Search

Genome-wide association studies (GWAS) seek to identify single nucleotide polymorphisms (SNP) causing phenotypic responses in individuals. Commonly, GWAS analyses are done by using single marker association testing (SMA) which investigates the effect of a single SNP at a time and selects a candidate set of SNPs using a strict multiple correction penalty. As SNPs are not independent but instead strongly correlated, SMA methods lead to such high false discovery rates (FDR) that the results are difficult to use by wet lab scientists. To address this, this dissertation proposes three different novel Bayesian methods: BICOSS, BGWAS, and IEB. From a Bayesian modeling point of view, SNP search can be seen as a variable selection problem in linear mixed models (LMMs) where $p$ is much larger than $n$. To deal with the $p>>n$ issue, our three proposed methods use novel Bayesian approaches based on two steps: a screening step and a model selection step. To control false discoveries, we link the screening and model selection steps through a common probability of a null SNP. To deal with model selection, we propose novel priors that are extensions for LMMs of nonlocal priors, Zellner-g prior, unit Information prior, and Zellner-Siow prior. For each method, extensive simulation studies and case studies show that these methods improve the recall of true causal SNPs and, more importantly, drastically decrease FDR. Because our Bayesian methods provide more focused and precise results, they may speed up discovery of important SNPs and significantly contribute to scientific progress in the areas of biology, agricultural productivity, and human health. Doctor of Philosophy Genome-wide association studies (GWAS) seek to identify locations in DNA known as single nucleotide polymorphisms (SNPs) that are the underlying cause of observable traits such as height or breast cancer. Commonly, GWAS analyses are performed by investigating each SNP individually and seeing which SNPs are highly correlated with the response. However, as the SNPs themselves are highly correlated, investigating each one individually leads to a high number of false positives. To address this, this dissertation proposes three different advanced statistical methods: BICOSS, BGWAS, and IEB. Through extensive simulations, our methods are shown to not only drastically reduce the number of falsely detected SNPs but also increase the detection rate of true causal SNPs. Because our novel methods provide more focused and precise results, they may speed up discovery of important SNPs and significantly contribute to scientific progress in the areas of biology, agricultural productivity, and human health.

High levels of fecal indicator bacteria (e.g. E. coli) are the leading cause of identified surface water impairments in the United States. The US Clean Water Act of 1972 requires that jurisdictions establish priority rankings for impaired waterways and develop a Total Maximum Daily Load (TMDL) plan for each. Although past research indicates that the risk of illness to humans varies by source of fecal contamination, current watershed assessments are developed according to total concentration of indicator bacteria, with all sources weighed equally. A stochastic model using Quantitative Microbial Risk assessment (QMRA) principles to translate source-specific (e.g. human, livestock) daily average concentrations of E.coli into a daily average risk of gastroenteritis infection was developed and applied to Pigg River, an impaired watershed in southern Virginia. Exposure was calculated by multiplying a ratio of source related reference pathogens to predicted concentrations of E.coli and a series of qualifying scalars. Risk of infection was then determined using appropriate dose response relationships. Overall, human and goose sources resulted in the greatest human health risk, despite larger overall E.coli loading associated with cattle. Bacterial load reductions specified in the Pigg River TMDL were applied using Hydrological Simulation Program- FORTRAN (HSPF) to assess the effect these reductions would have on the risk of infection attributed to each modeled bacterial source. Although individual risk sources (neglecting geese) were reduced below the EPA limit of 8 illnesses per 1000 exposures, the combined risk of illness varied between 0.006 and 64 illnesses per 1000 exposures. Master of Science

Prescribed burning (Rx) has become increasingly subjected to various regulations. Among these regulations are ordinances that restrict downwind impacts of smoke from prescribed fires. Regulations can severely limit burn managers and private landowners from using Rx as a forest management tool. This research can help us move away from these simplistic regulations, and help promote a regulatory environment in which scientific tools and knowledge are used to prohibit only activities for which the evidence suggests there will be adverse consequences. This research was divided into three parts that consisted of: (1) a pilot validation of the smoke emission model VSMOKE-GIS; (2) review of southeastern states' smoke management guidelines (SMG); and (3) a geographic analysis of Virginian's current SMG. VSMOKE-GIS showed good accuracy in predicting the PM2.5 concentration and location of the smoke plume downwind. Criteria were identified when managing Rx smoke and the strengths, weaknesses, and implications were discussed of the Rx programs. The geographic analysis demonstrated quantitatively how much area may be impacted with minimal apparent benefit. This research should provide a clearer spatial picture of the smoke management barriers associated with Rx on private woodlands in Virginia. These results should be a useful tool in developing a regulatory environment that encourages Rx when the conditions are optimal. We conclude with future recommendations for Virginia. Master of Science

For decades, the pavement engineering community has continued to drive improvements in accuracy and repeatability of terrain measurement systems. Traditional terrain measurement systems are tailored for a measuring a specific scale and resolution and hence application scope. These systems tend to focus on surface roughness alone and reject either fine macrotexture or large-scale global features. This work proposes a novel improvement to the terrain measurement system, by increasing the capability to measure the terrain surface at a variety of scales. By increasing the scales of measurement, desired aspects of the terrain profile can be accurately obtained for a wide variety of applications without having to omit large-scale features or macrotexture. In addition to increasing the capabilities of the traditional terrain measurement system, methods for addressing and minimizing sources of error within the system are developed. Major sources of error in terrain measurement systems, which compromise the accuracy and repeatability of the resulting measured terrain, include scanning laser uncertainty, inertial navigation system (INS) uncertainty and drift, triggering and time synchronization, system misalignment, and post-processing errors. These errors are addressed, and an improved Vehicle Terrain Measurement System (VTMS) is proposed. A triggering and time synchronization system is developed and insight into the development of this system for a terrain measurement system is gained. All three scanning lasers are individually assessed for linearity, with sample profiles analyzed for agreement. The improved VTMS represents a significant development in terrain measurement systems. Master of Science

This dissertation details the development of a novel reduced size axial mode helical antenna called the Stub Loaded Helix (SLH). The SLH achieves a significant reduction in helix size, both diameter and length, compared to the conventional axial mode helix antenna with only small compromises in performance. The SLH achieves this entirely through the use of a unique geometry. The performance characteristics of the SLH are explored through the use of computer simulations using NEC (Numerical Electromagnetics Code) to study the effects of design parameter variations. Based on those simulations, design guides are developed. The numerical simulations are verified though measurements of experimental prototypes. The program of experimental prototypes included the development of an appropriate impedance matching network for the SLH, which is also detailed. Ph. D.

The widespread contamination of our nation's groundwater has become a major threat to public health. The magnitude of this threat is escalated by our reliance upon groundwater. Groundwater is the primary drinking water source for over 50% of the U.S. population. Groundwater comprises more than 95% of all available freshwater in the United States. The objectives of this study were: (1) to make a preliminary assessment of the use of inhibitors to evaluate the biodegradation potential of soil, and (2) to evaluate the significance of each microbial group in the biodegradation process. Static microcosms containing Newport News and Blacksburg soil were used as the experimental environments. Butyrate and propionate were used as substrates. The metabolic inhibitors used were sodium molybdate (Na.7N1o04) and 2-Bromoethane sulfonic acid (BESA), which inhibit sulfate reduction and methanogenesis, respectively. Denitrification was suppressed by the absence Of sodium nitrate (NaNO3) in the microcosm dilution water. Both Newport News and Blacksburg soils were found to be dominated by denitrfying bacteria. In nitrate-amended Newport News microcosms, 300 mg/L butyrate degraded to .zero concentration in 5.5 days. This microcosm's degradation "rate" was 255% greater than non-nitrate amended microcosms, where sulfate reduction was predominant. Little or no methanogenic activity was observed in both the Newport News or Blacksburg soils. Advantages of this approach are its simplicity and directness in obtaining approximately the same information as more complicated enumeration procedures. A disadvantage of this approach is its reliance on metabolic inhibitors to suppress the microbial activity of specific microbial groups. Little is known on the effect of Na2MoO4 or BESA on the activities of denitrifiers, fermenters, or acetogens. Master of Science

