Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Daniel U. Thomson / Bovine viral diarrhea virus (BVDV) has emerged as one of the most important infectious diseases in cattle. One particular important manifestation, after successfully establishing an in utero infection of the fetus during the first trimester, is the development of a persistently-infected BVDV (PI-BVDV) calf. Persistently infected BVDV animals are a continuous source of virus and can shed the virus in virtually all secretions and excretions, including nasal discharges, saliva, semen, urine, tears, milk, and, to a lesser extent, feces. The objectives of this research were to determine: 1) the effects of short term exposure (13 – 18 days on feed (DOF)) to PI-BVDV feeder cattle; 2) the outcome of testing and removing PI-BVDV feeder calves at time of feedlot arrival on health, performance, and carcass characteristics; 3) the survival of BVDV on materials associated with livestock production; and 4) characterization of testing and longitudinal prevalences for PI-BVDV beef cattle. Testing and removing PI-BVDV calves at 13 to 18 DOF was too late to remove a morbidity effect due to PI-BVDV exposure. However, mortality, performance, and carcass characteristics were not different in cattle exposed to PI-BVDV cattle. Additionally, there were no harmful outcomes when newly arrived feeder cattle were exposed to a PI-BVDV animal for one to two days following feedlot entry. A non-cytopathic, Type 1b, BVDV was capable of surviving after application to various materials used in livestock production. BVDV tended to survive longer on non-porous materials than porous materials. When in the presence of mucus, BVDV was protected from degradation for longer periods of time than when not in the presence of mucus. There was no difference in overall PI-BVDV prevalence within cattle sampled in 2006 and 2007. Cattle that weighed less than 300 lbs. had a greater likelihood of being PI-positive than cattle with increased weights. Several months of the year had a greater likelihood of having PI-positive animals. Based on operation, cow-calf and stocker operations had a greater likelihood of having PI-positive animals than did feedlot operations.

Doctor of Philosophy / Department of Animal Sciences and Industry / Barry J. Bradford / The transition period in dairy cows is characterized by dramatic increases in nutrient requirements for lactation and substantial metabolic stress. The disturbed metabolic balance, coupled with suppressed immune function, contributes to markedly elevated incidence of health disorders. Several lines of evidence suggest that increased inflammation is common during the transition period. Unlike the classical inflammation associated with acute infection, the postpartum inflammatory state is low-grade and often of metabolic origin. This metabolic inflammation plays a key role in numerous disorders; an improved understanding of inflammatory pathways in transition cows may improve our ability to predict and prevent disorders. To mimic metabolic inflammation, in Experiment 1, we administered low amounts of recombinant bovine tumor necrosis factor-α (rbTNFα), a pro-inflammatory cytokine, to early lactation cows, and evaluated whether rbTNFα affects milk production, metabolism, and health. We found that rbTNFα administration increased systemic inflammation, decreased feed intake and milk yield, and increased incidence of disorders. Conversely, preventing excessive inflammation has the potential to improve productivity and health of dairy cows. To identify nutritional strategies that could enhance metabolism and immunity, we evaluated the efficacy of several feed additives. In Experiment 2, we evaluated effects of chromium propionate, rumen-protected lysine and methionine, or both on metabolism and immunity in lactating dairy cows, and found that supplementation of these nutrients may enhance neutrophil function. In Experiment 3, we determined whether supplementation of yeast product to transition cows could enhance production, metabolism, and immunity, and found that yeast product modulated feeding behavior, metabolism, immunity, and uterine inflammation. Overall, a greater understanding of the role of metabolic inflammation in the transition period and the nutritional strategies that could modulate these signals may improve the production and health of dairy cows.

