Transitioning cattle from a high-forage to a high-concentrate diet increases the risk for ruminal acidosis and is often related to decreased feed intake, which compromises animal health and performance. Since control of feed intake and rumen motility are closely related, we hypothesized that a reduction in rumen motility may be associated with a reduction in feed intake during this transition. Computer programs were created to analyze feed disappearance and rumen pressure data for feeding behavior as well as identification and characterization of rumen contractions, respectively. This method enabled timely analysis of large datasets and removed subjectivity associated with manual analysis. In the second part of this series, cattle were moderately transitioned from a 70% to a 90% concentrate diet, and SARA was induced. Although, reductions in feed intake were modest, on day 2 of high-grain feeding, animals slowed feed consumption rate and displayed a reduction in rumen contraction frequency, amplitude, and duration. Next, an abrupt transition from 50% to 90% concentrate was used to induce ruminal acidosis and cause some animals to stop eating. The abrupt increase in dietary concentrate was also associated with reductions in rumen motility. Patterns of ruminal pH, viscosity, and motility changes were related to when cattle reduced feed intake. Endotoxin quantification in blood samples from the ruminal vein, portal vein, and mesenteric artery suggested the point of endotoxin translocation into blood was across the ruminal epithelium. Additionally, the greater the concentration of endotoxin in the plasma, the more likely animals were to go “off-feed.” By understanding the physiological and behavioral mechanisms by which cattle adapt to high-grain diets, we can improve animal health and performance through these diet transitions.

The physical tumor microenvironment contributes significantly to carcinogenesis, cancer progression and metastatic dissemination. Two main components of the tumor microenvironment, hypoxia and acidosis, are present in nearly every solid tumor and act as powerful selection forces against the tumor. Hypoxia and acidosis promote tumor heterogeneity and contribute to chemotherapy and radiotherapy resistance. This dissertation interrogates methods to target the tumor microenvironment including two novel studies describing mechanisms of buffer therapy resistance and targeting tumor hypoxia with vasodilators to enhance the efficacy of a hypoxia activated prodrug, TH-302. In the first study, mechanisms of buffer therapy resistance were identified and detailed. Many studies have shown that the acidity of solid tumors contributes to local invasion and metastasis. Oral pH buffers can specifically neutralize the acidic pH of tumors and reduce the incidence of local invasion and metastatic formation in multiple murine models. However, this effect is not universal as we have previously observed that metastasis is not inhibited by buffers in some tumor models, regardless of the buffer used. B16-F10 (murine melanoma), LL/2 (murine lung) and HCT116 (human colon) tumors are resistant to treatment with lysine buffer therapy, whereas metastasis is potently inhibited by lysine buffers in MDA-MB-231 (human breast) and PC3M (human prostate) tumors. In the current work, I confirmed that sensitive cells utilized a pH-dependent mechanism for successful metastasis supported by a highly glycolytic phenotype that acidifies the local tumor microenvironment resulting in morphological changes. In contrast, buffer-resistant cell lines exhibited a pH-independent metastatic mechanism involving constitutive secretion of matrix degrading proteases without elevated glycolysis. These results have identified two distinct mechanisms of experimental metastasis, one of which is pH-dependent (buffer therapy sensitive cells) and one which is pH-independent (buffer therapy resistant cells). Further characterization of these models has potential for therapeutic benefit. In the second study, improving the efficacy of hypoxia activated prodrug, TH-302, through induction of hypoxia was investigated. Pancreatic ductal adenocarcinomas are desmoplastic and hypoxic tumors, both of which are associated with poor prognosis. Hypoxia activated prodrugs, such as TH-302, are specifically activated in hypoxic environments and are now in a Phase III clinical trial in pancreatic cancer. Using animal models, we show that tumor hypoxia can be exacerbated using a vasodilator, hydralazine, improving TH-302 efficacy. Hydralazine reduces tumor blood flow through the "Steal" phenomenon, where atonal immature tumor vasculature fails to dilate in coordination with normal vasculature. The current study shows that MiaPaCa-2 tumors exhibit a "Steal" effect in response to hydralazine, resulting in decreased tumor blood flow and subsequent tumor pH reduction. The effect is not observed in SU.86.86 tumors with mature tumor vasculature, as measured by CD31 and smooth muscle actin (SMA) immunohistochemistry staining. Combination therapy of hydralazine and TH-302 resulted in a reduction in MiaPaCa-2 tumor volume growth after 18 days of treatment. Further optimization of hypoxia-inducing agents and dosing regimens may lead to increased TH-302 activity, potentially improving clinical outcome. The data presented here demonstrate methods to effectively target the tumor microenvironment for therapeutic benefit. Further investigation into mechanisms of action and biomarkers for therapy response may have important implications on clinical treatment regimens for cancer patients.