There are thousands of species of mosquitoes, but only a handful of these species carry pathogens that cause human diseases. Here, we study two species, Aedes albopictus and Aedes aegypti, which transmit infections such as dengue, Zika, Mayaro virus, and La Crosse virus. Curtailing these diseases is a good reason to consider control of mosquito populations. However, mosquitoes are quite hardy and spraying of pesticides is typically a short-term solution. Thus, more long-term solutions require careful thought about mosquito populations, including early juvenile aquatic stages: egg, larva, and pupa. In this dissertation, we examine the factors that affect the dynamics of aquatic stages by creating mathematical models. The goal is to assess what key biological features most impact the total population. Both Aedes albopictus and Aedes aegypti lay eggs in small containers, producing limitations on space and food. We investigate how restricting resources changes development time, survival to adulthood, and body mass at emergence. The interactions between these changes are complicated, so to disentangle their effects we create three different mathematical models. The first model is discrete in time and focuses on the best way to incorporate the influence of larval density. We compare the impact of larval density by inputting seven different functional forms altering survival and development time. Larval density used in the model is determined from the average of the population size over the past one to thirty-six days. The second model is also discrete in time but focuses on the interaction between survival, development time, and mass. This model considers three levels of mass. Here, we use the density-dependent function determined from our first model and fit the maximum value for development time from experimental data. Survival values are fit using constants and a density-dependent function. Finally, growth is built in as a function of food. Food decreases at each time point as a function of the total larvae in the environment. We compare between model formulations with Akaike information criterion. The third model examines the ramifications of constricting resources on growth verses death. We employ a partial differential equation that has three independent variables: time, age, and mass. We find that density dependence is highly influential in the maturation of mosquitoes, and it is more essential to include its impacts on development time than on survival. These findings can be incorporated into a larger framework of disease dynamics, and give insight into better control of mosquitoes and disease spread. Doctor of Philosophy There are thousands of species of mosquitoes, but only a handful of these species spread human diseases. We specifically study mosquitoes that transmit diseases such as dengue and Zika, which plague large portions of the world. One way to reduce disease spread by mosquitoes is to eliminate the mosquito population, but mosquitoes are becoming resistant to commonly used insecticides. Thus, additional ways to combat mosquitoes are needed. To do so requires a better understanding of how mosquito populations change. This necessitates incorporating information from all life stages of the mosquito, including the juvenile stages that live in the water. In this dissertation, we examine various factors that affect the population size and characteristics of young mosquitoes by creating mathematical models. The goal is to assess what key biological features most impact the population. As young mosquitoes live in aquatic environments, there are limitations on space and food. We investigate how restricting resources changes the time it takes for mosquitoes to develop into adults, how many survive to adulthood, and what their body mass is as new adults. These outcomes interact in complicated way. To disentangle this, we create three different mathematical models. The first model focuses on the best way to incorporate the influence of limiting space or density on population size. The second model focuses on the interaction between mosquito body mass, survival, and time spent as a juvenile. The third model examines how resources alter growth and death and the ensuing mass of mosquitoes. Models are fit to experimental data and validated based on how accurately they describe known patterns. We find that population density is highly influential in the maturation of juvenile mosquitoes and its most important effect is on time spent as a juvenile mosquito. By correlating growth of juvenile mosquitoes to density and resources, our second model is better able to reproduce data. These findings provide important understanding on mosquito populations, which provides insight into how to better control mosquitoes and the diseases they spread.

Intramammary infections (IMI) are common in non-lactating heifer and dry cow mammary glands and occur during periods of appreciable mammary growth and development. The presence of these infections is expected to negatively impact mammary growth and development but has yet to be investigated. The works reported here investigated how IMI affects mammary tissue structure, cellularity, and the expression of integral mammogenic hormone receptors implicated in mammary growth. Non-pregnant non-lactating cows (n = 19) were administered estradiol and progesterone to stimulate mammary growth and 2 quarters of each cow were subsequently infused with either saline (n = 19) or Staphylococcus aureus (n = 19). Intramammary infusion of Staphylococcus aureus increased the number of immune cells present in gland secretions and also increased the proportion of neutrophils comprising these secretion somatic cells. Mammary tissues from quarters infused with Staphylococcus aureus contained more immune cells, less mammary epithelial tissue area, and greater tissue areas of intralobular stromal tissue than saline quarters. Staphylococcus aureus quarters also contained more apoptotic mammary epithelial cells and a lower proportion of apoptotic cells in the intralobular stroma compartment than saline infused quarters; this signified that Staphylococcus aureus quarters had less epithelial growth and experienced an expansion and/or lack of regression of stromal tissues. The number of cells expressing estrogen receptor α (ESR1) and progesterone receptor (PGR), as well as staining characteristics of ESR1 and PGR positive nuclei was also examined in these tissues. No appreciable differences were observed in any of the examined ESR1 and PGR measures between Staphylococcus aureus and saline mammary glands, but myoepithelial cells from Staphylococcus aureus glands had a greater nuclear staining area than saline quarters, indicating that these cells were affected by IMI. The results of these investigations indicate that IMI, in mammary glands that are concurrently stimulated to grow and develop, limits the growth of mammary epithelium and impairs regression of the stromal tissue, both of which are necessary for successful lactational performance. PHD Successful growth and development of the dairy cow udder (mammary gland) is important and has long-term impacts on milk production. Most mammary growth occurs during the first pregnancy but, at this same time, a bacterial infection can be present within the mammary gland and is expected to hinder normal growth and development. The studies conducted here sought to examine how a bacterial infection, within a cow’s udder, affects mammary gland growth and development. Overall, it was observed that a bacterial infection in the mammary gland reduced the amount of functional tissue that would eventually produce milk while simultaneously increasing the amount of connective tissue. Infected mammary glands also had a greater number of dying mammary cells, reducing the number of cells that would eventually produce milk. Estrogen and progesterone are known to be integral in supporting mammary growth, so an examination of the number of cells being able to receive signals from estrogen and progesterone was also undertaken; presence of an infection did not alter the number of cells able to receive estrogen and progesterone’s signal. This work furthered our understanding of how bacterial infections affect mammary tissue and alter normal developmental processes.

