Master of Science / Department of Animal Science and Industry / L.N. Edwards-Callaway / This thesis includes two studies that assessed pain responses to husbandry procedures in order develop mitigation tools. The objective of the first trial was to identify method-related differences in behavioral pain responses in calves of two ages (6 week and 6 months) subjected to castration: surgical cut (CP; n=18), surgical cut and emasculator (CC; n=20), rubber banding (BAND; n=18), or control manipulation of the scrotum (CONT; n=20). Behavior was evaluated pre- and post-castration to record foot stamps, tail flicks, kicks, elimination, in addition to standing and lying post-castration. For 6 week calves, the probability of kicking and eliminations was greater for surgical castrates and all castrated animals, respectively. The probability of kicking was greatest for all 6 month castrates while BAND and CONT had greater probability of elimination. Both age groups increased standing post-castration. Lying decreased in 6 week calves but was unchanged for 6 month calves. Six week calves displayed more tail flicks and tended to display less foot stamps than 6 month calves. Six week calves showed a decrease in tail flicks and foot stamps in response to castration while 6 month calves showed an increase in both behaviors. CP and CC, but not BAND, resulted in less tail flicks than CONT post castration. The second trial compared the effects of preemptive analgesics administered to calves subjected to dehorning with local anesthesia. Six month Holstein steers were randomly assigned to receive one of the following treatments (n= 8/group): meloxicam (1 mg/kg PO), gabapentin (15mg/kg PO), meloxicam (1 mg/kg) and gabapentin (15 mg/kg) PO, flunixin (2.2 mg/kg IV), or a placebo. Drug, cortisol, ex-vivo prostaglandin, haptoglobin, and substance P concentrations, ocular thermography, algometry, and average daily gain were evaluated. Analgesic-treated calves had lower plasma SP concentrations and improved ADG compared with controls. Flunixin calves had reduced cortisol and ex-vivo prostaglandin concentrations for 24h compared to controls. Meloxicam treated calves showed an increase in MNT at two horn bud sites compared with the other treatments. Overall, the results provide validation of responses to noxious stimuli that can be used to develop pain alleviation for livestock.

Doctor of Philosophy / Department of Anatomy and Physiology / David C. Poole / The overall aim of this dissertation is to better understand the mechanisms determining skeletal muscle oxygen delivery-utilization matching in health and disease. Emphasis is directed toward the role of nitric oxide (NO) bioavailability in modulating muscle microvascular oxygenation (PO2mv; the sole driving force for blood-myocyte oxygen flux) during transitions in metabolic demand. The first investigation of this dissertation (Chapter 2) demonstrates that alterations in NO bioavailability have a major impact on skeletal muscle PO2mv kinetics following both the onset and cessation of contractions. Specifically, increased NO levels (via the NO donor sodium nitroprusside; SNP) elevates whereas reduced NO levels (non-specific NOS inhibition with NG-nitro-L-arginine methyl ester; L-NAME) diminishes muscle PO2mv at the onset and during recovery from contractions in the spinotrapezius muscle of healthy young rats. Consistent with these results, inhibition of the neuronal NO synthase isoform (S-methyl-L-thiocitrulline; SMTC; Chapter 3) reveals alterations in NO-mediated regulation of skeletal muscle PO2mv with advanced age that likely contribute to exercise intolerance in this population. In Chapter 4 we observed that pronounced oxidative stress is implicated in these pathological responses seen in aged and diseased states. Transient elevations in the oxidant hydrogen peroxide to levels seen in the early stages of senescence and cardiovascular diseases promote detrimental effects on skeletal muscle contractile function (i.e., augmented oxygen cost of force production). Chapter 5 demonstrates that endurance exercise training improves skeletal muscle microvascular oxygenation (i.e., greater PO2mv and slower PO2mv kinetics) across the metabolic transient partly via enhanced NO-mediated function in healthy young individuals. These data carry important clinical implications given that exercise training may ameliorate NO-mediated function, muscle microvascular oxygenation deficits and consequently exercise intolerance in aged and diseased populations. In conclusion, alterations in NO bioavailability have a major impact on the dynamic balance between skeletal muscle oxygen delivery and utilization (i.e., PO2mv kinetics) in health and disease. While advanced age or the predations of disease impair considerably skeletal muscle microvascular oxygenation, exercise training-induced adaptations on the oxygen transport system constitute a non-pharmacological therapeutic intervention potentially capable of mitigating these microcirculatory deficits.

