Bovine respiratory disease (BRD) complex is a multiactorial disease syndrome that results from various individual contributions and interactions of pathogen, host, and environmental/management factors. Despite the efforts in research, prevention and treatment, BRD remains a leading cause of economic loss in the cattle industry. While advances in therapeutics and new vaccines have been developed over the past 20 – 25 years, the incidence of respiratory disease does not appear to be on the decline, rather it is appears to be increasing. While bacterial and viral pathogens, and various stressors associated with BRD have been characterized, there are no animal models that can reproduce similar presentation of symptoms observed for BRD in the industry. Based on the etiology of BRD, a series of projects were designed to provide a better understanding of the individual and multiple contributions for the factors associated with the complex. It is believed that the viral pathogens or stressors can suppress immune defenses allowing opportunistic bacteria the ability to colonize and cause an infection. Therefore, trials investigating the individual contribution that varying doses of infectious bovine rhinotracheitis virus and transportation stress have on cattle were conducted. A final project investigating the combination effect of the bacterial pathogen M. haemolytica and activation of the hypothalamic-pituitaryrenal axis to elicit glucocorticoid release was evaluated. Ultimately, the research projects were designed to build upon each other to understand each component in the etiology of this disease.

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Establishment and maintenance of pregnancy is a central concern to the cattle industry, as it strongly impacts efficiency and profitability of beef cow-calf operations. The objective of this study was to determine if nutrient restriction impacts ovulatory follicle size and corpus luteum (CL) development and function of Bos taurus cows enrolled in estrous synchronization. A total of 26 Angus cows were housed in a facility equipped with a Calan gate system for individual animal intake. Cows were stratified by body weight (BW), and randomly assigned one of two nutritional treatments: 1) 100% of nutrient requirements (MTN; n=13) or 2) 70% of nutrient requirements (REST; n=13). Individual daily intakes were measured and adjusted weekly based on BW. Cattle underwent an acclimation period of 14 days and were exposed to nutritional treatments for 30 days prior to estrous synchronization. Body weight was measured daily using an automated scale and a conventional livestock scale at the beginning and end of the experiment. Cows were synchronized using a 7-day CO-synch + CIDR protocol beginning on day -10. Ultrasonography of the ovaries was performed at each event of the estrous synchronization protocol on days -10, -3, 0, 5, and 7. Blood samples were taken on days -10, -3, and daily from day 0 through 7 to observe changes in progesterone (P4). Data were analyzed as repeated measures using the MIXED procedure of SAS. Initial BW tended to differ between treatments (P = 0.07; MTN 597 ± 32 kg, REST 604 ± 32 kg), but MTN had greater final BW (P < 0.001; 687 ± 24 and 556 ± 27 kg, respectively) and greater average daily gain (1.35 ± 0.18 and -0.72 ± 0.21 kg/d, respectively, P < 0.001) than REST. Diameter of the largest follicle was similar (P = 0.851) between treatments at CIDR insertion (12.6 ± 0.6 mm) and CIDR removal (12.9 ± 0.4 mm) but was greater (P < 0.05) for MTN than REST cows at 60 hrs after CIDR removal (14.01 ± 0.6 and 12.37 ± 0.5 mm, respectively). Volume of CL was similar (P > 0.1) at 5 (3211 ± 113 mm3) and 7 (5280.3 ± 212 mm3) days after ovulation. Concentration of P4 did not differ on days -10, -3, or 0-5. However, on days 6 and 7, P4 was greater (P < 0.05) for MTN than REST (2.07 ± 0.15 and 1.65 ± 0.15, and 2.27 ± 0.15 and 1.83 ± 0.15 ng/mL, respectively). In conclusion, nutrient restriction to 70% of maintenance during estrous synchronization negatively affects diameter of the ovulatory follicle and circulating P4, but it did not affect CL volume in multiparous Bos taurus beef cows. / Master of Science / Ensuring successful pregnancy in beef cow-calf operations is crucial for the efficiency and profitability of the cattle industry. This study investigates the effects of nutrient restriction on ovulatory follicle size and corpus luteum (CL) volume in Angus cows undergoing estrous synchronization. A total of 26 cows were subjected to either a maintenance diet meeting 100% of nutrient requirements (MTN) or a diet providing 70% of nutrient requirements (REST). Intakes were updated weekly using computer software. The cows underwent a 30-day nutritional treatment before synchronization of ovulation. Results revealed that cows on the maintenance diet exhibited greater final body weight and average daily gain compared to those on the restricted diet. While estrus expression showed a numerical increase in MTN cows, the impact was not statistically significant. Analysis of ovulatory follicle size demonstrated that MTN cows had larger follicles 60 hours after synchronization compared to REST cows. Surprisingly, corpus luteum volume did not differ between the two groups at 5 and 7 days after ovulation. Additionally, circulating progesterone (P4) levels were affected by nutrient restriction, with notable differences observed on days 6 and 7. In summary, nutrient restriction during ovulation synchronization negatively influenced ovulatory follicle size and P4 levels, but did not affect corpus luteum volume in mature Angus cows. These findings contribute valuable insights for the cattle industry, emphasizing the importance of proper nutrition for optimal reproductive health in beef cows.

