Master of Science / Department of Animal Sciences and Industry / J. Ernest Minton / Previous research evaluated a laboratory strain of Bacillus licheniformis (BL) in a model swine epithelium and found it exerted anti-inflammatory effects on Salmonella enterica serovar Typhimurium (S)-induced secretion of interleukin-8 (IL-8). The current investigation evaluated the anti-inflammatory actions of Bacillus bacteria available commercially as feed additives for the swine industry. Three isolates were obtained from the product, two Bacillus subtilis (BS1 and BS3) and one Bacillus licheniformis (BL2). Swine jejunal epithelial IPEC-J2 cells were seeded into wells on permeable membrane supports and allowed to form confluent monolayers. Treatments included apical pretreatment with BL, BS1, BL2, or BS3 for 17 h without S, and the same Bacillus treatments but with 10[superscript]8 CFU S added in the final 1 h of Bacillus incubation. Two additional treatments included negative control wells receiving no bacteria (C) and positive control wells receiving only S. Following bacterial incubation, wells were washed and fresh media containing gentamicin was added. Cells were incubated for an additional 5 h, after which apical and basolateral media were recovered for quantitation of IL-8 and bacitracin. In addition, inserts with epithelial cells that had received S were lysed and lysates cultured to determine treatment effects on S invasion. Exposure to S alone provoked an increase in IL-8 secretion from IPEC-J2 cells compared to C wells (P < 0.001 for both the apical and basolateral directions). Pre-treatment with each Bacillus isolate followed by challenge with S reduced S-induced IL-8 secretion in both apical and basolateral compartments compared to the wells receiving only S (P < 0.001; except for BS3 apical, P < 0.01). Secretion of bacitracin could only be detected in BL2 and BL2+S. Fewer S colonies could be cultured from lysates of BL2+S than S, BS1+S, and BS3+S treatments (P < 0.001). Results suggest that Bacillus subtilis and Bacillus licheniformis have the ability to intervene in secretion of the neutrophil chemoattractant IL-8 from swine intestinal epithelial cells. This effect on chemokine secretion by gastrointestinal epithelial cells in vitro could not be explained solely by production of bacitracin or reduced invasion of epithelial cells by S.

Master of Science / Clinical Sciences - Veterinary Medicine / Laurie A. Beard / Objective: To determine the pharmacokinetic properties of oral pergolide in normal mares. Animals: 6 horses, 3-17 years of age, 355-582 kg Procedures: In a randomized, cross over design six healthy adult female horses received pergolide (PO) 0.01mg/kg or placebo after 8 hours of fasting. Samples were taken over a period of 6 day for each portion of the study (treatment or placebo) with a two week minimum wash out period between study periods. Quantification of pergolide concentration was determined by UPLC-MS. Quantification of α-MSH was determined by radioimmunoassay validated for horses. Quantification of ACTH concentration was determined by chemiluminescent enzyme immunoassay. Results: Pergolide was detected in all treated horses. The relatively short time to peak concentration (0.5 hours) indicates a rapid absorption. Mean maximum concentration measured was 4.05 ng/ml + 2.02 with a median time to maximum concentration being 0.415 hours (range:0.33-1.0). The mean half life of pergolide was determined to be 5.86 hours + 3.42. Lower limits of quantitation for the UPLC-MS assay was 0.5 ng/ml. α-MSH results were evaluated using a multiple analysis of variance assay for repeated measures comparing treatment, time, and period. There was a significant treatment to period effect with p=0.02. The effect of period appears to be more significant (p=0.06) compared to the effect of treatment (p=0.77). No effect from pergolide was noted on ACTH concentrations. Conclusions and Clinical Relevance: Horses appear to absorb and eliminate pergolide more rapidly than previously expected. Based on this pharmacokinetic data the dosing strategies of pergolide may need to be altered. However, assay sensitivity does need to be improved prior to recommendations being made.