Increasing the milking frequency of early lactation dairy cows increases their milk yield, both during increased milking and after the cows are returned to a normal twice-daily milking schedule. When milked four times a day (4X) for only the first twenty-one days of lactation, the right udder half produces 3 kg/d more milk than the left half, which is milked only two times a day (2X) over the course of lactation (Hale et al., 2003). Alterations to this increased milking frequency (IMF) approach have been investigated in order to maximize production of the animals and determine the most efficient practice for producers. The aim of this study was to determine the appropriate duration of early lactation IMF treatment by increasing milking frequency of early lactation cows for various lengths of time, and subsequently increasing the use of this management practice on Virginia dairy farms. The right udder half of twenty-three primiparous and multiparous Holstein cows were milked 4X for 10, 20, or 40 days at the beginning of lactation, and the left udder half 2X for the entire lactation. Udder-half milk yields were measured at various time points throughout lactation and used to calculate the difference between right (4X) and left (2X) udder halves. Overall, treatment did not have a significant effect on milk yield difference throughout the entire lactation; the udder half differences for each group were -0.45 kg, 1.92 kg and 4.62 kg for the 10 d, 20 d and 40 d treatments (P > 0.05). In addition to the IMF portion of the experiment, two different methodologies were used to investigate the possible mechanism of local regulation of milk yield in response to IMF treatment. Milk fatty acid analysis was performed on milk samples obtained from the above experiment. Three different groups of fatty acids were analyzed to detect potential changes in the right udder half (4X) when compared to the left (2X); the three groups were denovo, C16, and preformed fatty acids. There was no significant effect of treatment on fatty acid composition of right and left udder halves for any of the three groups (P > 0.05). The second methodology used to explore a possible mechanism behind increased milk yield following IMF treatment was immunohistochemistry of mammary gland tissue samples obtained after IMF treatment in a previous experiment. The key target investigated was a component of the Hippo signaling pathway, Yes-associated protein (YAP). Intensity of YAP staining in the cytoplasmic area of mammary epithelial cells (MEC) and number of YAP-positive stained nuclei located in the MEC were quantified for each of the images obtained. There was no effect of treatment or day on intensity of staining (P > 0.05) with no difference in the intensity of staining between 4X and 2X samples or d 21 and d 60 samples. However, the interaction for treatment x day tended to be significant (P < 0.06), with the d 60 samples tending to have higher intensity of staining than d 21 samples. For YAP-positive nuclei, there was a significant effect of day (P < 0.05), with d 60 samples having significantly more YAP positive nuclei. There was not a significant effect of treatment or treatment x day interaction (P > 0.05) with 2X and 4X samples having the same number of YAP positive nuclei. Results from the first experiment reveal that 40 d of IMF during early lactation is sufficient to produce an increase in milk and component yields throughout lactation. This practice could be implemented on Virginia dairy farms as a way to increase efficiency and milk yield per cow. Results from the second half of this research indicates that further research is needed to investigate the fatty acid content of milk from cows subject to IMF treatment during early lactation. In addition, YAP potentially plays a role in the changes occurring in the mammary gland, with increased intensity of YAP staining and increased number of YAP positive nuclei observed at 60 DIM. Understanding of this protein and its involvement in the mammary gland could lead to identifying a mechanism for which this increase in milk yield and components following IMF is occurring. Further research needs to be done to provide results supporting the current experiment. MSLFS

Most mammary development is postnatal. Mammary growth that occurs before puberty is diminutive in amount but consequential for future milk production, especially in dairy heifers. With advanced knowledge on fundamental aspects that govern prepubertal mammary development, scientists and farmers alike can ensure that heifers perform their best once they become cows. The Hippo pathway has been identified as an evolutionarily conserved pathway that regulates organ size in many animal species; it might contribute to mammary growth in dairy heifers. This pathway is mediated by yes-associated protein (YAP) and through downstream gene transcription activation, results in cell proliferation. Because YAP has never been identified in bovine mammary tissue, questions examined in this body of work mainly focused on the abundance and localization of YAP in mammary tissue of prepubertal heifers. The first trial investigated effects of in vivo estradiol administration on YAP abundance and localization in prepubertal bovine mammary epithelial and myoepithelial cells. While YAP was present in nuclei and cytoplasm of both cell types, it was also discovered that estrogen did not influence YAP abundance or location. The second research trial focused on determining the effects of in vivo estradiol blockade on YAP abundance and localization in prepubertal bovine mammary epithelial and myoepithelial cells. Similar to the first experiment, results indicate that YAP abundance and localization was not influenced by estrogen blockade. Despite not being responsive to in vivo estradiol administration (experiment 1) or estradiol blockade (experiment 2) under the conditions of our experiments, YAP was present in nearly all mammary epithelial cells and myoepithelial cells of the 21 total prepubertal heifers examined. Its presence hints at an underlying biological function but that function was not ascertained here. It will be up to the next researcher to deduce what YAP contributes to mammary growth in prepubertal dairy heifers. MSLFS

Our collective knowledge of calf nutrition has evolved over the past 100+ years, but there are still areas of improvement that merit further scientific inquiry. The work described herein explored different aspects of calf nutrition with a central focus on rumen growth and development. The first study performed used 8 Holstein bull calves to determine if calf starters differing in starch and neutral detergent fiber (NDF) content would affect calf growth, intake, rumen metabolites, blood metabolites, and gross rumen measurements when fed along with milk replacer (MR). The experiment used completely pelleted calf starters consisting of ground and pelleted barley, wheat, and corn grains. Besides the high-starch starter resulting in lower rumen pH, the hypothesis that completely pelleted calf starter diets differing in NDF and starch level would alter intake, growth, rumen metabolism, and rumen measurements was not supported. However, calves fed the high-NDF starter were $5.71 less expensive per calf to raise. Findings suggest a form of feed effect in today's calf starter diets that might be of physiological and economic importance. The second study tested custom-built rumen infusion, sampling, and evacuation devices. The main objectives were to build and confirm the successful use of the devices in one Holstein bull calf at 62 days of age, which determined a liquid passage rate out of the rumen at 40.2% of ruminal fluid/h. The third and final study examined the effects of form of diet (MR only, n = 5; MR and starter, n = 6) on rumen growth and development. More specifically, isocaloric and isonitrogenous diets were fed to neonatal and ruminally cannulated Holstein calves for 6 week. The hypothesis of MR and starter calves having altered gross rumen measurements, epithelial stem and progenitor cell number, and epithelial proliferation status was supported, but hypothesized changes in volatile fatty acid (VFA) transporter abundance and VFA absorption rate were not supported. These results indicate that form of diet, even one that promotes rumen growth, does not equate to enhanced ability to absorb VFA, but there is an effect on rumen stem and progenitor cells as well as epithelial proliferation. PHD

The overall objective of this dissertation project was to identify factors and mechanisms that control bovine myoblast proliferation, differentiation, and fusion. Three studies were conducted during this project. The objective of the first study was to determine the effect of oxytocin (OXT) on myoblast proliferation, differentiation and fusion. Treating primary bovine myoblasts in culture with 10 nM and 100 nM OXT for 24 h increased their proliferation rate by 7% (P < 0.05) and 10% (P < 0.05), respectively. Treating bovine myoblasts with either concentration of OXT for 48 h had no effect on their differentiation and fusion, as indicated by no changes in mRNA expression of selected myoblast differentiation markers and fusion index. The objective of the second study was to determine the effects of arachidonic acid (AA) and its major metabolites prostaglandin E2 (PGE2), PGF2a, and PGI2 on myoblast proliferation, differentiation and fusion. Treating myoblasts with 10 μM AA, 1 μM PGE2, 1 μM PGF2α, and 1 μM PGI2 for 24 h each increased the number of proliferating cells by 13%, 24%, 16%, and 16%, respectively, compared to the control (P < 0.05). At the same concentrations, AA, PGE2, and PGF2a stimulated myoblast differentiation and PGE2 improved myoblast fusion (P < 0.05). Treating myoblasts with AA and the cyclooxygenase (COX)-1 and COX-2 inhibitor indomethacin or the COX-2-specific inhibitor NS-398 reversed the stimulatory effect of AA on myoblast proliferation (P < 0.05). The objective of the third study was to determine the role of the proteasome in bovine myoblast differentiation and fusion. It was found that the proteasome activity increased (P < 0.05) during myoblast differentiation and fusion. Adding 5 μM lactacystin, a specific inhibitor of the proteasome, to the differentiation medium nearly completely blocked myoblast differentiation and fusion. Inhibitor of DNA-binding 1 (ID1) is known to inhibit myoblast differentiation and to be degraded by the proteasome in some cells. Both ID1 protein and mRNA expression were found to decrease during myoblast differentiation and fusion, and the decrease in ID1 protein but not ID1 mRNA was reversed (P < 0.05) by treating the cells with lactacystin. In summary, this project reveals that OXT and AA are stimulators of bovine myoblast proliferation and that AA is a stimulator of bovine myoblast differentiation. This project also indicates that the proteasome plays a positive role in bovine myoblast differentiation and fusion, and that it does so perhaps by reducing the accumulation of the ID1 protein. Ph. D.

