts as a negative regulator to galectin-3 in vitro, decreasing its expression in murine and human melanoma cells and even in murine melanocytes. However, intermittent or acute hypoxia exposure did not alter galectin-3 expression in human melanoma cells in vitro. In addition, tumors originated from a mixture of positive and negative galectin-3 cells (mimicking heterogeneous tumors) showed higher growth rate compared to those derived from only galectin-3 positive or negative cells. Therefore, we showed evidences that galectin-3 intratumoral heterogeneity seems to be involved with the evolutionary success of melanoma and that acidosis may be the microenvironmental pressure responsible for the establishment and maintenance of galectin-3 negative cell fraction into the tumor bulk

Os aditivos alimentares monensina e óleos funcionais foram avaliados em dietas com elevada proporção de concentrado fornecida de forma abrupta a 12 novilhos canulados no rúmen. Foi utilizado delineamento de blocos ao acaso com medidas repetidas no tempo em dois períodos experimentais de 21 dias, com no mínimo seis semanas para readaptação dos animais à dieta volumosa (feno de Tifton). A dieta basal composta por 92,25% de concentrado (82,41% de milho quebrado, 6,78% de farelo de soja, 7,75% de feno de Tifton picado, 1,77% núcleo mineral e 1,29% ureia) foi fornecida no dia 1 de cada período experimental. Os animais receberam um dos aditivos alimentares, como seguem: sem aditivos (CTR), 400 mg de óleos funcionais de mamona e líquido da casca de caju/kg de MS ingerida (OF), 30 mg de monensina/kg de MS ingerida (M30) e 40 mg de monensina/kg de MS ingerida (M40). As variáveis ruminais e sanguíneas, o comportamento ingestivo e os microrganismos ruminais foram avaliados. Cada novilho foi considerado uma unidade experimental. Os dados foram analisados com o Proc Mixed, tendo o tempo após o fornecimento da alimentação como medidas repetidas. Animais alimentados com M40 reduziram a concentração total de ácidos graxos de cadeia curta (P = 0,017), a concentração de lactato (P = 0,0012) e osmolaridade ruminal (P = 0,04). Houve interação entre tratamento e dias após transição abrupta sobre a ingestão de matéria seca expressa em kg/dia (P = 0,008) e peso corporal (P = 0,045), bem como para o pH médio do rúmen (P = 0,04), a proporção molar de propionato (P = 0,034) e a proporção molar de valerato (P = 0,031). O tratamento M30 apresentou a menor relação acetato:propionato (P = 0,03). Os tratamentos não alteraram o comportamento ingestivo (P ≥ 0,05) ou as atividades comportamentais (P ≥ 0,05), bem como a expressão relativa de F. Succinogenes (P = 0,465), S. bovis (P = 0,781) e M. elsdenii (P = 0,972). O tratamento CTR apresentou maior propensão à ocorrência de desbalanço no sistema ácido-básico do sangue. Porém, o volume globular sanguíneo, o pH e a osmolaridade do sangue não foram alteradas pelos tratamentos (P ≥ 0,05). Os aditivos alimentares utilizados em dietas com elevada proporção de concentrado fornecidas de forma abrupta agiram sob formas distintas na redução da acidose subclínica. De forma geral, a inclusão de OF e M40 não resultou em melhoria expressiva no ambiente ruminal diante da situação desafiadora da dieta, especialmente na primeira semana, quando o tratamento M30 apresentou melhor capacidade de estabilizar a fermentação ruminal.