Doctor of Philosophy / Department of Animal Sciences and Industry / Evan C. Titgemeyer / Previous data suggest that greater amounts of postruminal protein increase small intestinal starch digestion in cattle. Duodenally and ileally cannulated steers were used in 5 studies to measure responses in small intestinal starch digestion to amino acids (AA) or casein. Flows of starch to the ileum from the diet were small. Small intestinal starch digestibility was 34.0% when raw cornstarch was continuously infused into the duodenum. Infusion of casein linearly increased (P ≤ 0.05) small intestinal starch digestibility, and small intestinal starch digestion adapted to infusion of casein in 6 d. Ethanol-soluble starch and unpolymerized glucose flowing to the ileum increased linearly (P ≤ 0.05) with increasing infusion of casein. Plasma cholecystokinin was not affected by casein infusion, but circulating levels of glucose increased linearly (P ≤ 0.05). In another study, 5 steers were fed a low-starch diet and provided continuous duodenal infusion of raw cornstarch in combination with AA or casein in order to measure response of small intestinal starch digestion. Duodenal infusion of casein increased (P ≤ 0.05) small intestinal starch digestion. When a mixture of AA with a profile similar to casein (CASAA) was infused, small intestinal starch digestion was similar (P = 0.30) to casein infusion. Infusion of only non-essential AA tended to increase (P = 0.14) small intestinal starch digestion relative to control; however, infusion of essential AA alone did not affect (P = 0.84) small intestinal starch digestion. Additionally, infusion of casein or essential AA increased ileal flows of ethanol-soluble starch, but non-essential AA alone were not different than the negative control. Duodenal infusion of Glu increased (P ≤ 0.05) small intestinal starch digestion, whereas a mixture of Phe, Trp, and Met (PTM) did not. Neither Glu nor PTM increased ileal flow of ethanol-soluble starch, but Glu and PTM provided together tended (P = 0.07) to increase ileal flows of ethanol-soluble starch. Our data suggest that Glu alone can increase small intestinal starch digestion in cattle similar to casein, but increases in small intestinal starch digestion in response to Glu are not associated with an increase in ileal flows of ethanol-soluble starch.

Doctor of Philosophy / Division of Biology / Sherry D. Fleming / Ischemia/reperfusion (IR) events result in severe tissue damage and often death. The complex network of molecular and cellular mechanisms that contributes to intestinal IR-induced pathology has hindered a comprehensive understanding of IR-induced injury and limited the success of medical intervention. Although several of the mechanisms contributing to intestinal IR-induced injury have been identified, the initiating event(s) remains unclear. Mouse models have been instrumental in the unraveling of the many components and interactions that ultimately result in tissue damage. It is clear that leukocyte infiltration, complement activation, eicosanoid and pro-inflammatory cytokine production are involved. Toll-like receptors and antibodies also play critical roles. Based on the literature, and especially data demonstrating a significant role for anti-phospholipid antibodies, we hypothesized that ischemia induces phospholipid alterations that result in the exposure of a neoantigen which is recognized by anti-phospholipid antibodies. Furthermore, we hypothesized that endothelial cells are the primary cell type involved in the initial molecular events that result in intestinal IR-induced pathology. A mouse model of intestinal IR as well as an in vitro cell culture system was used to explore these hypotheses. Mass spectrometry-based lipidomics was utilized to assess lipid responses to IR and hypoxia/re-oxygenation (HR). No inherent differences in intestinal phospholipid composition were found between wildtype and several strains of knock-out mice. It was determined that the lack of antibody production by Rag-1[superscript]-[superscript]/[superscript]- mice is responsible for protection against intestinal IR-induced injury, as antibody is needed to induce prostaglandin E[subscript]2 production, through up-regulation of cyclooxygenase 2 transcription. Unexpectedly, the presence or absence of toll-like receptor 9 was found to be inconsequential for tissue damage caused by intestinal IR. The results of several analyses point to endothelial cells as being directly involved in IR-induced pathology. Importantly, the activation of phospholipid scramblase 1 has been identified as a potential molecular mechanism by which subsequent molecular and cellular responses are elicited as a consequence of IR.