The majority of bovine mammary gland research focuses on the main cell types - mammary epithelial cells and fibroblasts. However, the extracellular matrix (ECM) within the mammary gland is also of importance for its ability to regulate cell shape, proliferation, polarity, differentiation, gene transcription, protein synthesis, and secretion. Decorin is an ECM proteoglycan known to impact mammary cell proliferation in humans and rodents. Prior to this work, very little was known about decorin in bovine mammary biology. A series of bovine mammary cell culture experiments was conducted. The first experiment demonstrated existence of decorin pathway molecules in immortalized bovine mammary cells, but stopped short of demonstrating mature decorin proteoglycan deposition into the extracellular space. During the investigation it was noted that when cultured under basal conditions, intracellular decorin core protein (DCP) localization patterns appeared to be coordinated with specific phases of the cell cycle. Therefore, the objective of the second set of experiments was to characterize DCP localization patterns in bovine mammary epithelial cells (BME) at known phases of the cell cycle. The work was carried out in two sequential experiments. The hypothesis of the first experiment was that DCP accumulates in BME during S-phase of the cell cycle; the research rejected this hypothesis. The hypothesis of the second experiment, formulated after completion of the first experiment for this objective, was that DCP accumulates in BME during metaphase of the cell cycle. However, the experiment was unable to confirm of reject this hypothesis. Major findings were that both BME and mammary fibroblasts produce DCP and known decorin pathway molecules. BME produce intracellular DCP, but it is not accumulated during the S-phase of the cell cycle. However, it is still unknown if DCP is accumulated in BME during metaphase. Future research should focus on further characterization of decorin and its associated pathway molecules to learn if decorin induces proliferation or apoptosis of bovine mammary epithelial cells. This is important because number and activity of mammary epithelial cells ultimately determine milk yield in dairy cows. Fundamental knowledge gained in this research area may one day be applied at the animal-level and lead to gains in milk production efficiency by altering the cellular composition of mammary glands. Ph. D.

Biological functions that use amino acids (AA) are limited by AA supply. This concept was likened to staves in a barrel, where the shortest stave determines the barrel's ability to hold water (Mitchell and Block, 1946). Inaccuracies in models that predict nutrient supply and requirements of dairy cows result in inefficient feeding, as under-prediction of requirements results in deficiency, and over-prediction results in excess. To avoid limitations in production due to AA deficiencies, protein is fed in quantities that likely exceed requirements. Overfeeding of AA results in increased expenses for producers and increased N excretion to the environment, providing economic and environmental incentives to increase N-efficiency. Work presented in the following chapters evaluated the impact of AA supplementation on milk production in dairy cattle, and evaluated the PREP10 and NRC (2001) nutrient requirement model predictions. In two feeding trials (Chapter 2 and Chapter 3), low protein diets did not result in decreased milk production, indicating that protein requirements were overestimated. Although supplementation of AA did not increase milk production, low protein diets resulted in greater N-efficiency, especially when supplemented with Histidine. Evaluation of the PREP10 and NRC (2001) models (Chapter 4) used production data from the literature to identify deficiencies in prediction equations, and found that correction of model bias would considerably reduce prediction errors. Model inaccuracies affect the inefficiency of dairy cow feeding, and must be evaluated to improve feed efficiency. Such improvements could act synergistically with AA supplementation to more closely match nutrient supply to requirements. Master of Science

Staphylococcus aureus (S. aureus) is a gram-positive bacterium that causes mastitis in bovines and leads to financial losses to the dairy industry. Although antibody response plays a role in immune defense against S. aureus, cellular responses are of interest for vaccine development. A vaccine that stimulates both antibody and cellular responses could promote memory cell formation and provide effective protection against S. aureus. The superantigens and virulence factors secreted by live S. aureus (LSA) can interfere with immune responses and memory cell formation. Because irradiation reduces the metabolic activity and secretion of proteins, including S. aureus superantigens and hemolysins, we hypothesized the irradiated S. aureus (ISA) could drive immune cell responses. Dendritic cells (DC) were co-cultured with lymphocytes to study the cellular responses to ISA and LSA. Dendritic cells present antigens and polarize lymphocytes into different helper T (Th) cell types that drive cellular immune responses. The DC loaded with either ISA or LSA induced increased mRNA transcription of Th17-related cytokines and cytotoxic effector memory cell formation during antigen recall experiments. Lymphocytes co-cultured with LSA-loaded DC exhibited a higher fold-change in interferon (IFN) γ mRNA compared to ISA-loaded DC, suggesting the secreted antigens and the metabolic activity of S. aureus play a role in Th1 polarization. Th1 polarization can drive excessive inflammation and suppress beneficial Th17 responses. Bovine DC were stimulated with a mutant α-toxin deletion S. aureus strain to evaluate if α-toxin-mediated NOD2 receptor signaling activates Th1 polarization in response to S. aureus, which revealed that NOD2 mRNA transcription in DC was independent of α-toxin and that the deletion of α-toxin had no effect on the transcription of the cytokine IL-12 or the production of IFNγ by lymphocytes, events that drive Th1 polarization, in co-cultures. The deletion of accessory gene regulator (agr), which controls α-toxin production, reduced IFNγ production in lymphocytes co-cultured with the S. aureus-loaded DC, indicating that agr controlled the ability of S. aureus antigens to drive the Th1 polarization of lymphocytes. Overall, this thesis demonstrates that ISA is a promising source of antigens that stimulate memory cells formation and Th17 polarization in bovine immune cells. The reduced Th1 cytokine response to S. aureus was not dependent on α-toxin, but other virulence factors controlled by agr should be screened to determine the source of Th1 stimulation. Ph. D.

Current data indicates that feeding dairy calves more nutrients in early life allows them to produce more milk in the future. Mechanisms responsible are poorly understood. Thirty-six Holstein heifer calves were fed either a restricted (R; 20.2% crude protein [CP], 19.8% fat, dry matter (DM) basis, fed at 0.44 kg/hd/d, DM basis) or an enhanced (EH; 28.9% CP, 26.2% fat, DM basis, fed at 1.08 kg/hd/day, DM basis) milk replacer (MR) and given either a placebo or estradiol (E2) implant to assess differential responses to E2. Our underlying hypothesis was that calves fed more nutrients are better able to respond to mammogenic stimuli and will have a more developed mammary gland as a result of imposed treatments. Enhanced-fed calves grew at a faster rate, were heavier at weaning, and had more functional mammary tissue (i. e., parenchyma; PAR) mass in the mammary gland at weaning (7.3-fold). Additionally, biochemical composition of the PAR was not impacted by the dietary treatments imposed. Furthermore, EH-fed calves had an increase in the number of actively dividing cells throughout the mammary PAR as well as increased intensity of estrogen receptor expression in the population of cells expressing the estrogen receptor. Enhanced-fed calves had an up-regulation of genes and pathways in the PAR related to metabolism, cellular signaling, and cellular growth. When given E2, EH-fed calves experienced the greatest overall mammary gland development and had the greatest PAR mass without compromised composition. When comparing EH- and R-fed calves given E2, differential expression of genes and pathways related to cell growth, cell signaling, and metabolism was observed. In summary, data indicates that enhanced feeding of calves in early life allows increased responsiveness to mammogenic stimuli and a corresponding increase in mammary development. We suggest that this may at least partly explain the improved future milk production in calves fed in this manner. Ph. D.