Monensin and functional oils were evaluated in high-concentrate diet abruptly fed to 12 ruminally canullated steers. A randomized complete block design with repeated measures over time within two experimental periods of 21 days each was used, with at least six weeks for readaptation of steers to forage diet. In the high-concentrate basal diet (92.25% of concentrate) the follow treatments were added: no additives (CTR); 400 mg of castor oil and cashew nut shell liquid/kg of DMI (FO); 30 mg monensin/kg of DMI (M30) and 40 mg monensin/kg of DMI (M40). The ruminal variables, blood metabolites, feeding behaviour and microbial variable were determined. Each steer was analysed as an experimental unit. Data were analysed by Proc Mixed with time after feeding used as repeated measures.Treatment M40 showed lower ruminal total short chain fatty acids (P = 0.017), lactate concentration (P = 0.0012) and osmolality (P = 0.04). The dry matter intake expressed by kg/day (P = 0.008) and porcenteage of body weight (P = 0.045), as well as mean rumen pH (P = 0.04), propionate (P = 0.034) and valerate (P = 0.031) molar proportion had significative interaction between treatment and day. The treatment M30 showed lower acetate:propionate ratio (P = 0.03). Treatments did not change the feed behaviour (P ≥ 0.05), behavioural activities (P ≥ 0.05), and relative expression of Fibrobacter succinogenes (P = 0.465), Streptococcus bovis (P = 0.781) and Megasphaera elsdenii (P = 0.972). The blood packed volume cell, osmolality and pH were unaffected by treatments (P ≥ 0.05), but high propensity to systemic acid-base imbalance were observed in CTR diet. Feed additives had different effects to reduce the subacute acidosis. The use of FO and M40 did not change most of the rumen fermentation variables, especially in the first week after abrupt transition, where M30 provided higher protection against acidosis.

Dissertação de Mestrado em Engenharia Zootécnica/Produção Animal
Subacute ruminal acidosis (SARA) is one of the most common metabolic diseases in dairy cows. The main cause for SARA is an excessive feeding of fermentable carbohydrates, mainly starch. To test the effect of varying starch levels in the diets on metabolic status and productive performance, six lactating Danish Holstein cows with ruminal cannulas and permanent intercostal artery catheters were used in a replicated 3 x 3 Latin square design. Animals were fed ad libitum with a control diet containing 20% starch (DM basis) during the adaptation period (6 days). At day 7 (D1), 9, 11 and 13 (D4) cows received one of the experimental diets containing either 28, 35, 42% starch (DM basis) corresponding to low, medium and high treatments. At day 8, 10, 12 and 14 cows were fed with the control diet. Rumen fluid (ventral and medial), blood, urine and milk samples were collected at day 6, 7 and 13, at -0.5, 1, 2.5, 4, 5.5 and 7 h relative to feeding. Additionally, milk yield, water intake and dry matter intake were recorded. In all samples, pH was measured. In blood samples, partial pressure of O2 and CO2 were measured. In milk samples, glucose, glucose-6-phosphate, β-hydroxybutyrate, lactate dehydrogenase, creatinine, malate, isocitrate and urate concentrations were measured using an enzymatic-fluorometric method. Daily milk samples were also measured for fat, protein, somatic cell count (SCC) and lactose using infrared spectrometry. The MIXED procedure of SAS was used with a model including starch level, sampling time and interaction between both as independent variables, and cow as a repeated effect. Production performance parameters (milk yield, fat, protein, SCC and lactose) were not affected by treatment nor were dry matter and water intake (P>0.05). Parameters affected by time were due to normal metabolic alterations during rumination. Ventral fluid pH was affected by treatment on D1, increasing from the medium treatment to the high treatment (6.19 ± 0.08 and 6.45 ± 0.08, respectively; P=0.01). Blood pH decreased in the medium and high treatments compared to the low treatment (7.50 ± 0.01 and 7.52 ± 0.01, respectively; P=0.02) on D4. Milk urate concentration increased in the medium treatment compared to the low treatment on D1 (129.6 ± 3.79 and 155.5 ± 8.48, respectively; P=0.01) and D4 (110.9 ± 3.79 and 105.7 ± 8.48, respectively; P=0.04). Milk pH decreased in the high treatment compared to the low treatment (6.75 ± 0.01 and 6.80 ± 0.01, respectively; P=0.02) on D1. Milk malate and creatinine were affected by the interaction on D4 (P<0.05) while isocitrate was affected on D1 (P=0.03). Our results demonstrate that all groups had some level of SARA. In general, cows had more metabolic imbalances on D1 than on D4 leading to the conclusion that the rumen microbiome adapted to the high starch content and cows also able to adapt the different metabolic pathways related to excretion of the acid, allowing cows to cope with high levels of dietary starch.