Doctor of Philosophy / Department of Animal Sciences and Industry / J. Ernest Minton / Toll-like receptors (TLR) are instrumental in discriminating between pathogenic and commensal bacteria and act as mediators, along with downstream chemokines, of subsequent innate and adaptive immune responses. However, little is known about the expression and regulation of TLR or chemokines in swine. The objectives of the experiments described herein were to characterize the expression of porcine TLR and to identify regulatory patterns in these receptors in the presence of live Salmonella enterica serovar Typhimurium (ST) or Choleraesuis (SC). The first two experiments evaluated the in vivo and in vitro expression of TLR2, 4, 5 and 9. Our results indicate that TLR2, 4, 5 and 9 are constitutively expressed in vitro in a porcine jejunal epithelial cell line (IPEC-J2), porcine mononuclear phagocytes (pMPs) and in vivo in the distal ileum. In IPEC-J2 cells, ST elicited an increase in TLR2 mRNA (P < 0.05), and both ST and SC increased TLR2 mRNA in pMPs (P < 0.05). In vivo, oral challenge with ST increased (P < 0.05) both TLR2 and TLR4 mRNA in the distal ileum. In addition, the second experiment evaluated interleukin 8 (IL8) and CC chemokine ligand 20 (CCL20) expression in IPEC-J2 cells in response to ST or purified bacterial flagellin (Flag). TLR5 was constitutively expressed in the ileum and in IPEC-J2 and pMP cells. Interestingly, IL8 and CCL20 mRNA and protein were increased (P < 0.05) by ST and Flag, even in the absence of changes in TLR5. In the third experiment, the expression of TLR and chemoattractive mediators were evaluated in a panel of tissues obtained from pigs challenged with ST and SC. All genes of interest were constitutively expressed; however, the effects of treatment were limited to isolated tissues and genes. Taken together, the data indicate that TLR and chemoattractive mediators are expressed in porcine tissues and cells and that the observations described represent novel evidence that pig pathogens may regulate TLR expression and activate chemokine secretion.

Master of Science / Department of Animal Sciences and Industry / J. Ernest Minton / Enteric pathogens are responsible for major economic losses in the swine industry. In the U.S., Salmonella enterica subspecies enterica serovar Typhimurium (ST) and serovar Choleraesuis (SC) account for essentially all cases of salmonellosis in swine. Previous studies documented that oral ST eroded growth and produced unmistakable changes in the endocrine stress and somatotropic axis of young growing pigs. However, these effects occurred in the absence of elevated systemic inflammatory cytokines that were previously thought to accompany disease-associated growth retardation. In the current study, it was hypothesized that SC would produce very different systemic inflammatory cytokine responses compared to ST given the likelihood of SC to produce systemic disease in pigs. Weaned pigs were housed two per pen with free access to feed and water during a 14 d experiment. On d 0, pigs were fed either 108 CFU SC or 108 ST, and bacteria were re-fed twice weekly through the course of the experiment. Control pigs were fed dough without bacteria. Serum was collected on d 0, 7, and 14 for determination of tumor necrosis factor alpha (TNFα), interleukin-1beta (IL-1β), and insulin-like growth factor-I (IGF-I) were determined. Rectal temperatures (RT) were monitored daily beginning 2 d prior to challenge with bacteria and until 7 d following the first bacterial feeding. Pigs were weighed initially, and at the conclusion of the study. Daily body weight gain was reduced by 25.4% in pigs fed SC (P<.0001) compared to control, while growth was similar between control pigs and those fed ST. Pigs fed SC had increased RT beginning on d 2 and continuing though d 7 (P < 0.05) with the greatest elevation spike on d 3 (P < 0.001) when compared to controls. On d 7, pigs fed SC had reduced IGF-I when compared to both control (P < 0.01) and ST pigs (P = 0.01). Despite the obvious febrile response, and the reductions in body weight gain and serum IGF-I, circulating TNFα and IL-1β were not affected by treatment. It was concluded that elevated TNFα and IL-1β are not obligatory correlates of SC-induced pathology and growth retardation in weaned pigs.