Bibliography: leaves 284-298 / 4 microfiches (339 fr.) : / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Summary: Shows that foetal acidosis is related to prolongation of the QT interval, a change which cannot be accounted for in foetal heart rate. Describes the configuration and time constants of the foetal electrocardiogram both during labour and at the time of delivery in normal and acidotic subjects. Confirms that foetal acidosis during labour is associated with acidosis at the time of delivery and with clinical depression of the newborn infant / Thesis (M.D.)--Dept. of Obstetrics and Gynaecology, University of Adelaide, 1970

Ruminal acidosis is a persisting digestive disorder in modern ruminant production; however, the susceptibility of cows to ruminal acidosis differs among cows fed a common diet. The overall objective of this research was to evaluate factors affecting the susceptibility of cows to ruminal acidosis. This research demonstrated that feeding sucrose in replacement for corn grain to Holstein cows in early lactation does not increase the risk for ruminal acidosis and may actually increase ruminal pH. However, regardless of dietary treatment, cows in early lactation were at risk for ruminal acidosis. In Study 2, diets differing in the forage-to-concentrate ratio were fed to Holstein cows to evaluate changes in the in vivo rate of short-chain fatty acid absorption (SCFA) and the expression of genes coding for transporters and enzymes involved in the absorption and metabolism of SCFA in ruminal tissue. Contrary to the hypothesis, the fractional rate of absorption and expression of genes involved in SCFA absorption and metabolism were not affected by the forage-to-concentrate ratio. Considerable variation among individual cows for the severity of ruminal acidosis was detected for cows on the diet containing the low forage-to-concentrate ratio. To determine the cause of this variation, a ruminal pH measurement system was developed to accurately and precisely measure ruminal pH in non-cannulated small ruminants. Sheep were then subjected to a ruminal acidosis challenge model in vivo, and the absorption of acetate and butyrate across the isolated ruminal epithelia was measured in vitro in Ussing chambers. The results of this study demonstrated that differences in the severity of ruminal pH depression among animals could largely iv be accounted for by differences in the absorptive capability of the ruminal epithelium. In summary, although ruminal acidosis is a common digestive disorder in dairy production systems, variation in the susceptibility to ruminal acidosis is common. The cause for much of this variation is due to differences in the absorptive capacity of the ruminal epithelia. / Animal Science

The mammalian Na+/H+ exchanger isoform 1 (NHE1) is a ubiquitously expressed membrane protein that regulates myocardial intracellular pH. Inhibition of NHE1 prevents hypertrophy and reduces ischemia-reperfusion (I/R) injury in animal models. To understand the regulation of NHE1 in the myocardium by phosphorylation we constructed adenoviruses, which express wild type or mutant cDNA for NHE1. Additionally, wild type and mutant NHE1 had mutations Leu163Phe/Gly174Ser, which increases NHE1 resistance to EMD87580 (NHE1 inhibitor) by 100-fold. This allowed measurement of exogenous NHE1 activity while inhibiting endogenous NHE1 activity. We examined the effects of a series of mutations of phosphorylation sites in the cytosolic domain of NHE1. Sustained intracellular acidosis and phenylephrine caused an ERK-dependent activation of NHE1 activity and phosphorylation levels. We demonstrated that amino acids Ser770 and Ser771 were essential for activation of NHE1 activity in isolated rat cardiomyocytes by sustained intracellular acidosis and phenylephrine. Furthermore, mutation of Ser770 and Ser771 to Ala prevented increased NHE1 phosphorylation by sustained intracellular acidosis and phenylephrine. This was found to occur in an ERK-dependent manner. Taken together, our results demonstrate that both sustained intracellular acidosis and phenylephrine rapidly activate the NHE1 protein in isolated cardiac cells via an ERK-dependent pathway that acts on the common amino acids Ser770 and Ser771 of the C-terminal tail of NHE1.