RESUMO - A acidose ruminal subaguda (ARS) é uma doença metabólica comum em vacas leiteiras. A principal causa é a alimentação com excesso de hidratos de carbono fermentescíveis, nomeadamente o amido. Para estudar o efeito da variação do conteúdo de amido nas dietas no estado metabólico e desempenho produtivo, foi realizado um ensaio com seis vacas Danish Holstein lactantes com cânulas ruminais e cateteres na artéria intercostal, num design quadrado latino replicado 3 x 3. A alimentação foi ad libitum com dieta controlo com 20% de amido (MS) durante o período de adaptação (6 dias). No dia 7 (D1), 9, 11 e 13 (D4), foi fornecida uma dieta experimental com 28, 35 ou 42% de amido (MS) correspondendo ao tratamento baixo, médio e alto. No dia 8, 10, 12 e 14 foi fornecida dieta controlo. Foram recolhidas amostras de fluído ruminal (ventral e medial), sangue, urina e leite nos dias 6, 7 e 13, às -0.5, 1, 2.5, 4, 5.5 e 7 h relativamente à hora de alimentação. Foram registados nestes dias o leite produzido, consumo de água e ingestão de matéria seca. Foi medido o pH de todas as amostras. A pressão parcial de O2 e CO2 nas amostras de sangue foi determinada. Foi medida a concentração de glicose, glicose-6-fosfato, β-hidroxibutirato, lactato desidrogenase, creatinina, malato, isocitrato e urato nas amostras de leite utilizando um método enzimático fluorométrico. Foi também determinada a gordura, proteína, contagem de células somáticas (CCS) e lactose nas amostras de leite utilizando espetrofotometria infravermelha. Foi utilizado o procedimento MIXED do SAS com um modelo que incluía nível de amido, hora de amostragem e interação entre os dois como variáveis independentes e a vaca como efeito repetido. O desempenho produtivo (quantidade, gordura, proteína, CCS e lactose), a ingestão de matéria seca e o consumo de água não foram afetados pelo tratamento (P>0.05). Os parâmetros afetados pelo tempo são resultado das alterações metabólicas durante a ruminação. O tratamento afetou o pH do fluído ventral em D1, aumentando do tratamento médio para o alto (6.19 ± 0.08 e 6.45 ± 0.08, resp.; P=0.01). O pH do sangue diminuiu nos tratamentos médio e alto em comparação com o baixo (7.50 ± 0.01 e 7.52 ± 0.01, resp.; P=0.02) em D4. A concentração de urato aumentou no tratamento médio em comparação com o baixo em D1 (129.6 ± 3.79 e 155.5 ± 8.48, resp.; P=0.01) e D4 (110.9 ± 3.79 e 105.7 ± 8.48, resp.; P=0.04). O pH do leite diminuiu no tratamento alto em comparação com o baixo (6.75 ± 0.01 e 6.80 ± 0.01, resp.; P=0.02) em D1. A concentração de malato e creatinina no leite foram afetados pela interação em D4 (P<0.05) e o isocitrato em D1 (P=0.03). Todos os grupos sofreram ARS. Em geral, as vacas demonstraram mais desequilíbrios metabólicos em D1 do que em D4, concluindo que o microbioma ruminal adapta-se ao tratamento e que os animais ajustam as vias metabólicas de excreção do ácido, desvalorizando o elevado conteúdo de amido na dieta.