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The bovine polymorphonuclear leukocyte (neutrophil) is a central component of the acute inflammatory response, and is capable of reacting to a myriad of pro-inflammatory chemical signals that have been characterized in the context of bovine respiratory disease (BRD). Human neutrophils and bovine macrophages are known to react to pro-inflammatory signals as well; however, they are also capable of responding to anti-inflammatory signals from the autonomic nervous system. In particular, activation of the beta2-adrenergic receptor on these cells decreases several aspects of inflammatory activity, including reactive oxygen species production, chemotaxis, degranulation, and inflammatory mediator production. Dysfunction of beta-adrenergic receptors is known to contribute to the pathophysiology of numerous diseases in both people and animals. For example, congestive heart failure, asthma, cystic fibrosis, atopic dermatitis, pheochromocytoma, myasthenia gravis, hypertension, and sepsis have all been linked to decreased beta1- / beta2-adrenergic receptor density (depending on the cell type) and / or uncoupling of the respective receptor from its effector enzyme, adenylyl cyclase. Dysfunction of the beta2-adrenergic receptor mechanism has also been described in pulmonary airway and vascular smooth muscle tissue from cattle, sheep, and rats exposed to Manheimia haemolytica, which provides insight into the pathophysiology of BRD. Despite the prominent role of the bovine neutrophil in the acute inflammatory stage of BRD, and despite the potential for dysfunction following excessive exposure to inflammatory stimuli, there are no reports that describe the presence of the beta2-adrenergic receptor on bovine neutrophils, nor function of the components responsible for its signal transduction cascade. Without complimentary work with bovine neutrophils, using data from human neutrophils to examine treatment options for the acute inflammatory stage of BRD is unrealistic. For this reason, the present dissertation proposed that 1) bovine neutrophils possess the beta2-adrenergic receptor mechanism, 2) components of the beta2-adrenergic receptor mechanism work in concert to increase bovine neutrophil adenosine 3,5-cyclic monophosphate (cAMP) levels and suppress superoxide anion production, and 3) the beta2-adrenergic receptor mechanism is dysfunctional following exposure to inflammatory stimuli. Using the nonselective beta1- / beta2-adrenergic receptor antagonist [3H]CGP-12177 we observed a maximum specific binding density (Bmax) value of 0.19 fmol per 100,000 bovine neutrophils. Although this value is approximately equal to what we observed with dairy cow neutrophils, human neutrophil Bmax values with this radioligand are anywhere from five to ten-fold greater, which suggests a significant species difference. We further defined the adrenergic receptor population on bovine neutrophils to be dominated by the beta2-subtype. Next, we characterized the function of beta2-adrenergic receptors by stimulating cAMP production with the beta2-adrenergic receptor agonist, terbutaline. The role of the beta2-subtype was confirmed when the terbutaline-mediated effect was negated by ICI-118,551, a beta2-adrenergic receptor antagonist. Also, the role of the phosphodiesterase enzyme in cAMP recycling in bovine neutrophils was illustrated, as the terbutaline-mediated rise in cAMP concentration was dependent upon phosphodiesterase inhibition by 3-isobutyl-1-methylxanthine (IBMX). This study confirms the anti-inflammatory nature of the beta2-adrenergic receptor on bovine neutrophils by demonstrating the ability of terbutaline and IBMX to decrease superoxide anion production in a dose-dependent manner. The synthetic cAMP analog, 8-bromo-cAMP also decreased superoxide anion production, but the effect was time-dependent because of its need to diffuse across the cell membrane. Moreover, IBMX exaggerated the terbutaline-mediated effect on superoxide anion production, while cAMP exaggerated the IBMX-mediated effect on superoxide anion, demonstrating that the beta2-adrenergic receptor acts in concert with adenylyl cyclase, while the phosphodiesterase enzyme functions to decrease their signal. By increasing the dose of the inflammatory stimulant opsonized zymosan eight-fold, we were able to eliminate the ability of various concentrations of terbutaline and IBMX to reduce superoxide anion production. We sought to provide a more specific demonstration of this phenomenon by activating protein kinase C (PKC) via phorbol 12-myristate 13-acetate (PMA) administration. However, preincubation with PMA actually increased terbutaline-mediated cAMP production, in a dose and time-dependent manner. At this time, we cannot explain why increasing the dose of opsonized zymosan and PMA had opposite effects on beta2-adrenergic receptor mechanism function. The answer may reside in the many reported functions of PKC isoforms. Additional studies that identify the PKC isoform repertoire in bovine neutrophils may illustrate the potential for selective inhibition, and may lead to more specific identification and treatment of beta2-adrenergic receptor mechanism dysfunction. Also, it remains to be seen how the various components of the bovine neutrophil beta2-adrenergic receptor mechanism function in-vivo during the acute inflammatory stage of BRD. / Ph. D.