Master of Science / Department of Biology / Anthony Joern / Temperature and food quality vary across broad latitudinal gradients, greatly affecting performance by insect herbivores. The contribution of each varies latitudinally so that geographically distinct populations are challenged by differences in nutritional needs and energetic demands. While there has been extensive work studying diet selectivity and nutritional ecology of insect herbivores, few studies have focused on how insect herbivores adapt across such vast environmental gradients. The generalist-feeding grasshopper, Melanoplus femurrubrum (DeGreer), has a broad geographic range that extends across much of North America, making this species ideal for comparative investigations of intrinsic performance responses to extensive but predictable patterns of environmental variation. I compared responses by six populations collected from populations located from Texas to North Dakota (USA) using a common garden experimental design to investigate clinal responses in grasshopper performance. I examined responses in: (1) body size, (2) thermoregulation and adaptive coloration, (3) developmental and growth rates, (4) metabolic rates, (5) total consumption and rates, (6) diet ratio selection, and (7) digestive processing efficiencies across the latitudinal gradient. Grasshopper body size followed the Converse Bergmann’s Rule with decreasing body size as latitude increased. Temperature influenced all other responses, but responses to diet were not always significant or directional. Latitudinal trends for development and growth rates were observed but mass-specific metabolic rates were similar for all populations. Total consumption was body size dependent but independent of diet type. Mass-specific consumption varied but no single directional trend was detected. There was a shift in carbohydrate-biased diet preference at low latitude toward protein-biased diet ratios at higher latitudes, suggesting adaptations to different energetic demands by these populations. However, post-ingestive (digestive) efficiencies demonstrated variable responses with northern populations observing highest efficiencies for some indices but not all. Overall, this research documents phenotypic plasticity to environmental variability to some degree for digestive efficiencies, but ecotypic responses in body size and diet preference among M. femurrubrum populations were observed.

Master of Science / Department of Animal Sciences and Industry / Jim L. Nelssen / Piglet birth weight (BtW) is inextricably associated with preweaning survival. The non-linear antagonistic relationship between BtW and mortality risk is more severe in pigs <1.11 kg BtW than in pigs with BtW >1.11 kg. Thus, our research categorized fetal pigs as small (SM), median (MD), or large (LG) size depending on relative crown-rump length at d-60 gestation or BW within litter at d-95 gestation to evaluate differences in fetal myogenesis and development. At both d-60 and d-95, brain weights did not differ but brain weight:liver weight ratio was larger (P<0.05) for SM compared MD and LG. Cross-sectional area of the Longissimus muscle increased with increasing fetal size so that LG and MD had larger (P<0.05) whole muscle cross-sectional areas than SM although number of primary and relative secondary muscle fibers and their respective cross-sectional areas did not differ. Day-60 mRNA abundance of both IGF-1 and IGF-2 in SM was greater (P<0.05) than in MD and LG. Fetal size had a marginally significant effect (P=0.103) on gene expression of IGF-2 receptor with expression least in LG. Small fetuses had greater (P<0.05) d-60 MyoD gene expression and d-95 serum IGF-1 levels than MD and LG. Two experiments were conducted to determine the effects of feeding low or high doses of chlortetracycline (CTC) and antibiotic alternatives (pharmacologic Zn, Cu, and essential oil), alone or in combination, on nursery pig growth performance. Pharmacologic Cu (125 ppm from CuSO[subscript]4), Zn (2,000-3,000 ppm from ZnO), or increasing CTC level (0-441 ppm) improved growth performance additively while Origanum essential oil (0.005%) elicited no benefits and decreased G:F. Interactive effects of supplemental Cu (125 ppm), Zn (150 ppm), and ractopamine HCl (10 ppm for 28 d) on finishing pig growth performance, carcass characteristics, and antimicrobial susceptibility of enteric bacteria was evaluated. Ractopamine increased (P<0.001) ADG, G:F, HCW, percentage carcass yield, loin depth, percent fat-free lean, and decreased (P=0.014) backfat. Copper and/or Zn did not improve ADG, ADFI, or carcass traits. Fecal E. coli and Enterococcus bacterial resistance to most antibiotics decreased (P<0.05) over time or was stable for those that had a low baseline percentage of resistance.

