Master of Science / Biomedical Sciences / Daniel U. Thomson / Two studies were conducted to evaluate two animal welfare issues in the beef industry today. The welfare of animals has become a major discussion among consumers and producers. The objective of these studies was to evaluate if certain production practices are beneficial to the wellbeing of the animals in a production setting. The first study evaluated if castration and implementation of growth promotion technologies of physically mature male beef cattle, which failed the breed soundness exam (BSE), improved carcass quality compared to male beef cattle left intact. Sixteen month old Angus bulls (n = 24; 606 + 11.4 kg) were stratified by weight and randomly assigned to 2 treatments: intact control (BULL) and castrated with growth promotion technology (STR) to evaluate performance and carcass quality. Cattle assigned to STR treatment were implanted with 120 mg trenbolone acetate (TBA) and 24 mg estradiol on d 0, and were fed ractopamine hydrochloride (300 mg/d) the final 28 d prior to slaughter. Cattle were fed a dry-rolled corn-based finishing diet (1.41 Mcal/kg NEg) for 62 d (final wt = 697 +/- 24.3 kg) then harvested at a commercial abattoir. Carcass characteristics were recorded and longissimus muscle samples were obtained. There were no differences between treatments for quality grade, yield grade, HCW, back fat thickness, or dressing percent. Steak tenderness values based on Warner Bratzler shear force (WBSF), and sensory panel evaluation showed no difference between BULL and STR steaks in myofibrillar tenderness, juiciness, beef flavor intensity, connective tissue, overall tenderness, and off flavor intensity. Cattle within the BULL treatment tended to have improved average daily gain (ADG) and feed efficiency, with no difference in carcass characteristics, WBSF, or sensory panel measurements compared to STR administered growth promotion technology. The second study evaluated if cohorts with horns within a pen lot of cattle caused an increase in carcass bruising, and to determine if horn tipping and dehorning is necessary. Carcasses from (n = 4,287) feedlot cattle were observed at one commercial beef packing plant in southwest Kansas to investigate the relationship between the presence and size of horns in cattle and the prevalence, anatomical location, and severity of bruising of carcasses. Horn measurements taken were the length of the longest horn from base to tip and the tip-to-tip distance between the tips of both horns. Bruises were evaluated by location and severity. Bruise severity was scored at 3 levels: minor: ≤ 5 cm, moderate: 5 to 15 cm, and severe: > 15 cm. Within pen lots of cattle, the percentage of cattle with horns ranged from 0 to 26%. There were 4,287 carcasses evaluated and 2,295 had one or more bruises for a total, overall bruise prevalence of 53.5%. Of the total number of bruises, 25.6% were severe, 35.6% were moderate, and 38.8% were minor. The majority of bruises (61.8%) occurred on the dorsal mid-line with similar rates of bruising occurring on the left (18.6%) and right (19.5%) sides. There was no relationship found between the prevalence of horns and prevalence of bruising in a pen lot of cattle (P = 0.90). These two studies conclude that feeding of bulls that fail the BSE could eliminate an animal welfare concern while removing the cost and management of growth promotion technology administration. Additionally to that there may be other factors causing carcass bruising at other than cohorts with horns.

Master of Science / Diagnostic Medicine and Pathobiology / Carol R. Wyatt / Currently, porcine reproductive and respiratory syndrome virus (PRRSV) is the most economically significant disease affecting the swine industry. PRRSV is known for its restricted cell tropism, primarily infecting porcine alveolar macrophages (PAM) via receptor-mediated endocytosis. PRRSV infects only a portion of the PAM population both in vivo and in vitro, which suggests that not every macrophage is PRRSV-permissive. Three surface proteins that can act as receptors for PRRSV have been identified on PAM, however, little else is known about the regulation of macrophage tropism. Factors determining cellular permissibility or resistance to PRRSV infection remain largely uncharacterized, although a recent study from our laboratory demonstrated that 1) permissiveness to PRRSV infection increased with time in culture, 2) macrophages from infected pigs could be superinfected, and 3) addition of actinomycin D, which inhibits mRNA synthesis, blocked infection. These data suggest that a PRRSV-permissive subpopulation of cells derives from a non-permissive precursor population and depends on new mRNA synthesis. The current studies were designed to examine the effects of PRRSV on both infected and uninfected PAM cells in vitro, specifically focusing on the expression of MHC I, MHC II, CD14, CD163 and CD172a surface proteins. The results show upregulation of MHC II, CD14, CD163 and CD172a expression in PRRSV-infected cells and a downregulation on the uninfected cells within the PRRSV-inoculated cultures. The role of apoptosis in the PRRSV-inoculated cultures was investigated, with results showing similar, low levels of apoptosis in control and infected PAM. PAM cytokine responses to PRRSV and LPS were also examined and, although they were uniquely different relative to control PAM, no trends were detected in the responses of PAM infected with PRRSV compared to uninfected and classically stimulated PAM. These data confirm that there are at least two subsets of macrophages within the alveolar population and suggests that the subsets are differentially affected by PRRS virus. We also demonstrated that MHC I becomes undetectable on PAM as a result of the freezing process, and that PRRSV-permissiveness is greater in the cell population after freezing.