Master of Science / Department of Clinical Sciences / James Roush / Objective – To measure the effect of cold and warm compress therapy on tissue temperature in healthy dogs. Design – Controlled, blinded, crossover study Animals – 10 healthy mixed breed dogs Procedures – Dogs were sedated with hydromorphone 0.1 mg/kg IV and diazepam 0.25 mg/kg IV. Thermocouple needles were inserted to 0.5 cm (superficial), 1.0 cm (mid) and 1.5 cm (deep) into a shaved, lumbar, epaxial region to measure tissue temperature. Cold 2° F (-16.8° C) and warm 117°F (47°C) compresses were applied with gravity dependence for periods of 5, 10 and 20 minutes. Control data was collected under identical sedation. Results – Mean temperature significantly decreased after 5 minutes of cold application at only the superficial depth. Application of cold for 10 and 20 minutes significantly reduced the temperature at all depths. Twenty minutes of cold application significantly decreased temperature at only the mid depth compared to 10 minutes of application. Warm compresses significantly increased temperature at all depths after 10 minutes of application. Temperatures associated with 20 minutes of warm application were not significantly different than 10 minutes of application. Conclusions – When utilizing these methods of cold and warm compression, minimum time of application should be 10 minutes. Minimal changes occur by increasing cold application to 20 minutes and no changes occur when increasing heat application to 20 minutes. There is minimal to no change at depths ≥ 1.5 cm when using this method of heat application. Changes in tissue temperature and side effects of application longer than 20 minutes and in the absence of mu agonist opioids require further evaluation.

Master of Science / Department of Animal Sciences and Industry / Jeffrey S. Stevenson / Our objective was to determine the optimal time to inseminate artificially cows following the standard CO-Synch protocol that also included a progesterone-releasing intravaginal controlled internal drug release (CIDR) insert. Lactating females from 3 Kansas locations were utilized. Crossbred Angus cows (n = 212) from the Agriculture Research Center in Hays (ARCH; location 1); Angus-Hereford crossbred cows (n = 249) from the Kansas State University Cow-Calf Unit (location 2); and purebred Angus, Hereford, and Simmental cows (n = 144) from the Kansas State University Purebred Beef Unit (location 3) were used in this study. Cows within each location were blocked by parity and assigned randomly within blocks to be artificially inseminated (AI) at 4 different times after the PGF2[Alpha] injection of the protocol: 48, 56, 64, or 72 h. Pregnancy diagnosis occurred at 32 and 63 d after insemination. Blood samples were collected 9 to 10 d and just before the first GnRH injection. Radioimmunoassays were performed on the blood sera samples to determine progesterone concentrations. Progesterone concentrations determined that approximately 60% of cows were cycling at the initiation of the study. A difference in cyclicity was observed with regards to age as well as body condition score. Pregnancies per AI (P/AI) at d 32 varied according to location and cycling status. Pregnancy loss between d 32 and 63 also was greatest for cows inseminated at 48 and 72 h. As pregnancy rates at d 63 increased with the 56- and 64-h treatments, pregnancy loss decreased. A significant difference in calving interval was detected among treatments, the shortest calving interval at 56 h. Results indicated that in most situations, the 56- and 64-h treatments presented the most desirable outcomes. The 56-h treatment presented the greatest number of P/AI for younger cows (≤ 3 yr), but for older cows, inseminations anytime 56 h or later produced the most P/AI. Overall pregnancy rates at d 63 were greatest for the 56-h treatment, with the fewest pregnancy losses. Given the interactions that seem to exist among location, cycling status, and age, further work is necessary to better define these relationships with the applied protocol.