This study assessed the value of triticale dried distillers grains with solubles(DDGS) in a feedlot finishing diet using 144 intact, and 16 ruminally cannulated crossbred yearling steers. Substituting triticale DDGS for a portion of dry-rolled barley grain (20% diet DM) decreased the prevalence of ruminal acidosis and tended to increase dry matter intake and fat deposition, but increased the incidence and severity of liver abscesses. Further substitution of triticale DDGS for barley silage (5 and 10% diet DM) increased the prevalence of ruminal acidosis, but tended to improve feed efficiency without affecting carcass characteristics. These findings suggest that feedlot finishing diets containing triticale DDGS allow producers to decrease dietary forage inclusion without affecting performance, but may require use of an antimicrobial to control liver abscesses. / Animal Science

Feeding diets rich in rapidly fermentable non-structural carbohydrates can lead to the development of ruminal acidosis. This study was conducted to determine if the duration of time that cattle are fed a high-grain diet affects their absorption of short-chain fatty acids (SCFA) and susceptibility to, and recovery from, ruminal acidosis. Sixteen Angus heifers (BW ± SEM, 261 ± 6.1 kg) were assigned to 1 of 4 blocks, and fed a backgrounding diet consisting of 60% barley silage, 30% barley grain, and 10% supplement (DM basis). Within block, cattle were randomly assigned to 1 of 2 treatments differing in the number of days they were fed the high-grain diet prior to an acidosis challenge: 34 d for long-adapted (LA) and 8 d for short-adapted (SA). All cattle were exposed to the same 20-d dietary transition using 5 dietary steps until achieving the final diet that contained 9% barley silage, 81% barley grain, and 10% supplement (DM basis). Data were collected during an 8-d baseline period (BASE), on the d of the acidosis challenge (CHAL), and during two consecutive 8 d recovery periods (REC1 and REC2). Ruminal acidosis was induced by restricting feed to 50% of DMI:BW for 24 h followed by an intraruminal infusion of ground barley at 10% DMI:BW. Cows were then given their regular diet allocation 1 h after the intraruminal infusion. The duration of time fed the high-grain diet did not affect ruminal pH, lactate, or SCFA concentrations (P > 0.050). However, during BASE and on the day of CHAL the SA heifers experienced greater linear (P = 0.031), quadratic (P = 0.016), and cubic (P = 0.008) between day change in the duration of time that pH was < 5.5 than LA heifers. Relative to BASE, inducing acidosis increased daily duration (531 to 1020 min/d; P < 0.001) and area (176 to 595 (min × pH)/d; P < 0.001) that pH was < 5.5. Inducing ruminal acidosis also increased the daily mean (0.3 to 11.4 mM; P = 0.013) and maximum (1.3 to 29.3 mM; P = 0.008) rumen fluid lactate concentrations relative to BASE, suggesting that an acute bout of ruminal acidosis was induced. In addition, a treatment × day interaction for the duration that pH was < 5.5 during REC1 suggests that LA cattle tended to recover from the CHAL more rapidly than SA cattle (P = 0.085). Indeed, analysis of covariance confirmed that the LA heifers experienced a quicker linear (P = 0.019) recovery over time from CHAL. The greater rate of recovery possibly resulted from the LA heifers having greater rates of both fractional butyrate (45 vs. 36 %/h; P = 0.019) and propionate absorption (42 vs. 34 %/h; P = 0.045), and tending to have greater rates, on an absolute basis, of butyrate absorption (94 vs. 79 mmol/h; P = 0.087) iii and, on a fractional basis, of total SCFA absorption (37 vs. 32 %/h; P = 0.100). Treatment × period interactions revealed that LA heifers had greater serum D-lactate concentrations (P = 0.003), and fractional rates of lactate absorption (P = 0.024) than SA heifers, during CHAL and REC1, respectively. When treatments were pooled, the absorption (%/h and mmol/h) of acetate, propionate, butyrate, and total SCFA increased between REC1 and REC2, with intermediate values for BASE (P ≤ 0.05). Corresponding to a reduction in absorption during REC1 (2 d post CHAL), saliva production (kg/h; P = 0.018) increased between BASE and REC1, with intermediate values for REC2. These results indicate that the duration of time cattle are fed a high-grain diet may stabilize rumen pH, both prior to and after an induced bout of acute ruminal acidosis, likely through increased ruminal absorptive capacity for SCFA and lactate. In addition, this study found evidence to suggest that beef cattle possess the ability to increase saliva secretion in order to compensate for decreased absorptive capacity.