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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
O objetivo deste estudo foi avaliar os efeitos dos períodos de 6, 9, 14 e 21 dias de adaptação de bovinos da raça Nelore confinados a dietas de alto teor de concentrado em relação ao desempenho, variação na ingestão de massa seca, energia líquida, perfil metabólico sanguíneo, comportamento ingestivo, saúde ruminal e características de carcaça. Os animais foram alimentados durante 88 dias, independentemente do período de adaptação adotado e o delineamento utilizado foi em blocos casualizados, com seis repetições cada, em que 96 bovinos da raça Nelore não castrados (391,1 ± 30,9 kg) foram alimentados em 24 baias (4 animais/baia), de acordo com os diferentes períodos de adaptação adotados: 6, 9, 14 e 21 dias. O programa de adaptação utilizado foi em escadas, com três dietas ao longo de períodos de adaptação com seguintes níveis de concentrado: 70, 75, 80,5 e 86% da massa seca na dieta. Contrastes ortogonais foram utilizados para avaliar a relação linear, quadrática e cúbica entre os dias de adaptação e a variável dependente, além da interação de tratamento e fase (P≤0,10). Os animais adaptados por 14 dias apresentaram resultados superiores em relação ao ganho de peso diário, peso vivo final e eficiência alimentar, bem como peso de carcaça quente (P=0,04) e área de olho de lombo final (P=0,01). A adaptação por 14 dias também proporcionou aos animais melhor desenvolvimento do epitélio ruminal, pois apresentaram maior área de superfície absortiva (P=0,02) e largura de papilas (P=0,06). Além disso, esses animais demonstraram que estavam adequadamente adaptados, pois a renovação celular do epitélio ruminal demonstrou-se estabilizada no índice de proliferação celular (P=0,003) e nos núcleos em morte celular (P=0,0004). Com base nesses resultados, recomenda-se adaptar bovinos da raça Nelore a dietas de alto concentrado com o protocolo em escadas por 14 dias, pois este proporcionou maior desempenho produtivo e desenvolvimento do epitélio ruminal.
This study was designed to determine the effects of adaptation periods of 6, 9, 14 and 21 days on feedlot performance, dry matter intake fluctuations, energy gain, blood metabolic profile, feeding behavior, rumen health and carcass traits of Nellore cattle. Cattle were fed for 88-d regardless of adaptation period adopted and the experiment was designed as a completely randomized block, replicated 6 times, in which ninety-six 20-mo-old yearling Nellore bulls (391.1 ± 30.9 kg) were fed in 24 pens (4 animals/pen) according to the different adaptation periods adopted: 6, 9, 14, and 21 days. The adaptation program consisted of ad libitum feeding of 3 diets over adaptation periods with concentrate level increasing from 70, 75, 80.5 and 86% of diet dry matter. Orthogonal contrasts were used to evaluate linear, quadratic, and cubic relationship between adaptation periods and the dependent variable, moreover the interaction the treatment and phase (P≤0.10). Cattle adapted for 14 days had significantly greater for average daily gain, final body weight, G:F ratio, hot carcass weight (P=0,04) and rib-eye area (P=0,01). The adaptation for 14 days the animals also provided better development of the rumen epithelium for a greater absorptive surface area (P=0.02) and width papillae (P=0.06). Moreover, these cattle showed that were properly adapted because the cell epithelium renovation proved to be stable in cell proliferation index (P=0.003) and cell death (P=0.0004). Thus, based on the results of this study, yearling Nellore bulls should be adapted for the step protocol for 14 days, because these provided greater performance and development of the rumen epithelium.
FAPESP: 2013/25403-0

L'acidose ruminale latente (ou acidose sub-clinique) est une préoccupation majeure pour la nutrition des ruminants à haut potentiel de production. Cet état se caractérise par une instabilité du microbiote et des fermentations ruminales qui s'orientent variablement vers le propionate et/ou le butyrate. L'une des stratégies de prévention de l'acidose latente consiste à distribuer dans l'alimentation des ruminants des probiotiques capables de rééquilibrer le microbiote et les fermentations ruminales (dans un sens favorable pour l'animal). L'analyse de la bibliographie montre toutefois que l'effet des probiotiques, et plus particulièrement des bactéries probiotiques (BP), est variable et parfois