Doctor of Philosophy / Department of Animal Sciences and Industry / J. Ernest Minton / The three experiments described below offer support of immune function by the swine gastrointestinal epithelium. Experiment one evaluated mediators that regulate the movement of macrophages (macrophage migration inhibitory factor; MIF), neutrophils (interleukin 8; IL8), dendritic cells (CC chemokine ligand 20; CCL20) and epithelial remodeling (osteopontin; OPN) in pigs challenged with Salmonella enterica serovar Typhimurium (ST) or Choleraesuis (SC). The proximal ileum had greater IL8 expression than the distal ileum (P < 0.05), and ST increased CCL20 (P < 0.05). In vitro, MIF, IL8, CCL20 and OPN mRNA expression induced by lipopolysaccharide (LPS), ST or SC using pig jejunal epithelial cells (IPEC-J2) resulted in increased IL8 secretion, and increased IL8 and CCL20 mRNA by ST and SC (P < 0.05). Experiment two evaluated how Lactobacillus reuteri (LR) and Bacillus licheniformis (BL) differed from ST or SC in their ability to regulate, stimulate, or modify IL8, CCL20, and tumor necrosis factor α (TNFα) in IPEC-J2 cells. ST stimulated an increase in IL8 secretion, with increases in IL8 mRNA (P < 0.05). BL increased IL8 mRNA (P < 0.0001). CCL20 mRNA was upregulated by ST (P < 0.05) and BL (P < 0.05). Only ST increased TNFα mRNA (P < 0.05). Another objective evaluated whether pre-exposure of IPEC-J2 cells to LR or BL modified ST induced IL8 secretion. IL8 secretion was increased by ST (P < 0.0001), and reduced by LR (P < 0.05). Only the BL/ST co-treated wells blunted basolateral IL8 secretion (P < 0.0001). Experiment three characterized the swine CCL20 mRNA sequence and evaluated tissue expression. Cloning of CCL20 from the porcine jejunum predicted a 97 amino acid peptide. All healthy tissues expressed CCL20 mRNA. In animals challenged with Salmonella spp., SC increased spleen and liver CCL20 expression. The data demonstrate that invasive bacterial pathogens in the pig gastrointestinal tract trigger upregulation of selected proinflammatory mediators; Salmonella spp. elicited differing patterns of activation in vitro and in vivo; IPEC-J2 cells increased IL-8 secretion in response to ST and BL, but not LR, while ST stimulated secretion was inhibited basolaterally by BL pre-exposure; and numerous porcine tissues are prominent sources CCL20.

Doctor of Philosophy / Department of Anatomy and Physiology / Michael J. Kenney / Important bidirectional interactions exist between the central nervous system and the immune system. Neural-immune interactions provide a regulatory system in the body and disturbances in these interactions may lead to disease. Although the sympathetic nervous system is thought to play a key role in mediating neural-immune interactions, central neural mechanisms mediating sympathetic-immune interactions and the effect of centrally-induced alterations in sympathetic nerve discharge on immune function is not known. We tested the hypothesis that central activation of sympathetic neural circuits alters splenic cytokine gene expression. In a separate study, we tested the hypothesis that hypothermia-induced changes in visceral sympathetic nerve discharge (SND) would be attenuated in middle-aged and aged compared with young rats. Previous studies have demonstrated that skin sympathoexcitatory responses to skin cooling are attenuated in aged compared with young subjects, suggesting that advancing age influences sympathetic nerve responsiveness to cooling. The effect of age on sympathetic nerves innervating other targets organs during acute cooling remains unknown. Central activation of splenic SND was produced using three different experimental interventions: increased core body temperature produced by acute heating, intracerebroventricular injection of angiotensin II (ANGII), and decreased core body temperature produced by acute cooling. Changes in gene expression profiles were analyzed using inflammatory cytokine-specific gene-array and further validated using real-time RT-PCR analysis. The following observations were made. 1)Splenic SNDincreased in response to each experimental intervention except in acute cooled young rats where there was a decrease in splenic SND. 2) Splenic cytokine gene expression of pro-inflammatory cytokines (e.g., IL-1β, IL-6, IL-2) and chemokines (GRO1, CXCL2, CCCL2 and, CXCL10) was increased in response to each experimental intervention. 3) Expression of splenic cytokine genes was reduced after splenic-denervation except in acute cooled rats. 4) Progressive hypothermia reduced splenic, renal, and adrenal SND in rats and was generally attenuated in middle-aged and aged rats. These results demonstrate the functional significance of changes in sympathetic nerve activity on splenic immune cell activation and the effect of age on SND responses to core body cooling.

