Master of Science / Department of Animal Sciences and Industry / Duane L. Davis / The common choice of medium for culturing pig umbilical cord matrix stem cells (PUCs) is high glucose Dulbecco’s Minimum Essential Medium (HG-DMEM) supplemented with fetal bovine serum (FBS). FBS is a chemically undefined supplement that encourages attachment of explants and cells and is useful for long-term proliferation in an undifferentiated state. Removing FBS from the culture medium would decrease the possibility of microbial contamination and might produce more consistent results. A defined medium would facilitate experiments to determine requirements for specific growth factors and nutrients. Starting PUCs in a FBS-free environment proved to be a challenge. The results of 15 experiments testing various media, supplements, and culture conditions indicate that PUCs initially plated in an FBS-free environment do not attach as readily as those in HG-DMEM supplemented with FBS. PUCs were collected using enzyme digestion of the whole cord or by plating explants from the cord in culture medium. In the final experiment PUCs were seeded in 24-well plates (5.0 * 10[superscript]4 viable cells per well) with a collagen coating and cultured in Knock-out DMEM (KO-DMEM) with basic fibroblast growth factor (5ng/mL) and platelet derived growth factor (5ng/mL) in a low oxygen atmosphere (5% O[subscript]2/ 5% CO[subscript]2/ 90% N[subscript]2). The total non-adherent cell count at passage 1 was 1.78 * 10[superscript]5 +or- 3.68 * 10[superscript]4 and the total adherent cells were 2.58 * 10[superscript]5 +or- 9.29 * 10[superscript]4. The well confluence during initial cell proliferation appeared similar to cells cultured in the control media with 20% FBS (total adherent cells = 6.40 * 10[superscript]5 +or- S.E. 1.61 * 10[superscript]5 and total non-adherent cells = 2.88 * 10[superscript]5 + 7.60 * 10[superscript]4). However the number of adherent cells recovered for passage 2 was considerably less for cultures in FBS-free media than for the control group. Serum may affect attachment by providing attachment factors or it could change expression of integrins or other attachment molecules on the PUCs that enhance attachment to plastic or other substrates. In future studies the requirements for attachment of PUCs should be further evaluated.

Master of Science / Department of Kinesiology / Timothy I. Musch / During exercise aged individuals exhibit endothelial dysfunction and decreased levels of whole-limb blood flow (BF), both of which may be linked mechanistically to age-related increases in reactive oxygen species (ROS). Ascorbic acid (AA) reduces levels of ROS and has been shown to alleviate vascular and hyperemic dysfunction at rest (Jablonski et al., 2007) and during small muscle mass exercise in humans (Kirby et al., 2009). However, the effect of AA on vascular function and BF to individual muscles during whole-body exercise is not known. PURPOSE: To test the hypothesis that a single high-dose infusion of AA would increase BF to the hindlimb musculature of old rats at rest and during treadmill running. METHODS: 18 old (~28 months) Fischer 344 x Brown Norway rats were randomized into rest (n=9) and exercise (n=9) groups. BF to the total hindlimb and individual muscles (28 individual muscles and muscle parts) was evaluated via radiolabeled microspheres before and after intra-arterial AA administration (76 mg/kg in 3 ml heparinized saline, 30 minute infusion) at rest and during submaximal treadmill running (20m/min, 5% grade). Total antioxidant capacity (TAC) and thiobarbituric acid reactive species (TBARS) were measured before and after AA to determine the ability of this specific dose of AA to increase levels of plasma antioxidants and decrease levels of ROS, respectively. RESULTS: At rest: AA increased TAC (~37%, P<0.05) but did not change TBARS (Pre: 6.8±0.7 vs Post: 7.0±1.0 µM, P>0.05). AA decreased total hindlimb BF (Pre: 25±3 vs Post: 16±2 ml/min/100g, P<0.05) and BF to 8 of the 28 muscles that were evaluated. During exercise: TAC was increased (~35%, P<0.05) and TBARS were decreased (Pre: 9.8±2.0 vs Post: 7.0±1.0 µM, P<0.05). However, there was no effect on either total hindlimb BF (Pre: 154±14 vs Post: 162±13, P>0.05) or BF to any of the individual muscles evaluated. CONCLUSIONS: Increased TAC via AA infusion reduces hindlimb muscle BF at rest but had no effect on BF during whole-body dynamic exercise. Thus, even though TBARS decreased, there was no evidence that AA supplementation increases blood flow to the locomotor muscles of old rats during whole-body exercise.