contradictoire ce qui serait probablement lié à l'instabilité du microbiote. Afin d'étudier la possibilité de prévenir l'acidose latente par les bactéries propioniques et/ou lactiques, nous avons émis l'hypothèse que leur efficacité dépend des orientations fermentaires dans le rumen. Des acidoses latentes butyrique et propionique caractérisées par des profils fermentaires et microbiens distincts ont été développées chez le mouton non productif et la vache laitière pour étudier l'effet et le mode d'action de la bactérie propionique P63 seule ou associée aux lactobacilles Lb. plantarum ou Lb. rhamnosus (P63, Lp + P63 et Lr + P63) sur le fonctionnement de l'écosystème ruminal et les performances animales. Chez le mouton en situation d'acidose propionique, les BP utilisées ont amélioré le pH ruminal via une réduction de la proportion en lactobacilles. Chez la vache laitière, la stabilisation du pH a été associée à une moindre disponibilité en hydrogène susceptible d'être transformé en protons, suite à une augmentation de la propionogenèse et/ou de la densité bactérienne, deux voies consommatrices d'hydrogène. Au cours de l'acidose latente butyrique, l'amélioration du pH n'a été observée que chez les moutons supplémentés avec Lp + P63. Cet effet semblait être dû à une diminution des acides gras volatils et de la proportion en S. bovis mais aussi à un pH initial faible (pH < 5,5) probablement optimal pour l'action des BP ; ce qui n'était pas le cas chez les vaches pour lesquelles le pH initial était compris entre 5,9 et 6,1. En revanche, l'efficacité digestive a été augmentée par l'association de P63 aux lactobacilles chez la vache laitière. L'association Lp + P63 a augmenté les activités fibrolytiques (cellulase, xylanase) et la digestibilité de la matière organique, tandis que Lr + P63 a amélioré la digestion des fibres et a diminué la production de méthane de 25%. Nous n'avons pas observé d'effet sur les performances zootechniques, ce qui serait probablement dû au dispositif expérimental en carré Latin qui n'est peut-être pas optimal pour mettre en évidence l'effet des BP. Nos résultats sont les premiers à démontrer l'efficacité des bactéries probiotiques pour sécuriser et/ou améliorer la digestion des rations et réduire la production de méthane chez le ruminant en acidose, et l'association de P63 avec les souches de Lactobacillus sont les plus efficaces. Enfin, nous avons validé notre hypothèse selon laquelle l'effet et le mode d'action des bactéries probiotiques pour prévenir l'acidose dépendent des orientations fermentaires dans le rumen.

Prostate cancer is the second most common cancer among men in the United States. While treatments for prostate cancer exist, none are curative. As a solid tumor, prostate cancer can grow beyond the diffusion limits of oxygen, thereby resulting in a hypoxic environment. While hypoxia can cause death to a variety of cell types, tumor cells can develop resistance to hypoxia and survive under minimal oxygen conditions. Hypoxia in tumor cells has also been associated with poor prognosis, increased metastasis, and decreased efficacy of chemotherapy. BNIP3, a BH-3 only proapoptotic Bcl-2 family member, has been shown to play an important role in cell death under hypoxic conditions in a variety of cell types. In normoxia, BNIP3 shows little to no expression in both cardiomyocytes and many cancer cell types, but is then upregulated under hypoxic conditions. Previous work in our laboratory provides evidence that hypoxia alone, as well as the concomitant increase in BNIP3 expression, cannot cause death of rat neonatal cardiomyocytes. Instead, our studies found that hypoxia with concomitant intracellular acidosis is required. Further studies indicated that BNIP3 is also necessary for hypoxia-acidosis associated cell death in cardiomyocytes. Our results in rat neonatal cardiomyocytes led us to hypothesize that cell death could be induced in hypoxic prostate cancer cells if concomitant acidosis could be induced. Additionally, our intention was to determine if BNIP3 was required for any prostate cancer cell death that may occur under hypoxia-acidosis conditions.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