Doctor of Philosophy / Department of Anatomy and Physiology / Bruce D. Schultz / The goal of this project was to define mechanisms responsible for Na+ transport in two hormonally-sensitive epithelium, the bovine mammary gland and porcine vas deferens. Glucocorticoid stimulation in these epithelia results in a significant increase in amiloride-sensitive ion transport, suggesting regulation of the epithelial Na+ channel, ENaC. ENaC has typically been described as a heteromultimeric ion channel with at least three different types of subunits, the most common being , β, and γ. Glucocorticoid-induced regulation of these subunits at the transcriptional level appears to be very different in the porcine vas deferens as compared to the bovine mammary gland. The aims of the study in mammary epithelium were to elucidate the mechanisms by which apical electrolytes and cytokines compromise barrier function in mammary epithelium. The long term goal is to better understand and manage the interaction between ionic composition of milk and breakdown of the gland epithelium that occurs during mastitis. Our results suggest a causal link between changes in milk electrical conductivity and epithelial barrier breakdown that has not been appreciated previously. Results will provide benefits to dairy farmers by characterizing steps that might prevent the development of mastitis or hasten recovery. The aims of the study using porcine vas deferens epithelial cells include determining the time course, concentration- and structure-dependency for regulation of amiloride-sensitive ion flux by corticosteroids. Corticosteroids caused a concentration-dependent increase in amiloride-sensitive Isc with a rank order of potency of dexamethasone>prednisolone>cortisol. Hill analysis indicates steep concentration dependency. The corticosteroid-induced, amiloride-sensitive current is Na+ absorption as indicated by radiotracer flux measurements. Studies employing selective antagonists (spironolactone, mifepristone) define glucocorticoid receptor mediation. These results suggest that vas deferens epithelia are exquisitely sensitive to corticosteroid exposure. Observed changes in epithelial function in response to corticosteroid exposure would rapidly and chronically affect the luminal environment to which sperm are exposed. Thus, physiological and pharmacological corticosteroid exposure is expected to affect male fertility.

Master of Science / Department of Animal Sciences and Industry / Duane L. Davis / Intestinal absorption of colostral leukocytes (CL), peripheral blood mononuclear cells (PBMC), and porcine umbilical cord matrix stem cells (PUC) was analyzed in neonatal pigs. Maternal CL have previously been demonstrated in pigs, and maternal PBMC have been observed in calves to enter neonatal circulation after ingestion. PUC are primitive stem cells that are easily isolated from Wharton's jelly of the porcine umbilical cord. These cells do not have an immunogenic effect on the host upon initial transplantation. The general characteristics of PUC may allow them to serve as a delivery system to the neonate. Cellular migration through the duodenum, jejunum, and ileum was assessed using confocal microscopy. In vitro experiments utilized an organ explant culture system to determine the trafficking of labeled cells. Small-intestine tissue was collected from stillborn and sacrificed neonates. All three cell types (CL, PBMC, and PUC) were detected below the luminal surface, after 72 h of culture with media, and regardless of whether explants were from stillborns or live-born pigs. In vivo trafficking was assessed using neonatal pigs that were fed PBMC isolated from their mother or PUC from an unrelated pig. The effect of prior exposure to 25% acellular colostrum (AC) in medium was evaluated for both cell types. Piglets were euthanized 8 h or 24 h post feeding and sections of the small intestine collected. Both PBMC and PUC were found in all intestinal samples. Exposure to AC had no detected effect on the ability of either cell type to attach and migrate into the tissue. Labeled PUC were detected on the surface of the epithelium and in the lamina propria 8 h post treatment. PBMC were observed on the surface of the epithelium, in the lamina propria, and superficial submucosa 8 h following ingestion. In neonates sacrificed 24 h post treatment, both PUC and PBMC were observed on the surface of the epithelium, in the lamina propria, superficial submucosa, and deep submucosa of the small intestine. PUC and PBMC were noted at the apex, intermediate between the apex and the base, or at the base of the villus.