Master of Science / Department of Clinical Sciences / Mike D. Apley / Lameness is a common, costly, and painful affliction in cattle at all production levels. There are currently no compounds specifically approved for analgesia in cattle in the United States. We hypothesized that intra-articular amphotericin B produces a controlled, transient synovitis-arthritis in cattle and that this model would allow characterization of the analgesic effects of intravenous sodium salicylate. This study examined the efficacy of sodium salicylate for providing analgesia in an amphotericin B-induced bovine synovitis/arthritis model utilizing ten male Holstein calves, 4-6 months old, and weighing approximately 250 kg. The study used a repeated measures partial cross-over design with 2 phases consisting of 3 treatment periods within each phase. Calves were blocked by weight and randomly assigned to sodium salicylate (50mg/kg intravenously) or placebo group for phase 1. In period 1, lameness induction was simulated with a needle-prick of the coronary band, followed by drug or placebo administration. At predetermined timepoints, serial blood samples for cortisol and salicylate concentrations, electrodermal activity measurements, heart rates, and pressure mat data were collected. Visual lameness scores were recorded by a blinded observer. In period 2, lameness was induced with injection of amphotericin B into the distal interphalangeal joint followed by drug or placebo administration with sample collection as previously described. In period 3, drug or placebo was administered to the respective calves with sample collection. After a 10-day washout, Phase 2 was conducted with treatments crossed over between groups. Cortisol and salicylate samples were analyzed by competitive chemiluminescent immunoassay and fluorescence polarization immunoassay, respectively. The pharmacokinetic data were analyzed using compartmental analysis. Mean intravenous salicylate apparent volume of distribution (V[subscript]d) was 0.2 ± 0.005 L/kg, total body clearance (CL[subscript]B) was 4.3 ± 0.2 mL/min*kg, and elimination half life (T[subscript]1/2 el) was 36.9 ± 1.2 minutes. The repeated measures data were analyzed based on a univariate split-plot approach with a random effects-mixed model. Differences in stance phase duration and serum cortisol concentration values were seen between both periods and treatment group*periods; differences in heart rate, contact surface area, and contact pressure values were seen between periods, suggesting that our lameness model was effective. No differences were seen between treatment groups. When analyzed by visual lameness score, differences were seen in heart rate, contact surface area, contact pressure, and cortisol concentrations. Area under the time-effect curves, determined using the trapezoidal rule, had results similar to the repeated measures data, except for a difference in period for electrodermal activity. This amphoterecin B-induced synovitis/arthritis model is a useful tool for studying changes associated with lameness in cattle. Sodium salicylate was not effective in providing analgesia following lameness.

Master of Science / Department of Diagnostic Medicine and Pathobiology / Melinda J. Wilkerson / Failure of passive transfer (FPT) is one of the main reasons for increased mortality rate in newborn calves and diagnosis is dependent on determination of serum IgG concentrations (diagnosis is based on < 1 g/dL of total IgG). Several qualitative assays are available, but the reference method, single radial immunodiffusion assay (SRID), albeit quantitative measures only one subclass at a time. We set out to develop a competitive multiplex microsphere flow cytometry assay to measure bovine IgG1 and IgG2 concentrations in 30 serum samples acquired from newborn Holstein calves prior to and 24 hours after ingestion of colostrum and to compare the values with SRID. A triplex bead assay was created by mixing three distinct sets of Quantum plex carboxylated fluorescent microspheres that were coated with purified bovine IgG1, IgG2 or albumin using a two step chemical reaction. The triplex protein coated beads were reacted with a cocktail of sheep anti-bovine IgG1 and IgG2. Evaluation of analytical specificity demonstrated cross reactivity between anti-bovine IgG2 and IgG1 coated beads that precluded determination of IgG2 > 0.5 g/dL. Cross reactivity between anti-IgG1 and IgG2 coated beads was minimal and did not affect IgG1 concentrations between 0.15 to 1.2 g/dL. A competitive linear decrease in the fluorescence intensity was observed in the triplex assay when 2-fold dilutions spanning a concentration range of 12 mg/dL – 100 mg/dL of either purified bovine IgG1 or IgG2 were included as a competitive inhibitor of the reaction. Precolostral serum samples from 29 calves were determined to be < 0.4 g/dL by SRID. Standard calibrants for the flow assay were prepared from two fold serial dilutions of purified bovine IgG (stock concentration 10 g/dL) using a precolostral calf serum pool as the diluent. The standard calibrants (IgG1 was 1.0- 0.16 g/dL and IgG2 was 3.4 – 0.22 g/dL) were used as the inhibitors in a triplex assay to develop a standard curve for unknown samples. Dilutions of bovine reference serum containing known amounts of IgG1 (1.2 – 0.15 g/dL) and IgG2 (1.6 – 0.2 g/dL) was used as positive control. The intra Intra-assay and inter-assay precision of the mutiplex assay was good (coefficient of variation < 10%). Since the IgG2 concentrations of post colostral samples were below detection limit, only IgG1 values were compared to the SRID. The agreement between triplex microsphere assay and SRID for IgG1 was poor with a mean bias of 0.743 g/dL towards triplex microsphere assay (95% confidence interval of 0.382 to 1.105 g/dL). Method comparison studies between total IgG determined by SRID and the gamma-globulin fraction determined by serum electrophoresis indicated that the SRID calculated higher values than the protein method (mean bias of -1.4 g/dL, 95% confidence interval was -1.8 to -1.05 g/dL). We hypothesized that the positive bias for the microsphere assay was explained in part by the use of dilution factors, use of standards that had a low analytical range, and erroneously high standards used in the SRID method.