In vitro maturation of dog oocytes has always been the main obstacle preventing reproductive biologist from producing canine in vitro cultured embryos. The unsuccessful oocyte maturation in canine species originates from their unique physiological and biological specifications. Ovulation of dominant follicles in bitch (6-12 in each oestrous cycle) occurs at prophase I stage of oocyte nucleus and meiotic resumption develops during 3-5 days of oviductal transition. During this PhD thesis, studies were designed in order to speculate characteristics of canine oocyte maturation in vitro in terms of maturation media components, gas composition of the incubator and hormonal requirements. Level of oxidative stress during 72h (culture period) of in vitro maturation showed that 5%O2, 5% CO2 and 90% N2 composition improves meiotic resumption and reduces degeneration rate significantly compared to 5% CO2 in air. Utilization of caffeine as a non specific phosphodiesterase inhibitor at 10mM for 12h at the beginning of the 72h culture (12+60) also improved MII maturation rate (16.9% ± 2.4; P < 0.05). Among several hormonal treatments recombinant porcine Growth Hormone (PGH) at 100ng/ml and Melatonin (MTN) at 100nM concentrations had outstanding improvement over meiotic resumption (28.9% ±10.0 and 56.2% ±8.6 respectively; P < 0.05). Attempts were made to study developmental potentials of optimally matured oocytes by parthenogenetic activation (PA) and in vitro fertilization (IVF) using chilled semen. Partial digestion of the zona pellucida prior to IVF improved the cleavage rate at 48h 6.4% ± 0.3 and resulted in production of a single 8 cell embryo. Moreover; canine follicular cells were culture in order to characterize their primary culture morphology and steroidogenic responsiveness to physiological and pharmaceutical substances. Immunolocalization of aromatase (CYP19) positive cell clumps, presumptive oestrogen producing colonies, was identified. This primary culture also maintained its steroidogenic machinery up to 96h (measured by radioimmunoassay) with a significant increase in production of estradiol and progesterone after 72h compare to the start of the culture.

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.

The purpose of this study was to examine the effect of a six-week water-running program on the maintenance of cardiorespiratory performance ($\rm\dot VO\sb2$ max, ventilatory threshold, running economy); metabolic measurements of blood glucose, blood lactate and plasma norepinephrine; and body composition. Sixteen trained male runners ($\rm\dot VO\sb2$ max = $\rm 58.6\pm 3.6 ml\cdot kg\sp{-1}\cdot min\sp{-l})$ were assigned to one of two groups matched by $\rm\dot VO\sb2$ max, treadmill run (R) or water run (WR). Subjects participated in their respective training programs consisting of workouts of either 30 min at 90-100% $\rm\dot VO\sb2$ max or 60 min at 70-75% $\rm\dot VO\sb2$ max which were alternated daily, five days a week. Workouts for the R group were conducted on motorized treadmills in an air conditioned environment; WR subjects performed workouts in a swimming pool $\rm({\sim} 27\sp\circ C)$ while wearing a commercial flotation belt. Workout intensity was monitored by heart rate telemetry. / Following six weeks of workouts, no significant difference (p $>$ 0.05) was observed for treadmill $\rm\dot VO\sb2$ max between R $\rm (60.1\pm 3.6 ml{\cdot} kg\sp{-1}{\cdot} min\sp{-1})$ and WR $\rm(59.6\pm 5.4 ml{\cdot} kg\sp{-1}{\cdot} min\sp{-1}).$ Similarly, ventilatory threshold $\rm(48.2\pm 3.3 vs 47.4\pm 6.7 ml{\cdot} kg\sp{-1}{\cdot} min\sp{-1})$ and running economy $\rm(50.7\pm 2.0 vs 48.9\pm 2.2 ml{\cdot} kg\sp{-1}{\cdot} min\sp{-1}$ at 255 $\rm m{\cdot} min\sp{-1})$ were not different (p $>$ 0.05) between R and WR. Postexercise blood glucose (R = 8.5 $\pm$ 1.0; WR = 7.4 $\pm$ 1.1 mM) and blood lactate (R = 8.5 $\pm$ 1.9; WR = 8.3 $\pm$ 2.0 mM) were similar (p $>$ 0.05) between the two groups following six weeks of workouts, as was plasma norepinephrine (R = 7.4 $\pm$ 0.3; WR = 7.0 $\pm$ 0.5 ng $\cdot$ ml$\sp{-1}$). No significant group differences (p $>$ 0.05) were observed for total body weight, lean body mass or fat weight upon completion of the six-week exercise period. / It is concluded that water running may serve as an effective training alternative to land based running for the maintenance of physiological determinants of aerobic performance for up to six weeks among trained endurance athletes. / Source: Dissertation Abstracts International, Volume: 55-08, Section: B, page: 3186. / Major Professor: Robert J. Moffatt. / Thesis (Ph.D.)--The Florida State University, 1994.