The key transcription factor sterol regulatory element binding protein-1 (SREBP1) plays a central role in milk fat synthesis. SREBP1 stimulates the transcription of genes encoding lipogenic enzymes. The overall objective of these studies was to investigate the mechanisms of SREBP1 regulation by nutrients. In the first study, chromatin immunoprecipitation (ChIP) accompanied with deep-sequencing was employed to investigate the potential sterol regulatory elements (SRE) in the promoter of SREBP1-target genes. The SRE in three known SREBP1-target genes SREBP1, fatty acid synthase (FASN) and stearoyl-CoA desaturase (SCD) were first validated in a bovine mammary epithelial cell line (MacT) and in bovine mammary tissues. At least one or two SRE binding sites in 24 selected lipogenic genes were identified within 50,000 base pair to the 5'-transcription start site through ChIP-seq. The genes closest to the highest enriched peaks were involved in cell integrity, defense or signal transduction whereas lipogenic genes were not among the top enrichment leading to the questions about the success of the ChIP. The second study was conducted to determine the effect of t10, c12-conjugated linoleic acid (CLA) on insulin induced gene-1 (Insig1), an endoplasmic reticulum (ER) protein that anchors SREBP1 and prevents proteolytic activation of SREBP1. MacT cells were treated with increasing levels of t10, c12-CLA. High concentration of t10, c12-CLA inhibited Insig1 degradation therefore decreased SREBP1 maturation. Furthermore, immunoprecipitation (IP) confirmed that t10, c12-CLA reduced Insig1 proteasomal degradation by disrupting the interaction between Insig1 and UBX domain-containing protein 8 (Ubxd8), which is part of a degradation complex that removes Insig1 from the ER. In the third study, three potential regulators of SREBP1 activation and their pathways were investigated in insulin, t10, c12-CLA or glucose treated MacT cells. Insulin-induced mammalian target of rapamycin (mTOR) signaling stimulated lipogenesis via activation of SREBP1 and the stimulatory effect was based on the regulation on cAMP response element binding protein coactivator 2 (CRTC2) phosphorylation, Lipin1 translocation and glycogen synthase kinase-3 (GSK3)-dependent proteasomal degradation. t10, c12-CLA inhibited SREBP1 through AMP-activated protein kinase (AMPK) phosphorylation, a key protein kinase in energy homeostasis. Glucose stabilized the SREBP1 chaperone protein SCAP and facilitated SREBP1 activation. Overall, SREBP1 activation is under specific regulation of t10, c12-CLA and interacts with multiple major cellular signaling pathways in response to hormonal stimulation and nutrient availability. Ph. D.

The SH3 and cysteine rich domain 3 (Stac3) gene is expressed specifically in skeletal muscle and essential for skeletal muscle contraction and postnatal life in mice. In this dissertation project, I conducted two studies to further understand the role of STAC3 in skeletal muscle development, growth, and contraction. In the first study, I compared the contractile responses of hindlimb muscles of Stac3 knockout and control mice to electrical stimulation, high [K+]-induced membrane depolarization, and caffeine and 4-chloro-m-cresol (4-CMC) activation of ryanodine receptor (RyR). Frequent electrostimulation-, high [K+]-, 4-CMC- and caffeine-induced maximal tensions in Stac3-deleted muscles were approximately 20%, 29%, 58% and 55% of those in control muscles, respectively. 4-CMC- and caffeine-induced increases in intracellular calcium were not different between Stac3-deleted and control myotubes. Myosin-ATPase and NADH-tetrazolium reductase staining as well as gene expression analyses revealed that Stac3-deleted hindlimb muscles contained more slow type-like fibers than control muscles. These data together confirm a role of STAC3 in EC coupling but also suggest that defective EC coupling is only partially responsible for the significantly reduced contractility in Stac3-deleted hindlimb muscles. In the second study, I determined the potential role of STAC3 in postnatal skeletal muscle growth, fiber composition, and contraction by disrupting Stac3 gene expression in postnatal mice through the Flp-FRT and tamoxifen-inducible Cre-loxP systems. Postnatal Stac3 deletion inhibited body and limb muscle mass gains. Histological staining and gene expression analyses revealed that postnatal Stac3 deletion decreased the size of myofibers and increased the percentage of myofibers containing centralized nuclei without affecting the total myofiber number. Postnatal Stac3 deletion decreased limb muscle strength. Postnatal Stac3 deletion reduced electrostimulation- but not caffeine-induced maximal force output in limb muscles. Similarly, postnatal Stac3 deletion reduced electrostimulation- but not caffeine-induced calcium release from the sarcoplasmic reticulum. These results demonstrate that STAC3 is important to myofiber hypertrophy, myofiber type composition, contraction, and EC coupling in postnatal skeletal muscle. Ph. D.

Volatile fatty acids (VFA) are important products of ruminal fermentation. The VFA are not only the major source of energy to the ruminant animals but also influence methane production in the rumen. Therefore it is important to understand mechanism controlling VFA production and to depict VFA production in a model. This will allow us to devise strategies to enhance energy utilization and reduce methane production in ruminant livestock. An evaluation of a mechanistic model in predicting VFA production was conducted and equations were introduced into the model to improve the predictions. Later a continuous culture experiment was conducted to test the hypothesis on which those equations were based on. A mechanistic model -" Molly, was evaluated using a dataset with reported VFA production rates. The results of residual error analysis indicated that the root mean square prediction errors (RMSPE) were 63, 63, and 49% for acetate, propionate and butyrate, respectively. An assessment from two studies reporting VFA production revealed a potential of reducing errors of prediction by representing interconversion among VFA. In the second study, equations based on thermodynamics influence of pH and VFA concentration were introduced in the model to represent interconversion among VFA. The parameters for de novo VFA production and VFA absorption were re derived with (VFAInt) and without (BASE) the new interconversion equations. There were some improvements in the VFA concentration predictions but the improvements were both in VFAInt and BASE models. The RMSPE of VFA production were still above 50% for acetate, propionate and butyrate. The larger errors of predictions were attributed to measurement variation in VFA production literature, or possible incorrect rate constants for interconversion equations. Finally, a third study was conducted to assess the effect of pH, and VFA concentration on VFA and methane production in continuous culture. The treatments consisted of control, 20 mmol/d acetate infusion (INFAC), 7 mmol/d propionate infusion (INFPR), and low pH (LOWPH). Individual isotopes of acetate, propionate and butyrate were infused in the fermenters to estimate interconversions among VFA. With LOWPH treatment methane emission was reduced whereas production of propionate was increased. Hydrogen production was higher in INFAC indicating that some of the acetate could have been degraded to CO2 and H2. It was estimated that around 3 % of de novo acetate was converted to propionate and 9 % to butyrate. Exchange between propionate and butyrate was insignificant and below 1% of de novo production of either VFA. However, treatments did not affect interconversion rates among VFA. These results indicated that pH and VFA concentration do not have thermodynamic influence on VFA interconversion as hypothesized. Ph. D.

Pre-pubertal bovine mammary development involves ductal and stromal tissue changes. In mice, this process is impacted by presence of innate immune cells. Whether or not such immune cells are present or involved in bovine mammary development is unknown. We studied the presence, location and changes in numbers of eosinophils, mast cells and macrophages in pre-pubertal bovine mammary tissue. Chemical stains and immunofluorescence were used to identify the cells in formalin fixed, paraffin embedded mammary tissue. The first set (ONT) included samples (n=4/week) from birth to 6 weeks of age. Another set (OVX) determined the influence of ovaries, 19 animals were intact or ovariectomized 30 days before sampling. They were 90, 120 or 150 days old at examination. The third set (EST) allowed examination of the potential influence of exogenous estrogen on innate immune cells in the mammary gland. Samples were from calves given estrogen implants (n=6) or placebo (n=4) at 56 days old, and sampled at 70 days old. We examined 20 images each of NEAR and FAR stroma from every animal. More eosinophils were observed in NEAR versus FAR in the ONT and OVX , more mast cells observed in NEAR versus FAR in ONT. More macrophages were observed in NEAR versus FAR in ONT and EST. We show, for the first time, that innate immune cells are present in prepubertal bovine mammary tissue and that abundance is related to the epithelial structure. We suggest a possible role for these cells in control of bovine mammary development. Master of Science