Master of Science / Department of Kinesiology / Craig A. Harms / Diaphragmatic fatigue is known to limit endurance performance during heavy exercise in humans. Previous reports have shown that diaphragmatic fatigue is reduced in rats with N-acetylcysteine (NAC; a nonspecific antioxidant) infusion, suggesting that oxidative stress contributes to this fatigue. However, it is not known if oral supplementation of NAC will reduce respiratory muscle fatigue during heavy exercise in humans. Therefore, the purpose of this study was to determine the effect of an acute oral dose of NAC on respiratory muscle fatigue during whole body heavy exercise. Eight healthy, non-smoking men (22+/-2 yrs), with no history of cardiovascular or lung disease, completed baseline pulmonary function tests followed by an incremental cycle VO[subscript 2peak] test. A randomized, double blind crossover design was then used where subjects were given either placebo (PLA) or NAC (1800 mg) 45 min prior to a 30 minute constant load (85% VO[subscript 2peak]) discontinuous (six-five minute stages) or continuous (cycle until volitional exhaustion) exercise test. Tests were separated by approximately one week. Maximum pressures (inspiratory, PImax; expiratory, PEmax) and venous blood samples (plasma lactate and total plasma glutathione) were made prior to- and following each 5-min of exercise in discontinuous tests and pre- and post-exercise in continuous tests. Subject's VO[subscript 2peak] was 43+/-5 ml/kg/min. There was no difference (p>0.05) in PImax between NAC (127.9+/-34.1 cmH[subscript2]O) or PLA (134.1+/-28.1 cmH2O) at rest. During exercise, PImax was significantly lower ([similar to]14%) in 6 of 8 subjects with PLA compared to NAC at minutes 25 and 30 of the discontinuous test indicating respiratory muscle fatigue. With NAC, PImax did not change (p>0.05) from rest throughout exercise indicating no respiratory muscle fatigue. There was no difference (p>0.05) in PEmax, plasma glutathione, lactate, oxygen uptake (VO[subscript 2]), ventilation (VE), heart rate (HR), or rating of perceived exertion between PLA and NAC at rest or during exercise. Time to exhaustion was not different (p>0.05) during the continuous tests (PLA: 1263 + 334 sec; NAC: 1047 + 136 sec). These results suggest that an acute dose of NAC reduces respiratory muscle fatigue during high intensity exercise but does not alter other ventilatory or metabolic indices. The significance of this reduced respiratory muscle fatigue with NAC on whole body exercise performance remains to be determined.

Master of Science / Department of Kinesiology / Thomas J. Barstow / Background and Aim: Long-term microgravity exposure, via spaceflight or -6° head-down tilt bedrest, has been shown to produce significant cardiovascular deconditioning and decreases in exercise performance. However, there is little known about how acute microgravity exposure influences the cardiovascular system’s ability to adjust to increases in physical work. Therefore, the aim of this study was to compare cardiovascular and exercise performance during acute upright, supine and -6° head-down tilt positions. Methods: Seven healthy inactive men performed maximal cycle exercise (VO2peak) tests in the upright, supine, and -6° head-down tilt on separate days. Oxygen consumption and heart rate were measured continuously throughout the testing procedures. Cardiac output (acetylene exhalation technique) was measured periodically and interpolated to the 100-watt work rate. Stroke volume was calculated from cardiac output and heart rate data. Results: Peak oxygen uptake and heart rate were significantly decreased in the supine and -6° head-down tilt positions compared to the upright (VO2peak 2.01±0.46, 2.01±0.51 versus 2.32±0.61 L/min respectively; peak heart rate 161±13, 160±14 versus 172±11 bmp). However, cardiac output at 100-watts was similar in all three-exercise positions. Calculated stroke volume at 100-watts was significantly higher in the -6° head-down tilt position compared to the upright position (76.6±4.7 versus 71.2±4.5, ml). Conclusion: These results suggest that exercise capacity is immediately decreased upon exposure to a microgravity environment, prior to any cardiovascular deconditioning. Therefore, an astronaut’s exercise performance should be evaluated with exercise tests in the -6° head-down tilt position prior to space flight in order to establish a baseline response.

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.