Doctor of Philosophy / Department of Anatomy and Physiology / Thomas J. Barstow / The overall aim of this dissertation is to better understand the dynamic matching between O2 delivery and uptake following the onset of exercise. The first study of this dissertation (Chapter 2) revealed that: i) the dynamics of muscle oxygenation were determined primarily by the QO2–VO2 interaction during the initial phase of QO2 response (first 15-20 s); and ii) absolute values in the steady state used to calculate blood flow from VO2 and O2 extraction did not affect the dynamics of blood flow response. Consistent with these predictions, using pulmonary gas exchange and near-infrared spectroscopy in humans (Chapter 3) we observed that the estimated kinetics of capillary blood flow (moderate exercise 25.4 ± 9.1 s and heavy exercise 25.7 ± 7.7 s) were not significantly different from the kinetics of muscle VO2 (moderate exercise 25.5 ± 8.8 s and heavy exercise 25.6 ± 7.2 s). In Chapter 4 we observed that nitric oxide (NO) is essential to maintain microvascular O2 pressure (PO2mv ~ QO2/VO2) of contracting rat muscles. Blockade of NO synthase with L-NAME accelerated the kinetics [ΔMean response time(L-NAME–CONTROL) = -6.5 ± 6.6 s, P< 0.05] and reduced the contracting steady-state PO2mv [ΔPO2mv(L-NAME–CONTROL) = -5.0 ± 1.0 mmHg; P < 0.05] compared to control. In Chapter 5 we focused on the kinetics of bulk limb blood flow (LBF) to show that a low-pass filter (LPFILTER) developed for LBF data improved the confidence of kinetic analysis by decreasing the standard error of the estimate (SEE ~ 95% confidence interval) for all kinetics parameters compared to the Beat-by-Beat method (e.g., time-constant phase 2: Beat-by-Beat = 16 ± 5 s; LPFILTER = 1.1 ± 0.5 s). In conclusion, the early increase in QO2 is the main determinant of muscle oxygenation dynamics and NO is essential to maintain the tight coupling of QO2 and VO2 kinetics during exercise. In this context, application of a LPFILTER to LBF data provides the best confidence for kinetic analysis of bulk QO2 that should facilitate investigations integrating bulk and microvascular QO2/VO2 matching in a variety of settings in health and disease.

Doctor of Philosophy / Department of Anatomy and Physiology / Thomas J. Barstow / The interaction between dynamic muscle contractions and the associated muscle blood flow is very intriguing leading to questions regarding the net effect of these contractions on oxygen delivery and utilization by the working muscle. Study 1 examined the impact of contractions on muscle blood flow at the level of the femoral artery. We demonstrated that muscle contractions had either a facilitory, neutral, or net impedance effect during upright knee extension exercise as intensity increased from very light to ~70% peak work rate. This led to the question of what impact a change in contraction frequency might have on the coupling of blood flow to metabolic rate during cycling exercise. The blood flow/VO2 relationship has been shown to be linear and robust at both the central (i.e., cardiac output/pulmonary VO2) and peripheral (leg blood flow/leg VO2) levels. However, an increase in contraction frequency has been reported to either decrease, have no effect, or increase the blood flow response during exercise. Study 2 determined if the steady state coupling between muscle blood flow and metabolic rate (centrally and/or peripherally) would be altered by varying contraction frequency. Our results indicate that both central and peripheral blood flow/VO2 relationships are robust and remain tightly coupled regardless of changes in contraction frequency. Study 3 examined muscle microvascular hemoglobin concentration and oxygenation within the contraction/relaxation cycle to determine if microvascular RBC volume was preserved and if oxygen extraction occurred during contractions. We concluded that microvascular RBC volume was preserved during muscle contractions (i.e., RBCs remained in the capillaries), which could facilitate continued oxygen delivery. Further, there was a cyclic pattern of deoxygenation/oxygenation that corresponded with the contraction/relaxation phases of the contraction cycle, with deoxyhemoglobin increasing significantly during the contractile phase. These data suggest that oxygen extraction continues to occur during muscle contractions. Significant insight has been gained on the impact of muscle contractions on oxygen delivery to and exchange in active skeletal muscle. This series of studies forms a base of knowledge that furthers our understanding of the mechanisms which govern the control of skeletal muscle blood flow and its coupling to muscle metabolic rate.