The use of antibiotics in dairy cattle has the potential to stimulate the development and subsequent fecal dissemination of antibiotic resistance genes (ARGs) in bacteria. The objectives were to use metagenomic techniques to evaluate the effect of antibiotic treatment on ARG prevalence in the fecal microbiome of the dairy cow and to determine the temporal excretion pattern of ARGs. Twelve Holstein cows were assigned to one of four antibiotic treatments: control, pirlimycin, ceftiofur, or cephapirin. Fecal samples were collected on d -1, 1, 3, 5, 7, 14, 21, and 28. Samples were freeze-dried and subjected to DNA extraction followed by Illumina paired-end HiSeq sequencing and quantitative polymerase chain reaction (qPCR). Illumina sequences were analyzed using MG-RAST and the Antibiotic Resistance Gene Database (ARDB) via BLAST. Abundance of ampC, ermB, tetO, tetW, and 16S rRNA genes were determined using qPCR. All data were statistically analyzed with PROC GLIMMIX in SAS. Antibiotic treatment resulted in a shift in bacterial cell functions. Sequences associated with 'resistance to antibiotics and toxic compounds' were higher in ceftiofur-treated cows than control cows. Ceftiofur-treated cows had a higher abundance of 𝛽-lactam and multidrug resistance sequences than control cows. There was no effect of treatment or day on fecal tetO and ermB excretion. The relative abundances of tetW and ampC were higher on d 3 post-treatment than d 5 and d 28. In conclusion, antibiotic use in dairy cattle shifted bacterial cell functions and temporarily increased antibiotic resistance in the fecal microbiome. Master of Science

Growth hormone (GH) has growth-stimulating effects on skeletal muscle and liver but a growth-inhibitory effect on adipose tissue. The mechanisms underlying these actions of GH are not fully understood. Two studies were conducted to achieve the following objectives: 1) to determine the cellular mechanism by which GH stimulates liver growth; 2) to determine the effects of GH on the commitment of mesenchymal stem cells (MSCs) to myogenic and adipogenic lineages. In the first study, the GH-deficient lit/lit male mice were injected (s.c.) daily with rbGH or vehicle for two weeks. GH-injected lit/lit mice tended to have a greater liver/body weight percentage than lit/lit control mice. GH injection did not alter the percentage of proliferating cells in the liver. However, GH-injected lit/lit mice had 18% larger hepatocytes and 16% less DNA per unit liver weight than those of lit/lit control mice. These data together indicate that GH stimulates liver growth in mice by increasing the size, not by increasing the number of hepatocytes. In the second study, we treated the MSC cell line C3H10T1/2 cells with or without 5'-azacytidine and rbGH for 4 days. We assessed the myogenic or adipogenic potential by determining the ability of these cells to differentiate into myotubes or adipocytes, respectively. C3H10T1/2 cells treated with 5'-azacytidine and GH formed more myotubes, myoblasts, and fewer adipocytes compared to cells treated with 5'-azacytidine alone. Taken together, these results suggest that GH enhances 5'-azacytidine-induced myogenic commitment but inhibits 5'-azacytidine-induced adipogenic commitment in C3H10T1/2 cells. Master of Science

Our purpose was to determine the effects on mammary gland development in prepubertal heifers given the anti-estrogen tamoxifen. Sixteen Holstein calves were randomly assigned to one of two treatment groups: tamoxifen-injected (TAM) or control (CON). Calves were subcutaneously injected daily from 28 to 120 days of age with 0.3 mg/kg tamoxifen or carrier. At 120 days calves were euthanized and udders removed. Weight of trimmed parenchymal tissue (left rear quarter) was dramatically lower in TAM calves than in CON calves (p < 0.0003; 16.1 vs. 34.8 g). Parenchymal samples from three regions of the left rear quarter (lower, middle and outer regions) were processed for immunohistochemical staining for Estrogen Receptor α and Progesterone Receptor, myoepithelial cells, and label retaining cells. Overall, the proportion of neither ER nor PR labeled cells was impacted by TAM treatment. However, imaging analysis indicated a markedly higher intensity of ER expression in CON calves. TAM caused an increase in myoepithelial cell differentiation similar to what is seen in ovariectomy. We were able to effectively use a new technique of multispectral imaging to identify label retaining cells, which led to the discovery of an increase in the percentage of label retaining cells in TAM compared to CON. While treatment with the anti-estrogen tamoxifen reduced mammary parenchymal mass similarly to OVX, the mechanism(s) involved appear to differ. This suggests that the impacts of ovariectomy are only partially explained by the absence of estrogen. Master of Science

Growth hormone (GH) is a poly-peptide hormone produced by the anterior pituitary. Growth hormone not only stimulates body and muscle growth but also inhibits adipose tissue growth. The overall objective of this study was to determine the mechanisms by which GH inhibits adipose tissue growth. Three studies were conducted to achieve this objective. The first study was conducted to determine if GH inhibits fat tissue growth by stimulating lipolysis. In this study, adipose tissue weight and adipocyte size were compared between GH-deficient growth hormone releasing hormone receptor (Ghrhr) homozygous mutant mice (i.e., lit/lit mice), lit/+ mice, and lit/lit mice injected with GH. lit/lit mice had less body weight but more subcutaneous fat and larger adipocytes compared to lit/+ mice at the same ages. GH treatment to lit/lit mice for four weeks partially reversed these differences. These data suggest that GH inhibits adipose tissue growth in mice at least in part by stimulating lipolysis. Additional data from this study suggest that GH indirectly stimulates lipolysis in vivo and this indirect mechanism is independent of " adrenergic receptors in the adipose tissue. The second study was conducted to investigate if GH inhibits fat tissue growth also by inhibiting adipogenesis. In this study, stromal vascular fraction (SVF) cells were isolated from subcutaneous fat of lit/+ and lit/lit mice and were induced to differentiate into adipocytes in vitro. Oil Red O staining and gene expression analysis revealed that the SVF cells from lit/lit mice had greater adipogenic potential than from lit/+ mice. This suggests that GH inhibits adipose tissue growth also through inhibition of adipogenesis. Additional data from this study suggest that GH may inhibit adipogenesis by inhibiting the formation of adipogenic precursor cells in adipose tissue in mice. The third study was conducted to determine the role of the central component of GH receptor signaling, STAT5, in GH inhibition of differentiation of bovine preadipocytes. In this study, preadipocytes were isolated from subcutaneous fat of adult cattle and were induced to differentiate with or without GH. Based on Oil Red O staining, gene expression, glycerol-3-phosphate dehydrogenase (G3PDH) activity and acetate incorporation assays, GH inhibited differentiation of bovine preadipocytes into adipocytes. GH induced phosphorylation of STAT5 in differentiating bovine preadipocytes. Overexpression of constitutively active STAT5 through adenovirus mimicked the effect of GH on differentiation of bovine preadipocytes. These data support a role of STAT5 in mediating the inhibitory effect of GH on differentiation of bovine preadipocytes into adipocytes. Overall, GH inhibits adipose tissue by both stimulating lipolysis and inhibiting adipogenesis; GH stimulates lipolysis through an indirect mechanism that is independent of the " adrenergic receptors; GH inhibits adipogenesis through a direct mechanism that may involve the transcription factor STAT5. Ph. D.

The aims of the first study were to determine the effects of early weaning followed by a period of high-grain feeding on signaling protein phosphorylation in muscle and on acetate clearance in growing steers. Twelve Angus X Simmental steers were used in the study. Six steers were weaned early (EW) and fed a high-gain diet for 148 d and the remainder were weaned at the normal age (NW). Longissimus muscle tissue biopsies were collected to determine total and phosphorylated forms of AMP kinase and downstream proteins of the mTOR signaling pathway. Of the 12, 8 steers were used to assess acetate clearance at different age points. During early grain feeding, phosphorylated:total ratios of rpS6 and S6K1 were greater in EW steers and they had lower acetate clearance rates and greater synthesis rates. The objectives of second study were to assess the effects of early grain feeding on acetate and glucose turnover rates, palmitate synthesis, and on acetate and glucose preference by subcutaneous, intramuscular and visceral adipose tissues in finishing steers. Sixteen Angus x Simmental steers were infused continuously with [2H3] acetate (n = 8) or [U-13C6] glucose (n = 8), over a 12 h period immediately prior to harvest. Plasma acetate and glucose enrichment, and palmitate enrichment in different adipose tissue locations were determined. There were no treatment effects on acetate or glucose turnover rates or palmitate fractional synthesis rates (FSR). Acetate turnover and palmitate FSR from acetate were greater than the corresponding rates from glucose. There were no differences in preference for acetate or glucose among the fat depots. In conclusion, phosphorylation ratios of signaling proteins were not affected treatment. Acetate clearance increased when steers were heavier and older. Thus older calves are able to clear more acetate per unit of time and body mass than younger calves reflective of an enhanced ability to utilize the substrate. There were no differences in preference for acetate and glucose among the major fat depots. Thus diets leading to high glucose supply will not preferentially direct energy storage to intramuscular stores. Ph. D.