O presente estudo teve o objetivo de avaliar o impacto da flutuação natural da ingestão de massa seca no desempenho, características de carcaça, comportamento ingestivo, perfil metabólico sanguíneo e aspectos morfofisiológicos ruminais de bovinos alimentados com dietas de alta inclusão de concentrado, por meio de estudo meta-analítico. Os experimentos contidos na meta-análise utilizaram animais com grupamento genético similar ao utilizado em confinamentos comerciais brasileiros. A meta-análise teve como base de dados os resultados de dez experimentos conduzidos no Confinamento Experimental da Faculdade de Medicina Veterinária e Zootecnia da UNESP, em Botucatu, entre os anos de 2006 e 2015. Os dados foram coletados a partir 838 bovinos, divididos em 238 baias, consideradas as unidades experimentais para o estudo. Os valores de flutuação de ingestão de massa seca na baia foram calculados pela diferença da ingestão de massa seca entre dois dias consecutivos e dividido pelo dia anterior para encontrar o valor em porcentagem. Com base na mediana geral de 5,62%, os animais foram divididos em dois grupos: alta e baixa flutuação de massa seca. As análises estatísticas foram feitas com auxílio do programa computacional R (versão 3.2.3). Utilizou-se um modelo de regressão linear misto para analisar os dados, no qual os experimentos e baias foram considerados como variáveis aleatórias e grupos de flutuação como variável fixa. Os grupos de baixa e alta flutuação apresentaram flutuação média de 4,79 e 6,47%, respectivamente. O grupo de baixa flutuação de ingestão de massa seca apresentou maior ingestão de massa seca em % do peso vivo (P < 0,01), maior ganho de peso diário (P = 0,05), maior ingestão de massa seca em kg (P = 0,06), maior ganho de peso total (P = 0,08), além de maior deposição em área de olho de lombo (P = 0,03) e menor força de cisalhamento (P = 0,03). Com relação ao comportamento ingestivo, o grupo de baixa flutuação de ingestão de massa seca apresentou maior taxa de eficiência da alimentação da massa seca, maior taxa da eficiência da ruminação da massa seca (P = 0,09), maior tempo de ruminação (P = 0,04) e menor tempo em ócio (P = 0,06), além de menor número de idas ao bebedouro (P = 0,04). Não foram observadas diferenças no perfil metabólico sanguíneo, incidência de rumenites e morfologia das papilas ruminais entre os dois grupos avaliados. Menor flutuação na ingestão de massa seca melhora o desempenho e ganho em musculatura de bovinos confinados.
This study aimed to evaluate the inpact of natural dry matter intake fluctuation on performance, carcass characteristics, feeding behavior, blood metabolic profile and morphophysiological ruminal aspects of cattle fed with diets of high inclusion of concentrate. The experiments used for this meta-analysis used cattle with genetic group similar to used in Brazilian commercial feedlots. The meta-analysis was conducted using a database of results of ten experiments conducted in Experimental Feedlot of Faculty of Veterinary Medicine and Animal Science, UNESP, Botucatu, between the years 2006 and 2015. Data were collected from 838 bulls, divided into 238 pens, considered the experimental units for the study. The values of dry matter intake fluctuation were calculated by the difference in dry matter intake between consecutive days and divided by the previous day to find the value in percentage. Based on the overall median of 5.62% cattle were classified into two groups: high- or low-fluctuation. All data analysis was performed in R using a mixed model approach where pens and year were random variables and fluctuation group fixed. The low and high fluctuation groups presented a dry matter intake fluctuation average of 4.79% and 6.74%, respectively. Low fluctuation group showed higher dry matter intake as percent of body weight (P <0.01), higher average daily gain (P = 0.05), higher dry matter intake in kg (P = 0.06), greater total weight gain (P = 0.08), as well as greater deposition in rib eye area (P = 0.03), and lower shear force (P = 0.03). In relation to feeding behavior, low dry matter intake variation group showed higher intake efficiency rate of dry matter, higher rumination (P = 0.04) and less resting time (P = 0.06), as well as fewer visits to the drinker (P = 0.04). There were no differences in blood metabolic profile, incidence of rumenites and morphology of rumen papillae between two groups. Low dry matter intake fluctuation improves feedlot cattle performance and gain muscle.

Dissertation (Ph.D.)--University of Toledo, 2008.
Typescript. "Submitted as partial fulfillment of the requirements for The Doctor of Philosophy degree in Exercise Science." Bibliography: leaves 5-7, 24-34, 61-66, 88-92.