Master of Science / Department of Anatomy and Physiology / Bruce D. Schultz / Epithelial cells lining the vas deferens modify the luminal contents to which sperm are exposed in response to neuroendocrine, autocrine and lumicrine transmitters. The role and identity of vas deferens epithelial potassium channels that provide the correct luminal environment for sperm maturation and delivery have not yet been determined. Cultures of vas deferens epithelial cells isolated from adult pigs were employed to investigate contributions of selected ion channels to net flux. A two-pore potassium channel, TASK-2, was identified on the apical membrane of cultured primary porcine vas deferens epithelial cells (1°PVD). Bupivacaine, a known TASK-2 inhibitor, when added to the apical bathing solution, inhibited forskolin- stimulated short circuit current, Isc, in a concentration dependent manner with a maximum inhibition of 72 ± 6% and an IC50 of 7.4 ± 2.2 µM. Apical exposure of 1°PVD cells to quinidine, lidocaine, and clofilium (other known TASK-2 blockers) inhibited forskolin-stimulated Isc in a concentration dependent manner. Fitting a modified Michalis-Menten function to the data revealed IC50 values of 274 µM, 531 µM, and 925 µM, respectively. Riluzole, a two-pore potassium channel activator, stimulated bupivacaine-sensitive Isc, further confirming the contribution of TASK-2 to net ion flux. Western blotting demonstrated the presence of TASK-2 immunoreactivity in 1°PVD cell lysates, while immunocytochemistry demonstrated apical localization of the targeted epitope in virtually all cells lining native porcine vas deferens. These results suggest that TASK-2 likely plays a role in vas deferens epithelial ion transport that may account for the reportedly high concentration of potassium in the male reproductive duct lumen. TASK-2 likely contributes to male fertility as an integral member of the regulated transport processes that account for the luminal environment to which sperm are exposed.

Doctor of Philosophy / Department of Anatomy and Physiology / David C. Poole / The components of the O2 transport pathway can be divided into (along with their respective circulations) the pulmonary, cardiovascular, and skeletal muscle systems. They must operate in tight conjunction with one another, especially during dynamic exercise, to sustain ATP production within muscle mitochondria. Any limitation placed on the O2 transport pathway will result in decreased performance. The purpose of this dissertation is to present four novel studies which examine specific limitations on (1) the pulmonary system (i.e. lungs and circulation) within the highly athletic Thoroughbred horse (Studies A & B), and (2) within the peripheral circulation (i.e. microcirculation) within a disease model of Type II diabetes, the Goto-Kakizaki (GK) rat (Studies C & D). Study A demonstrates that locomotory respiratory coupling (LRC) is not requisite for the horse to achieve maximal minute ventilation (VE) during galloping exercise because VE remains at the peak exercising levels over the first ~13 s of trotting recovery (VE at end exercise: 1391±88; VE at 13 s: 1330±112 L/sec; P > 0.05). The horse also experiences exercise-induced pulmonary hemorrhage (EIPH) which has been linked mechanistically to increased pulmonary artery pressure (Ppa) during high intensity exercise. Therefore, in Study B, we hypothesized that endothelin-1 (ET-1), a powerful vasoconstricting hormone, would play a role in the augmented Ppa and therefore, EIPH. However, contrary to our hypothesis, an ET-1 receptor antagonist did not decrease Ppa nor prevent or reduce EIPH. Studies C and D examine potential mechanisms behind the exercise intolerance observed in humans with Type II diabetes. Utilizing phosphorescence quenching techniques (Study C) within the GK spinotrapezius muscle, we found lowered microvascular PO2 (PO2mv; Control: 28.8±2.0; GK: 18.4±1.8 mmHg; P<0.05) at rest and a PO2mv “undershoot” during muscle contractions. After conducting intravital microscopy within the same muscle (Study D), we discovered the percentage of RBC-perfused capillaries was decreased (Control: 93±3; GK: 66±5 %; P<0.05) and all three major hemodynamic variables (i.e. RBC velocity, flux, and capillary tube hematocrit) were significantly attenuated. Both studies (C & D) indicate that there is reduced O2 availability (via decreased O2 delivery; i.e. ↓QO2/VO2) within Type II diabetic muscle.