The objective of these studies was to investigate the milk fat synthesis regulation by transcription factors. In the first study, bovine mammary epithelial (MAC-T) cells were treated with sterol regulatory element binding protein-1 (SREBP-1) specific siRNA. The mRNA and protein expression of SREBP-1 were decreased by more than 90% by siRNA. Fatty acid (FA) synthesis, uptake, and selected lipogenic enzyme expression were reduced in cells treated with SREBP-1 siRNA. Therefore, SREBP-1 plays an important role in integrated regulation of lipid synthesis in MAC-T cells through regulation of key enzymes. In the second study, MAC-T cells treated with hormones or FA were transfected with luciferase reporter constructs containing response elements for SREBP-1, peroxisome proliferator-activated receptor γ (PPARγ), or liver X receptor (LXR). The activation of PPARγ and SREBP-1 were stimulated by insulin and insulin combined with leptin, respectively. Trans-10, cis-12 conjugated linoleic acid (CLA) inhibited SREBP-1 activation, and this inhibition was not attenuated by insulin and leptin. Neither trans-10 nor cis-12 double bond inhibited SREBP-1 activation. Taken together, trans-10 and cis-12 double bonds need to be conjugated in CLA to reduce SREBP-1 activation and this inhibition cannot be overcome by insulin and leptin combination in MAC-T cells. In the third study, lactating dairy cows were intravenously infused with 0.625 g/h trans-10, cis-12 CLA for 14 h. We confirmed the appearance of trans-10, cis-12 CLA in the milk of CLA treated cows. Milk and component yield were not affected by the CLA treatment. The desaturation of stearic acid was reduced by CLA. The mRNA and protein expression of transcription factors or lipogenic enzymes were not affected by trans-10, cis-12 CLA. DNA-binding activities for PPARγ and LXR and the activation of SREBP-1 to its mature form were not changed by the treatment. The infusion time in this study was probably too short to induce any changes in transcription factors and lipogenic enzymes. We confirmed DNA-binding activities of PPARγ and LXR in bovine mammary gland. Overall, a prominent role for SREBP-1 in mammary epithelial cell lipid synthetic pathways was described and regulation of transcription factor activation by trans-10, cis-12 CLA was specific to SREBP-1. Ph. D.

Excess nitrogen (N) excretion from animal agriculture results in reduced air and water quality, and poses a risk to human health. Although the dairy industry utilizes milk urea N (MUN) to monitor protein feeding and N excretion, phenotypic diversity among cows may influence MUN and thus bias feed management. An initial study using data from 2 previously published research trials and a field trial, observed that cow had a significant effect on MUN variation. Regression models, utilized to predict MUN, corrected for dietary nutrients and some animal effects, and thus the observed effect of cow on MUN variation may reflect genetic selection decisions of animals with either poor or efficient urea transport. A second trial observed that MUN and PUN concentrations were positively correlated with gut urea clearance, providing evidence for differences in urea transport activity among cows. The presence of urea transport variation suggests that current protein recommendations may not estimate true requirements. A third trial observed that animals fed sub-NRC levels of RDP and RUP had reduced N intake and excretion of fecal N, urinary urea-N, and MUN. Animals maximized N efficiency and had no loss in milk production, suggesting a possible overestimation of RDP and RUP in the current NRC prediction model. The present project provides evidence for phenotypic variation among cows, which may be partially explained by differences in urea transport activity. Future work confirming genetic variation among urea transporters may provide an opportunity to improve feeding management if cow urea efficiency is known. Master of Science

Marine oils are used as ration additives to provide omega-3 fatty acids to dairy cows. Supplementing dairy cows with omega-3 fatty acid-rich feeds does not easily increase quantities in milk fat of dairy cows because polyunsaturated fatty acids are biohydrogenated in the rumen. Lipid encapsulation of omega-3 fatty acids provides protection from biohydrogenation in the rumen and allows them to be available for absorption and utilization in the small intestine. Lactating cows were supplemented with rumen protected algae biomass or algal oil in a 4 × 4 Latin Square. Feeding lipid encapsulated algae supplements increased docosahexaenoic acid content in milk fat while not adversely impacting milk fat yield; however, docosahexaenoic acid was preferentially esterified into plasma phospholipid, limiting its incorporation into milk fat. In the second study, triglyceride emulsions of oils enriched in either oleic, linoleic, linolenic, or docosahexaenoic acids were intravenously infused to avoid confounding effects of triglyceride esterification patterns in the small intestine and to compare mammary uptake. Milk transfer of fatty acids delivered as intravenous triglyceride emulsions was reduced with increased chain length and unsaturation. Increased target fatty acids were evident in plasma phospholipid, suggesting re-esterification in the liver. Transfer efficiencies were 37.8, 27.6, and 10.9±5.4% for linoleic, linolenic, and docosahexaenoic acid. Both liver and mammary mechanisms may regulate transfer of long-chain polyunsaturates. Intracellular fatty acid binding proteins (FABP) are cytoplasmic proteins that are hypothesized to be essential for fatty acid transport and metabolism by accelerating longchain fatty acid uptake and targeting to intracellular organelles, such as the endoplasmic reticulum for triglyceride esterification. FABP3 mRNA is highly expressed in bovine mammary and heart tissue, but is not present in MAC-T cells, a bovine mammary epithelial cell line. When overexpressed in MAC-T cells, FABP3 does not appear to be rate-limiting for fatty acid uptake in vitro and did not alter lipid metabolism. The function of FABP3 in the mammary gland remains unclear. Ph. D.

A study was conducted to examine the correlation between a novel behavior monitoring system and a validated data logger. We concluded that the behavior monitoring system was valid for tracking daily rest time in dairy cows (R=0.96); however the correlation values for rest bouts and rest duration were relatively low, (R=0.64) (R=0.47), respectively. Daily monitoring of animal activity and milk components can be used to detect mastitis prior to clinical onset. Data from 268 cases with clinical mastitis and respective controls (n=268) from Virginia Tech and the University of Florida dairy herds were examined. Variables collected included daily milk yield, electrical conductivity, milk fat, protein, and lactose percent, as well as activity measurements including daily rest time, daily rest duration, daily rest bouts, and daily steps taken. Variables were collected for case and control cows in the 14 d prior to and after clinical diagnosis, for a total 29 d monitoring period. A milk sample was aseptically collected upon detection of clinical signs as observed by milker's at both farms. A statistical method (candisc discriminant analysis) was used to combine all measurements and sensitivity and specificity was calculated. Virginia Tech cows on d -1 (sensitivity=95%, specificity=95%), Virginia Tech and University of Florida cows on d -1 (sensitivity=88%, specificity=90). Overall, daily monitoring of animal activity and milk components can detect mastitis prior to onset of clinical signs of disease. This may allow producers to intervene and make proactive management decisions regarding herd health prior to clinical diagnosis. Master of Science

Staphylococcus aureus causes nosocomial- and community- acquired infections. This versatile pathogen expresses virulence factors (VF) that enhance establishment of infection and immune evasion. Our research focused on defining the roles of S. aureus VF on host immune responses during intracellular or extracellular infections. Accessory gene regulator (agr) controls VF expression and intracellular survival. Our goal was to determine mammary epithelial cells (MEC) responses to intracellular infection and subsequent polymorphonuclear leukocyte (PMN) responses. Intracellular S. aureus increased thrombomodulin expression by MEC and activated protein C (APC) production. APC inhibited PMN chemotaxis. Findings depicted an indirect role for VF on PMN responses, so next we determined signaling pathways and cytokine responses of PMN to S. aureus toxins. Live S. aureus infections increased activation of stress signaling pathways and highlighted a role for agr-regulated genes in MAPK p38 phosphorylation and α-hemolysin in ERK phosphorylation and IL-8 expression in PMN. Continuing our studies of VF, chemotaxis inhibitory protein of S. aureus (CHIPS) inhibits monocyte chemotaxis. We hypothesized that CHIPS inhibited C5a receptor (C5aR) signaling. Monocytes pretreated with CHIPS did not inhibit C5aR signaling. Nevertheless, signaling pathways can reduce PMN function in models such as glucocorticoid treatment. Immunosuppressive effects of glucocorticoids on PMN are restored with OmniGen-AF® supplementation. Glucocorticoid receptor and Toll-like receptor signaling potentially crosstalk to restore PMN function. OmniGen-AF® supplementation restored dexamethasone-induced immunosuppression in a MyD88-dependent manner. Overall, this research focused on characterizing immune responses to S. aureus infections and PMN signaling pathways and how it is key to understanding pathogenesis. Ph. D.