[Truncated abstract] Antimicrobial growth promoters are added to feed to prevent lactic acidosis in ruminant animals by selectively inhibiting rumen bacteria that produce lactic acid. However, recently imposed or impending bans on the use of antimicrobial growth promoters in animal production have lead to a critical need to find practical alternatives that are safe for the animal and consumer and that obtain similar production benefits. I investigated bioactive plants of Australia for their potential to prevent lactic acidosis in ruminants. The unifying hypothesis tested was that plants would be identified that selectively inhibit lactic acid-producing bacteria and consequently protect against lactic acidosis. This hypothesis was tested in a three phase process: phase 1, plant selection and collection; phase 2, a three stage protocol for screening plants and essential oils; phase 3, in vivo experiments and chemical fractionation of the most promising plant. I developed an in vitro bioassay that simulated acidosis by adding glucose to rumen fluid in Bellco tubes and incubating for 5 h (Chapter 4). The pH and gas production were used as indicators of acidosis and fermentation activity. I used this bioassay to screen ninety-five plants (dried and ground material from 79 species) and ten essential oils and included a negative control (oaten chaff) and a positive control (virginiamycin). One plant, Eremophila glabra, produced a similar pH (5.63) to the positive control (5.43) although it inhibited gas production to a moderate extent (P < 0.05). ... Seven serrulatane diterpenes were identified to be the major secondary metabolites in E. glabra. The metabolites were screened using a broth dilution and microtitre spectrophotometry method and were selective against S. bovis at between 320 and 1077 [mu]g/ mL. The serrulatanes from E. glabra were probably responsible for the activity against acidosis that I observed in vitro, because they selectively inhibited lactateproducing bacteria. It is also possible that a synergy between serrulatanes and possibly other metabolites are responsible for the activity observed in vitro. The results from my experiments support the role that bioactive plants may have to replace the antibiotics that are added to livestock feed. Australian plants were identified containing compounds that were active against the bacterial processes responsible for ruminant acidosis. To my knowledge this is the first work undertaken to identify bioactive plants of Australia for their potential to prevent acidosis. I developed in vitro screening bioassays that targeted key indicators of acidosis. These bioassays enabled me to identify 5 plants from the 104 screened that could potentially control acidosis. One of these plants in particular, E. glabra, showed a level of activity in vitro that was comparable to antibiotic protection against acidosis. The exciting in vitro results were not demonstrated in vivo but only one dose level of E. glabra was used, which was based on the in vitro work. In contrast to the in vitro system the rumen is a continuous flow system with greater complexity and it is possible that the concentration of E. glabra that I used in vivo was not optimum. This places importance on future dose response experiments to confirm the efficacy of E. glabra in vivo.

Abstract High-sucrose diet and metabolic acidosis have some similar effects on bone and they both reduce the formation of dentine. This series of experiments was conducted in order to get information about the effects of acidosis and alkalosis on dentine during primary dentinogenesis and also to ascertain if high-sucrose diet affects dentine formation via acidosis. Chronic metabolic acidosis (0.25 mol/L of NH4Cl in drinking water), chronic metabolic alkalosis (0.25 mol/L of NaHCO3 in drinking water) and chronic respiratory alkalosis (atmospheric pressure equivalent to an altitude of 3000 m) were induced in the rats immediately after weaning for 6 and 7 weeks. One subgroup from each of the main groups was fed a high-sucrose (43%) diet and one a standard maintenance diet, each ad libitum. The control groups had the same diets, but normal drinking water and atmospheric pressure. All the rats were injected with tetracycline (to mark the onset of the experiment in dentine) and inoculated orally with Streptococcus sobrinus. The acid-base status was verified by blood gas analysis at the end of the experiments. After sacrifice, fissure caries was scored with Schiff reagent and the areas of dentinal lesions and tetracycline-marked new dentine were measured from sagittally sectioned mandibular molars. The mineral elements (Ca, Mg, F, Na, P and total mineral contents) of the dentine formed before and during the experiment were measured with an electron probe microanalyzer. With the high-sucrose diet, respiratory alkalosis and metabolic acidosis promoted the initiation and progression of caries while metabolic alkalosis slightly retarded it. With the standard diet, all the experimental conditions slowed the rate of dentine formation and metabolic acidosis had the most pronounced effect. The mineral analysis revealed a totally different pattern of mineralization when the rats with metabolic acidosis (increased calcium and total mineral content) were compared to the previously reported rats with a high-sucrose diet (decreased calcium and total mineral content). Besides this, metabolic alkalosis did not correct the effects of the dietary sucrose on dentine formation and blood gas analysis showed no acid-base disturbances in the sucrose diet group. Therefore, a high amount of sucrose in the diet slows the rate of dentine formation and reduces the ability of teeth to resist caries attack by mechanisms different from those of metabolic acidosis. Nevertheless, metabolic acidosis was found to be the most harmful state of disturbance in acid-base balance for the teeth of young rats, especially with a diet containing a high amount of sucrose.