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Das Ziel dieser Arbeit war es, den Einfluss unterschiedlich zusammengesetzter Tränken auf den Säuren-Basen-Haushalt bei Kälbern mit experimentell induzierter metabolischer Azidose zu untersuchen. Bei gesunden Kälbern konnte nachgewiesen werden, dass die Fütterung von milchbasierten ORL mit einer [SID3] ≥92 mmol/l eine Erhöhung der Plasma-[SID3] zur Folge hat (BACHMANN et al. 2009b) und damit eine alkalische Wirkung auf den SBS. Zu prüfen galt die Hypothese, ob es bei Kälbern mit experimentell induzierter metabolischer Azidose zu einem stärkeren alkalisierenden Effekt nach Gabe einer Tränke mit einer hohen [SID3] kommt. Bessere alkalisierende Eigenschaften einer Tränke könnten potentiell genutzt werden, um den Genesungsprozess von Kälbern mit metabolischer Azidose zu beschleunigen. Material und Methoden Zur Verfügung standen zwölf Kälber der Rasse Holstein-Friesian im Alter von weniger als vier Lebenswochen. Unter Verwendung von in der Literatur beschriebenen Induktionsprotokollen konnte per Infusion bei jeweils sechs Tieren eine manifeste hyperchlorämische Azidose und bei weiteren sechs Kälbern eine D-/L-Laktatazidose ausgelöst werden. Die Tiere wurden im Anschluss mit Milchaustauscher, wasser- oder milchaustauscher-basierter oraler Rehydratationslösung getränkt bzw. blieben im nüchternen Zustand. Zur Bestimmung von Stewart-Parametern des Säuren-Basen-Haushaltes im Plasma wurden nach einem festgelegten zeitlichen Schema vor und nach Induktion sowie vor und nach der Tränkegabe venöse Blutproben entnommen. Ergebnisse Bedingt durch die Einleitungsprotokolle war die Interpretation der Messergebnisse durch den starken Anstieg des Plasmavolumens bedeutend erschwert. Die eingesetzten Induktionsprotokolle sind nicht für Untersuchungen des Säuren-Basen-Haushaltes bei Kälbern nach unterschiedlicher Fütterung geeignet. Aus diesem Grund kann die aufgestellte Hypothese, dass die Verabreichung von milchaustauscher-basierter ORL zu einer besseren alkalischen Wirkung bei Kälbern mit metabolischer Azidose führt, anhand der vorliegenden Ergebnisse weder abgelehnt noch bestätigt werden. Einige Untersuchungsergebnisse zeigten positive Effekte auf Parameter des SBS bei Kälbern, denen eine Tränke verabreicht wurde, im Vergleich zu nüchternen Tieren. Diese Parameter waren die Plasma-D-Laktatkonzentration, die Strong Ion Difference [SID3] und [SID4] und ionisiertes Calcium [Ca2+]. Die Effekte waren nicht auf eine bestimmte der drei eingesetzten Tränkevarianten zurückzuführen. Weiterhin konnte gezeigt werden, dass ein quadratischer Zusammenhang zwischen der berechneten Variable Strong Ion Gap [SIGAlb/TP] und den gemessenen D-/L-Laktatkonzentrationen im Plasma besteht. Dies könnte zukünftig genutzt werden, um Faktoren zu etablieren, welche die Ableitung der Plasma-D-/L-Laktatkonzentration bei durchfallkranken Kälbern mit ZNS-Symptomatik aus gängigen Parametern ermöglicht, ohne die Laktatkonzentration direkt messen zu müssen. Schlussfolgerungen Für zukünftige Untersuchungen dieser Art wäre ein Induktionsprotokoll wünschenswert, das sowohl eine metabolische Azidose bei gleichzeitiger Dehydratation der Kälber vereint und so die metabolischen Bedingungen durchfallkranker Kälber simuliert. Die Gabe von ORL als direkte Einmischung in Milch bzw. MAT wird kritisch betrachtet. Ein negativer Effekt bei der Gabe von MAT-basierter ORL konnte bei diesen Untersuchungen auf keinen der bestimmten Parameter des SBS im Vergleich zu den anderen Tränkezusammensetzungen festgestellt werden. Klinische Nebenwirkungen Bei dieser Untersuchung traten nach der mehrfachen Infusion von D-/L-Laktat unerwartete Nebenwirkungen auf. Die Verabreichung führte zu starken Irritationen der Vena jugularis externa und zu Ödembildungen. Erhöhte D-Laktatkonzentrationen werden beim Menschen und verschiedenen Tierarten als Marker für traumatische Prozesse, Ischämie, Diabetes, gastrointestinale und neurologische Störungen diskutiert. Die weitere gezielte Untersuchung des Einflusses der wiederholten oder anhaltenden Einwirkung von D-Laktat auf Epithelzellen könnte nach den vorliegenden Untersuchungsergebnissen einen weiteren Anhaltspunkt für die Aufklärung der genauen pathologischen Mechanismen des D-Laktates bieten.

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