Master of Science / Department of Clinical Sciences / Bradley J. White / Castration and dehorning are common husbandry practices in the United States. The objective of this research was to evaluate behavioral responses to these painful procedures. Two studies were conducted using three dimensional accelerometers to measure behavioral differences in cattle following dehorning and castration. The first study evaluated the potential of different analgesics to mitigate pain following castration and dehorning compared to negative controls as judged by behavioral measures. Holstein-Fresian calves given one of three different analgesic protocols (sodium salicylate, a combination of xylazine, ketamine and butorphanol, and both treatments together) or received no analgesia. All cattle were surgically castrated and dehorned. All treatment groups spent more time lying down and less time walking in the post-surgery than pre-surgery, and significant interactions were found between treatment and time relative to surgery (P<0.05). The second study evaluated behavioral changes following castration and dehorning performed independently or concurrently when compared to a negative control (no surgery). Accelerometers recorded behavior in Holstein-Fresian calves in which differing surgical procedures were performed: castration only (CO), dehorning only (DO), castration and dehorning (CD), and no surgical procedure performed (CON). Behavioral data was measured for 6 days following surgery. Significant interactions were found between treatment and time (P<0.05) in both walking and lying behavior. Calves that were dehorned and castrated spent less time walking one day after the procedure compared to controls, but very few other behavioral differences were identified. These studies illustrate that cattle behavior changes following painful procedures such as castration and dehorning.

Master of Science / Department of Clinical Sciences / Daniel Thomson / A total of 400 head of naturally parasitized commercial feedyard cattle (subset of 40 hd/pen; 10 pens) were utilized to compare trade name ivermectin pour-on and a generic ivermectin pour-on. The efficacy of each product was measured by obtaining rectal fecal egg counts on day 0 and day 14 using a Modified Wisconsin sugar float with centrifugation from rectal fecal samples and calculating fecal egg reduction post treatment. There were no differences in net egg count reduction between treatments (P= 0.15) at 14 days post-treatment application. Regardless of treatment, only 26% of animals had a fecal egg count reduction of >90% and only 35% achieved a FECR of >80% which is low considering 90% reduction is the accepted efficacy level. Cattle treated with the generic pour-on had improved average daily gains (ADG) compared to cattle treated with the trade name pour-on P = 0.02. This study demonstrated decreased efficacy of both products with a FECR of less than 90% within a commercial feedyard environment.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Raymond R. R. Rowland / Infectious cDNA clones of PRRSV make it possible to construct marker viruses for the study of virus replication and pathogenesis. The nonstructural protein 2 (nsp2) of porcine reproductive and respiratory syndrome virus (PRRSV) is the single largest protein produced during virus replication. The cDNA of the pCMV-129 infectious PRRSV clone was modified by creating unique Mlu I and SgrA I restrictions sites at nucleotide (nt) positions 3,219 and 3,614, respectively: both located within the C-terminal region of nsp2. cDNAs coding for oligo- and polypeptide tags, including FLAG, enhanced green fluorescent protein (EGFP) and firefly luciferase were inserted into the newly created restriction sites. The results showed that only the EGFP-containing genomes were properly expressed and produced virus. EGFP fluorescence, but not EGFP immunoreactivity, was lost during passage of recombinant EGFP viruses in culture. Sequencing of a fluorescence-negative EGFP virus showed that the EGFP remained intact, except for the appearance of mutations that may affect chromophore formation. The results show that nsp2 can be a site for the expression of foreign proteins. Removal of the region between Mlu I and SgrA I sites resulted in a virus that contained a 131 amino acid deletion. The deleted region was replaced with EGFP or an eight amino acid influenza hemagglutanin (HA) tag. Recombinant viruses were used to infect pigs. Gross and micro-histopathology showed reduced pathogenesis when compared to the parent wild-type virus. The 131 amino acid peptide, when expressed as a recombinant protein and coated onto enzyme linked immunosorbent assay (ELISA) plates, was recognized by sera from pigs infected with wild-type virus, but not the deletion mutants. The results from this study show that nsp2 is a potential target for the development of marker vaccines that can differentiate infected from vaccinated animals (DIVA) and for virus attenuation.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Gary A. Anderson / Foot-and-mouth disease (FMD) is a highly infectious viral disease of cattle, pigs, sheep, goats, buffalo, and artiodactyl wildlife species. Foot-and-mouth disease virus is endemic and periodic devastating epidemics have occurred and caused heavy economic losses in Iraq for a long time. The first official cases of FMD were recorded in 1937, while the first record of a specific FMD serotype in Iraq was serotype A in 1952. Other serotypes have been reported since then; serotypes O, SAT-1 and Asia1 were recorded in 1957, 1962, and 1975, respectively. Veterinary Services in Iraq has been severely weakened over the past two decades, and its infrastructure has been devastated as a consequence of previous political conflicts, wars and international sanctions. The breakdown of Veterinary Services led to the disruption of disease control strategies, collapse of disease surveillance and monitoring, and weakening of response systems. The destruction of the Al-Dora FMD laboratories for diagnosis and vaccine production by the United Nation in 1996, and the restrictions placed on the importation of vaccines have strongly affected the FMD control program. A severe epidemic of FMD occurred in Iraq in 1998, affecting 2.5 million ruminants and causing heavy losses in newly born animals. It is estimated to have killed about 550,000 animals. The outbreak was due to the serotype O1 Middle East strain which has affected large and small ruminants. In 2009, Iraq was severely affected by new serotype A (subtype A Iran 05). The major efforts of Veterinary Services in Iraq have been directed towards control of FMD by vaccination strategies. Two types of vaccine have been used, trivalent vaccine (O, A 22, and Asia 1) for cattle and buffalo and monovalent vaccine (O Manisa) for sheep and goats. Vaccination has been implemented once yearly on a voluntary basis. Sometimes other limited control measures have accompanied vaccination, which include quarantine, movement control, focused vaccination, disinfection, and public awareness programs. The FMD control program in Iraq has been confronted by many challenges: deficits in FMD surveillance and emergency preparedness, limited diagnostic capabilities, difficulties in restricting animal movement, and lack and irregular supply of appropriate vaccines.