Ten healthy eumenorrheic white females (26.4 $\pm$ 5.2 yrs) were studied during the regular menstrual cycle (25 to 37 days) to determine the effects of the different phases of the cycle on plasma lipid and lipoprotein concentrations. Three blood samples were collected throughout the phases of the menstrual cycle. The first blood sample was collected in the early follicular phase (days 4-7) after the onset of the menses; the second blood sample in the ovulatory phase (days 12-14) approximately 24-48 hrs after ovulation; and the third blood sample in the luteal phase (days 24-26) nearly 6-9 days after ovulation. A repeated measures MANOVA was utilized for the analysis of the data with Tukey post-hoc employed to locate significant differences between means. A significant increase (12.4% and 24.4%) in plasma total cholesterol levels and (20.2% and 30.9%) in LDL-C levels were demonstrated during the follicular phase compared to the ovulatory and luteal phases of the cycle, respectively. During the ovulatory phase of the cycle, total cholesterol levels also increased significantly (10.7%) relative to the luteal phase. In addition, during the follicular phase there was a significant increase in plasma levels of HDL-C (19.2%) and HDL$\sb3$-C (13.9%) compared to the luteal phase. Moreover, a significant increase (74.2%) in plasma HDL$\sb2$-C levels were demonstrated during the ovulatory phase versus the luteal phase. Furthermore, HDL-C levels were significantly higher (20.8%) during the ovulatory phase compared to the luteal phase. There were no statistically significant changes in plasma triglyceride concentrations among cycle phases. The results of this study suggest that the fluctuations in plasma lipid and lipoprotein concentrations throughout the different phases of the normal menstrual cycle seems of vital importance to define exact days and phases of the / cycle in which blood samples are to be collected for analyzing plasma lipid and lipoprotein levels in premenopausal women. / Source: Dissertation Abstracts International, Volume: 55-12, Section: B, page: 5292. / Major Professor: Robert Moffatt. / Thesis (Ph.D.)--The Florida State University, 1994.

Os insetos possuem uma película que reveste o intestino médio (membrana peritrófica, MP), que é composta por quitina e proteínas (peritrofinas). Além de possuir as funções de proteção contra abrasão causada pela comida e contra microrganismos, a MP possui algumas funções específicas que dependem do fato de que essa estrutura compartimentaliza o lúmen do intestino médio em duas regiões denominadas espaço endoperitrófico e espaço ectoperitrófico. As funções específicas da MP são baseadas em evidências indiretas e incluem o impedimento da ligação não específica de comida na superfície celular, o decréscimo na excreção das enzimas digestivas através da sua reciclagem e, em insetos mais evoluídos, o impedimento de oligômero- e dímero-hidrolases de penetrarem no espaço endoperitrófico. As proteínas da membrana microvilar intestinal e uma peritrofina (proteína da membrana peritrófica) de Spodoptera frugiperda foram isoladas e utilizadas para a produção de anticorpos em coelho. Esses anticorpos, juntamente com um anticorpo anti-amilase de Tenebrio molitor (que reconhece as amilases de S. frugiperda), foram utilizados em estudos de imunocitolocalização realizados com a ajuda de anticorpos secundários acoplados a uma proteína fluorescente ou a partículas de ouro coloidal. Os resultados mostraram que a peritrofina de S. frugiperda é secretada pelas células colunares da região anterior do intestino médio através de vesículas que se destacam das microvilosidades (secreção microapócrina). As vesículas com dupla membrana (uma da própria vesícula e a outra da microvilosidade) tornam-se vesículas com membrana simples através da fusão entre membranas e, nesse processo, a peritrofina e parte da amilase e tripsina são liberadas. As membranas remanescentes das vesículas, ainda contendo proteínas microvilares, amilase e tripsina ligadas, são incorporadas a um material com consistência de gel que forma parte da MP. Larvas alimentadas com calcoflúor tiveram a sua MP desestruturada e, em razão disso, perderam o gradiente decrescente antero-posterior de tripsina e quimotripsina observado ao longo do intestino médio das larvas controle. Esse gradiente é presumivelmente formado por um contrafluxo de fluidos (no espaço entre a MP e o epitélio) que permite a reciclagem de enzimas. / Insects have a film-like anatomical structure (peritrophic membrane, PM) which lines the midgut. It is composed of chitin and proteins (peritrophins). Besides the functions of protection against food abrasion and microrganisms, PM has specific functions that depend on the fact that this structure compartimentalizes the midgut lumen into an endoperitrophic and an ectoperitrophic space. Knowledge on these specific functions are based only in indirect evidence and include: prevention of non-specific food binding onto cell surface; prevention of enzyme excretion by allowing enzyme recycling and restriction of oligomer hydrolases to ectoperitrophic space. A peritrophin from Spodoptera frugiperda PM, as well as microvillar proteins from S. frugiperda anterior midgut, were isolated and used to raise antibodies in a rabbit. These antibodies, a Tenebrio molitor anti-amylase antibody that cross-reacts with S. frugiperda amylases, and wheat-germ aglutinin were used in immunolocalization experiments performed with the aid of confocal fluorescence and immunogold techniques. The results showed that the peritrophin is secreted by anterior midgut columnar cells in vesicles that pinched-off the microvilli (microapocrine secretion). The resulting double membrane vesicles become single membrane vesicles by membrane fusion, releasing peritrophin and part of the amylase and trypsin. The remaining vesicle membranes (still containing microvillar proteins and membrane-bound amylase and trypsin) are incorpored into a jelly-like material associated with PM. Calcofluor-treated larvae lacking a PM were shown to lose the trypsin and chymotrypsin decreasing gradient observed along the midgut of control larvae. This gradient is thought to be formed by a countercurrent flux of fluid (in the space between PM and midgut cells) that powers enzyme recycling.