Doctor of Philosophy / Department of Anatomy and Physiology / Thomas J. Barstow / The overall aims of this dissertation are to determine the incidence and magnitude of insulin resistance (IR) in a cohort of normoglycemic college-age subjects with a family history of type 2 diabetes, and to ascertain if there is early macro- and microvascular dysfunction relative to IR. Study 1 (Chapter 2) revealed a 7-fold range in IR in healthy college subjects concomitant with measures of insulin, both fasted and during an oral glucose tolerance test, but not related with any measure of plasma glucose. These results emphasize that early in the etiology of carbohydrate dysregulation, abnormalities first occur with regard to insulin sensitivity. Using brachial artery blood flow (BABF, Doppler fluxometry) and near-infrared spectroscopy (NIRS) (Chapter 3) we extended the understanding of the use of these non-invasive tools to assess forearm resting metabolic rate and to compare the parameters of both the NIRS oxy-hemoglobin signal, as a index of perfusion in the microcirculation, and BABF, as an independent measure of microvascular reactivity during post occlusive reactive hyperemia (PORH). Resting metabolic rate ranged ~ 2 fold (2.83-5.15 [Mu]MO[subscript2]/min/100g) similar to direct measures. Amplitude, but not kinetic parameters for NIRS variables correlated with comparable parameters for BABF, providing evidence for the possible utility of NIRS in examining microvascular reactivity. In study 3 (Chapter 4), utilizing our extended understanding of hemodynamics garnered from the results of study 2, we assessed the influence of IR on macro- and microvascular reactivity. We observed that i) the magnitude of IR was significantly correlated with attenuation of endothelium-dependent vasodilation of the brachial artery (P< .01) indicating the possibility of a reduced nitric oxide bioavailability and an enhanced atherogenic milieu. Additionally we found ii) BABF at rest and during reactive hyperemia to be strongly correlated with conductance (reduced downstream resistance—an indicator of microvascular control abnormalities) independent of forearm metabolic rate, and iii) parameters of BABF (microvascular response) were also strongly correlated with brachial artery vasoreactivity (macrovascular response). In conclusion, this body of work furthers our insight into the need for earlier identification of "disease" earlier in the progression to type 2 diabetes, and provides direction for future investigations into prevention / intervention to improve microvessel functionality and to slow the atherosclerotic process in larger vessels.

Doctor of Philosophy / Department of Anatomy and Physiology / Deryl L. Troyer / Umbilical cord matrix stem (UCMS) cells are derived from Wharton's jelly and have been shown to express genes characteristic of primitive stem cells. They can be isolated in large numbers in a short time and thus potentially represent an abundant source of cells for therapeutic use. We investigated the migratory nature of human UCMS cells towards MDA 231 human breast carcinoma cells in an in vitro model of cell migration; UCMS cells cultured with or without MDA 231 cells for 24 hours. Next, we evaluated the effect of chemokines, stromal derived factor 1 (SDF-1) and vascular endothelial growth factor (VEGF) on human UCMS cells by treating with increasing doses of SDF-1 and VEGF. UCMS cells were found to migrate towards MDA 231 cells in a dose dependent manner. Both SDF-1 and VEGF induced migration of UCMS cells in a dose dependent manner. These results suggest that MDA 231 cells might be releasing chemokine factors, such as SDF-1 and VEGF, which promote UCMS cell migration towards the tumor cells in vitro. Stem cells that migrate to tumors may allow targeted delivery of therapeutic agents that otherwise may have severe side effects. To evaluate the selective engraftment and therapeutic efficiency of human UCMS cells that were engineered to express interferon beta (UCMS-IFN-beta) MDA 231 cells (2,000,000) were intravenously injected into severe combined immune deficient (SCID) mice, followed by three weekly intravenous injections of fluorescently labeled UCMS-IFN-beta cells (500,000). To evaluate the synergistic effect of 5-Fluorouracil (5-FU) and IFN-beta, MDA 231 cells were intravenously injected into SCID mice, followed by three weekly intravenous injections of fluorescently labeled UCMS-IFN-beta cells and three weekly intra peritoneal injections of 5-FU. In both of the above experiments, mice were euthanized one week after the last UCMS cell transplant and lung weights were compared to the controls to determine the differences in tumor burden. After transplantation of UCMS-IFN-beta cells into MDA 231 tumor-bearing mice, UCMS cells were found near or within metastatic lung tumors but not in other tissues, and in these animals, the lung weight was significantly less than MDA 231 tumor-bearing animals that received saline injections. Histologically, there was significant reduction in the tumor area in MDA 231 tumor bearing lungs after UCMS-IFN-beta treatment. When 5-FU was given along with UCMS-IFN-beta cells, there was further reduction in tumor area. These results indicate that UCMS cells can potentially be used for targeted delivery of cancer therapeutics.