Master of Science / Department of Clinical Sciences / Walter C. Renberg / Objective: To compare the in vitro elution characteristics of six common analgesic medications from a commercially available absorbable gelatin sponge. Study Design: Experimental study. Methods: Gelatin sponges were loaded with various analgesic medications, including two opioids, preservative-free morphine and fentanyl, two local anesthestics, bupivacaine and lidocaine, and two α2-adrenergic agonists, dexmedetomidine and xylazine. The loaded sponges were placed in dishes containing phosphate-buffered saline (PBS) and maintained at 37° C with constant agitation. Concentrations of each drug were determined at various time points up to 24 hours post-inoculation using high-pressure liquid chromatography. Two phases were conducted, utilizing undried loaded sponges (phase one) and dried loaded sponges (phase two). Results: In both phases, all analgesic medications eluted from the gelatin sponge and equilibrated rapidly with the PBS, achieving maximal concentration within 30 minutes. In phase two, the rapid nature of the release was captured by increasing sampling within the initial 30 minutes. Results were consistent for each medication with minimal variation. Steady state concentrations were significantly higher in phase two with four out of six medications. Conclusions: Analgesic medication elution from an absorbable gelatin sponge was rapid and consistent. Drying the loaded sponge prior to use will likely substantially increase the amount of medication eluted but not prolong release. Clinical Relevance: The rapid release of analgesic medications from the gelatin sponge makes a prolonged analgesic effect unlikely without further modification. Toxicity may be a concern. Further study is required to investigate efficacy in vivo, safe dosing regimens and prolongation of duration of action.