Consumer demand for milk fat has declined due to the increased risk of cardiovascular disease associated with consuming a high saturated fat diet. Milk fat synthesis is energetically expensive for the dairy cow, especially during early lactation or periods of poor nutrition. Thus, manipulating milk fat production and composition may promote the synthesis of more market-valuable milk components and improve energy utilization in dairy cows during periods of increased energy demand. Therefore, the objective of the present studies was to identify molecular proteins that regulate fatty acid synthesis in bovine mammary epithelial cells. The regulation of lipogenic genes including acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) is controlled by transcription factors including sterol regulatory element binding protein-1 (SREBP1) and liver X receptor (LXR). In vivo, diet-induced milk fat depression or supplementing diets with polyunsaturated fatty acids inhibits milk fat synthesis by regulating SREBP1 expression. Results confirm that polyunsaturated fatty acids inhibit fatty acid synthesis in bovine mammary epithelial cells by regulating the expression of SREBP1. In hepatocytes, LXR can regulate the transcription of SREBP1 in addition to ACC and FAS. Results confirm that LXR activation enhanced synthesis of fatty acids in bovine mammary epithelial cells by promoting the transcription of FAS and SREBP1. Activation of LXR was unable to prevent the inhibitory effect of polyunsaturated fatty acids on fatty acid synthesis. In the lactating mammary gland, LXR may contribute to the synthesis of fatty acids by regulating the expression of SREBP1. In addition to modifying the expression of lipogenic genes, some enzymes can be phosphorylated by AMP-activated protein kinase (AMPK), an energy-sensing protein, inhibiting their activity. Presence of AMPK mRNA was identified in bovine mammary epithelial cells and activation of AMPK dramatically decreased fatty acid synthesis in bovine mammary epithelial cells. In the lactating mammary gland, AMPK may sense energy availability and regulate milk fat synthesis to control energy utilization. Identification of SREBP1, LXR, and AMPK as regulators of fatty acid synthesis in bovine mammary epithelial cells may lead to the development of technologies allowing dairy producers to modify milk fat production and composition to meet consumer demand and maximize profitability. / Ph. D.

Three studies were conducted to assess if the ability of Enterococcus spp. surviving the ensiling process and bovine gastrointestinal tract could impact risk of bovine mastitis. The first study determined ability of enterococci to survive 3 wk ensiling. Grass and corn crops were divided into 3 treatments: 2 commercial silage inoculants, 1 negative control. After wk 1, 2, and 3 of ensiling, dry matter and bacterial enumeration were performed. Addition of silage inoculant led to greater levels of enterococci in grass silage compared with negative control levels, but showed less difference in inoculated corn silage. The second study quantified enterococci shedding rates in lactating dairy cows. Using a 4 x 4 Latin Square design, lactating, ruminally fistulated Holsteins were inoculated with enterococcal isolates from silage inoculants, ensiled forages, or clinical mastitis cases. Over the 4-period study, each period consisted of rumen and fecal sampling (2 wk) followed by a wash period (10 d). There were no significant differences in rumen or fecal enterococci levels between the 4 treatments. Both rumen and fecal enterococci levels showed significant differences between baseline and treatment periods (period 3, 4). The third study analyzed similarity in enterococcal isolates of silage and bovine origin using pulsed-field gel electrophoresis patterns from SmaI restrictions. Dendogram analysis showed none of the isolates met or were greater than a 75% genetic similarity and produced a genetically diverse population. Results suggest Enterococcus spp. from silage inoculants are not likely to contribute to an increased risk of enterococcal bovine mastitis. / Master of Science

The objective of this study was to examine the effects of presynchronization and recombinant bovine somatotropin (rbST) on conception rates following a timed insemination (TAI) protocol in lactating dairy cows. A further objective included the evaluation of the efficacy of the Early Conception Factor (ECF) test kit. Recombinant bST may offer some benefit when used in conjunction with estrus synchronization and TAI. Presynchronization treatment consisted of two injections of PGF2α given 14 d apart, with the second dose administered 14 d prior to the initiation of a TAI protocol. A total of 216 lactating Holstein cows were presynchronized with PGF2α and then received GnRH (100μg) at 67 ± 7 d post partum (PP), administration of PGF2α (25 mg) 7 d later, another GnRH (100μg) administration 2 d after PGF2α, and were inseminated 8-18h later (OvSynch). First service conception rate (CR) was determined by rectal palpation at 42 ± 7 d after artificial insemination (AI). Treated