Master of Science / Department of Clinical Sciences / David E. Anderson / Lameness in cattle is a common cause of pain however there are no approved cattle analgesic drugs. Flunixin meglumine, the only non-steroidal anti-inflammatory drug approved for use in adult dairy cattle, is labeled for pyrexia associated with bovine respiratory disease, endotoxemia, acute mastitis and associated inflammation. There is currently a lack of objective data regarding the analgesic efficacy of flunixin meglumine in cattle. The objectives of this study were to characterize an amphotericin B-induced lameness model and to ascertain the analgesic effects of flunixin meglumine using multimodal assessment. We hypothesized that flunixin meglumine would provide analgesia as evidenced by increased activity levels as well as increased exerted force and contact area on the affected limb in flunixin treated steers. Amphotericin B-induced synovitis arthritis was induced in the distal interphalangeal joint of 10 dairy steers. The cattle were randomly allocated between a treatment and a control group. The treatment steers received flunixin meglumine at the time of arthritis induction and at 12 hours post-induction. Accelerometric, gait, pressure mat, vital parameter and plasma cortisol data were gathered in the pre and post-induction phases. The data were analyzed using linear mixed models with treatment and time designated as fixed effects. Induction of amphotericin B arthritis produced a moderate, transient lameness. Control steers were more than twice as likely to be lame as flunixin meglumine treated steers using visual lameness assessment (92.2% ± 8.1 versus 40.7% ± 2.5) (P<0.03). Flunixin meglumine treated steers placed significantly greater force and contact area on the affected foot. Control steers also placed significantly greater force, impulse and contact area on the paired claw as compared to control steers. Flunixin treated steers spent considerably less time in recumbency than their control counterparts, particularly in the immediate post-induction time period. This is one of the first studies to document the character of an amphotericin B-induced synovitis arthritis model in cattle as well as to document analgesic efficacy of a nonsteroidal anti-inflammatory drug in an induced lameness model. Flunixin meglumine was efficacious in providing analgesia in an amphotericin B-induced lameness model in dairy steers.

Master of Science / Department of Clinical Sciences / Amy Rankin / Objective- The purpose was to compare the efficacy and duration of 0.5% proparacaine, 0.5% bupivacaine, 2% lidocaine, and 2% mepivacaine on corneal sensitivity in clinically normal horses. Animals- 68 clinically normal horses Procedures- In group 1, 60 horses from the Kansas State University horse unit were assigned to receive one topical anesthetic in a completely randomized design. In group 2, 8 privately owned horses were sequentially treated with each of the topical anesthetics in random order with a one week washout period between drugs. Corneal sensitivity was assessed by corneal touch threshold (CTT) measurements which were taken with a Cochet-Bonnet aesthesiometer before anesthetic application (T0), 1 minute after (T1), every 5 minutes until 60 minutes (T5-T60), and then every 10 minutes until 90 minutes (T70-T90) after application. General linear mixed models were fitted to CTT in each design in order to assess the effects of topical anesthetics over time, accounting for repeated observations within individual horses. Results- Corneal sensitivity, as determined by CTT measurements, decreased immediately following application of the topical anesthetic, with persisting effects until T35 for proparacaine and mepivacaine, T45 for lidocaine, and T60 for bupivacaine. Maximal CTT reduction was achieved following application of bupivacaine or proparacaine, while mepivacaine was least effective. Conclusions and Clinical Relevance- All topical anesthetics reduced corneal sensitivity, though maximal anesthesia and effect of duration differed between drugs. For brief corneal anesthesia, 0.5% proparacaine or 2% lidocaine appeared adequate, while 0.5% bupivacaine may be most appropriate for procedures requiring longer periods of corneal anesthesia.

Master of Science / Department of Anatomy and Physiology / Mark Weiss / Artificial insemination and cryopreservation of sperm are important components of any transgenic animal facility because they allow for the reduction in animal colony size and the safe storage of germplasm from valuable strains. In addition, they allow long-term storage of these strains and easy transportation of the genetic material to other research facilities internationally. Thus far, only one laboratory has created live rat pups after sperm cryopreservation and intrauterine insemination. Another laboratory made advances in cryopreservation media that improved sperm motility post-thawing, but no pups resulted from this work. In my study, these two cryopreservation media were utilized to perform intrauterine inseminations with both fresh samples of rat sperm as well as samples that were cryopreserved in liquid nitrogen to replicate and extend these studies. Pharmacoejaculation was tested as a means to obtain spermatozoa without euthanizing the male to collect the epididymis, but results were inconsistent and the samples were not useful for intrauterine inseminations or cryopreservation. Epidiymal sperm was then collected into the various media and frozen in liquid nitrogen. In my hands, the frozen/thawed rat sperm achieved motility of less than 1%. Next, the impact of altering the freezing rate on sperm motility was evaluated. Epididymal sperm was collected and processed using a modified protocol and were then frozen at 2, 4 or 6 cm above the level of liquid nitrogen. Four to six days after freezing, samples were thawed and post-thaw sperm motility was evaluated. Sperm motility was measured prior to freezing as well as after-thawing. The sperm motility was correlated with LIVE/DEAD® staining. Sperm motility did not differ between the groups as a result of the freezing rate (Friedman test p=0.23). The published techniques are not robust and require further development to improve the motility of rat sperm after cryopreservation and achieve pregnancy via intrauterine insemination.