cows (n=113) received rbST 67 ± 7 d PP whereas control cows (n = 113) were presynchronized without rbST. The cycling status of all cows was determined by paired milk P4 levels at 53 and 67 ± 7 d PP. No differences (P > 0.10) in conception rate were observed between control and rbST treated cows (44.7 and 40.7% respectively), nor was there any interaction of cyclicity and rbST. Milk samples were collected 7 d following AI for use in ECF test kit evaluation. Samples were stored at -20ºC (n=216) and at 5ºC (n=113) until assayed. Test results for frozen and refrigerated samples were compared to conception rates determined by rectal palpation at 42 ± 7 d after AI. The rate of false positive and negative results for frozen milk samples were 36.1 and 14.8% respectively, and 40.7 and 7.1% for refrigerated milk samples. Treatment with rbST at the time of the first GnRH injection of an OvSynch protocol did not significantly alter first service conception rates. Additionally, an acceptable 92.9% accuracy of the ECF test for the detection of open cows 7 d after AI using milk samples stored at 5ºC was obtained. / Master of Science

The liver is one of the most frequently injured abdominal organs in motor vehicle collisions. Although previous studies have quantified the tensile failure properties of human liver parenchyma at 48hrs postmortem, it is currently unknown how the material properties change between time of death and 48hrs postmortem. Therefore, the purpose of this study was to quantify the effects of postmortem degradation on the tensile material properties of bovine liver parenchyma when stored in DMEM or saline. Fourteen fresh bovine livers were obtained from a local slaughter house and stored in either DMEM or saline as large blocks, small blocks, or slices of tissue. Multiple parenchyma dog-bone samples from each liver were tested once to failure at three time points: ~6hrs, ~24hrs, and ~48hrs postmortem. The data were then analyzed to determine if there were significant changes in the material properties with respect to postmortem time. The results showed that the failure strain decreased significantly between 6hrs and 48hrs after death when stored as large blocks in saline. Conversely, neither the failure stress nor failure strain changed significantly with respect to postmortem time when stored as large blocks in DMEM. The modulus did not significantly change for tissue stored as large blocks in either fluid. Preliminary results indicated that reducing the tissue storage size had a negative effect on the material properties and cellular architecture. Overall, this study illustrated that the effects of postmortem liver degradation varied with respect to the preservation fluid, storage time, and storage block size. / MS / Although the liver is one of the most frequently injured abdominal organs in motor vehicle collisions (MVCs), currently accepted anthropomorphic test devices are unable to predict abdominal organ injury risk. Consequently, finite element models are becoming an important tool for assessing abdominal organ injury risk in MVCs. However, these models must be validated based on biomechanical data in order to accurately assess injury risk. Given that previous studies that have quantified the tensile failure properties of human liver parenchyma have been limited to testing at 48hrs postmortem, it is currently unknown how the material properties change between time of death and 48hrs postmortem. Therefore, the purpose of this study was to quantify the effects of postmortem degradation on the tensile material properties of bovine liver parenchyma with increasing postmortem time when stored in DMEM or saline. A total of 148 uniaxial tension tests were successfully conducted on parenchyma samples of fourteen bovine livers acquired immediately after death. Tissue was immersed in DMEM or saline and kept cool during preparation and storage. Twelve livers were stored as large blocks of tissue, while two livers were stored as small blocks and slices. Multiple dog-bone samples from each liver were tested once to failure at three time points: ~6hrs, ~24hrs, and ~48hrs after death. The data were then analyzed using a Linear Mixed Effect Model to determine if there were significant changes in the failure stress, failure strain, and modulus with respect to postmortem time. The results of the current study showed that the failure strain of bovine liver parenchyma decreased significantly between 6hrs and 48hrs after death when stored as large blocks in saline and refrigerated. Conversely, neither the failure stress nor failure strain changed significantly with respect to postmortem time when stored as large blocks in DMEM. The modulus did not significantly change for tissue stored as large blocks in either saline or DMEM. In addition, preliminary results indicated that reducing the tissue storage size had a negative effect on the material properties and cellular architecture. Overall, this study illustrated that the effects of postmortem liver degradation varied with respect to the preservation fluid, storage time, and storage block size.