Staphylococcus aureus (S. aureus) is the etiological agent of food-borne diseases, skin infections in humans and mastitis in bovines. S. aureus is also known to exist as a commensal on skin, nose and other mucosal surfaces of the host. This symbiotic association is a result of immune dampening or tolerance induced in the host by this pathogen. We proposed the variation in protein expression by commensal and pathogenic strain as an important factor behind the difference in pathogenicity. The identification of differentially expressed proteins was carried out using a quantitative mass spectrometry (MS)-based proteomic approach, known as stable isotope labeling of amino acids in cell culture (SILAC). Four commensal and pathogenic strains each were grown in the SILAC minimal media (RPMI 1640), containing light (12C) and heavy (13C) form of lysine, respectively, until early stationary growth phase. Various protein fractions, including cell wall, membrane and secreted, were extracted from the bacterial cultures and mixed in a 1:1 ratio. The relative abundance of proteins present in light and heavy labeled samples was determined using MS analysis. From a total of 151 differentially expressed proteins, 58 were found to be upregulated in the pathogenic strains. These proteins are involved in a variety of cellular functions, including immune modulation, iron-binding, cellular transport, redox reactions, and metabolic enzymes. The differentially expressed proteins can serve as putative candidates to improve current approach towards development of a vaccine against S. aureus. Master of Science

Phosphorus (P) is an instrumental nutrient in numerous physiological processes, but can have detrimental environmental impact if fed in excess. Increased P intake in dairy cows leads to increased fecal excretion of P and a reduction in efficiency of use. Variability in P concentration or availability in feedstuffs can exacerbate P excretion. To investigate variability in P between and within feedstuffs, 170 feed samples (forages, concentrates, and by-products), were collected from across the U.S., classified by region fed, and analyzed for total P, inorganic P, and phytate. Forages contained a greater proportion of P in the inorganic form and less total P and phytate as compared to concentrates and by-products. The majority of total P (71.2, 81.8, and 81.9% of total P in forages, concentrates, and by-products, respectively) was associated with inorganic P and phytate. The enzyme phytase has been used successfully in swine and poultry nutrition, as a feed additive, to increase available P and reduce the need for supplemental inorganic P. An experiment was conducted to investigate the effect of phytase use and forage particle length, using a 2 x 2 factorial, on P availability in lactating dairy cows. Total P intake of the four diets was similar (P > 0.15). Total tract digestibility of total P tended (P < 0.10) to be reduced and total P excretion was increased (P < 0.05) with phytase supplementation. Milk fat yield, protein yield, 3.5% FCM, and ECM were increased (P < 0.05) with addition of exogenous phytase to the diet. This indicates that phytate may contain some anti-nutritional factors that reduce availability in other nutrients used for milk production. Variation in P compounds between feeds, and variation in P digestion and production performance with exogenous phytase suggests opportunity for improvement in prediction of P availability from feeds for lactating cows. Ph. D.

Agricultural runoff is the largest source of nitrogen and phosphorus pollution entering the Chesapeake Bay, contributing 38% of nitrogen and 45% of phosphorus (USEPA, 2010). Since agricultural runoff is the number one contributing source of nitrogen and phosphorus entering the Chesapeake Bay, action needs to be taken to reduce nitrogen and phosphorus on agriculture production facilities, such as dairy farms. The impact of feed management software on whole-farm nutrient balance was studied on 18 dairy farms located in Virginia from 2006 to 2010. Nine farms began using the TMR Tracker feed management software in 2006 and were compared to 9 control farms not using feed management software. Each of the treatment farms were visited on a monthly basis to collect ration and feed ingredient samples and feed management data. Whole-farm nutrient balance was calculated using University of Nebraska software. Herd sizes and crop hectares averaged 314 and 366 for treatment and 298 and 261 for control farms. Milk production averaged 3,226 and 2,650 tonnes per year respectively. Measures of surplus (input-output) and use efficiency (input/output) for nitrogen and phosphorus were analyzed over a four year time span and did not differ between treatment and control farms whether expressed on a per farms, cow or hectare basis. Due to the large variation in feeding accuracy within farms, the use of feed management software did not influence whole-farm nutrient balance. Sources of variation that contributed to loading errors were investigated within the feed management data. Percent load deviation increased over time from 2007 to 2009 from 0.94 ± 0.53 to 2.37 ± 0.50 percent of the actual load weight. Effects of month, day of the week and time of day on percent load deviation were not significant. There was no effect of percent load deviation on milk production. No relationship was observed between percent load deviation and whole-farm nutrient balance. Master of Science

Staphylococcus aureus is a versatile opportunistic pathogen causing a wide spectrum of diseases in both humans and animals. My research focused on characterization of the immune responses of monocyte derived dendritic cells (DC) to S. aureus. We initially evaluated the potential of circulating monocytes to serve as precursors for DC during S. aureus infection. The CD14⁺ monocytes, when stimulated with irradiated (ISA) or live S. aureus (LSA), differentiated into CD11chigh CD11bhigh DC (MonoDC) in an autocrine fashion. This was associated with the up- regulation of granulocyte-macrophage colony stimulating factor (GMCSF) and tumor necrosis factor-α (TNF-α) gene transcription. We continued our studies to identify the role of TNF-α in the LSA induced differentiation of monocyte to MonoDC. Blocking TNF-α reduced the expression of CD11c and increased the expression of CD14 on LSA stimulated monocyte derived MonoDC. Stimulated monocytes were able to secrete monocyte chemotactic protein-1 (MCP-1), a chemokine that recruits monocytes to the site of infection/injury and induces the expression of β₂ integrins on DC. Characterization of the response of DC derived from monocytes using GMCSF and IL-4 revealed that, intact S. aureus rather than its purified structural components were efficient in DC activation. In response to ISA or LSA stimulation, DC induced proliferation of T cells collected from the peripheral circulation of cows with a history of S. aureus mastitis. Subsequent characterization of the proliferating T cells identified the presence of memory T cells. Finally, we identified a unique population of DEC205⁺CD8a+ in monocyte derived DC. We further elucidated the role of DC DEC205, a C-type lectin, in S. aureus uptake. Blocking of receptor mediated endocytosis resulted in reduced uptake of S. aureus by DC. Confocal microscopy confirmed a role for DEC205 in S. aureus internalization and delivery to endosomes. DEC205 DC upon stimulation with S. aureus displayed enhanced maturation and antigen presentation. In conclusion, monocyte derived DC can uptake S. aureus and elicit cell mediated immune responses. Ph. D.

Influenza A virus (IAV) is a significant problem worldwide, and respiratory disease is further complicated by secondary bacterial infection. The emergence of highly pathogenic strains of IAV in conjunction with the increase of antibiotic-resistant bacteria threatens human health. A large-animal model effective for study of polymicrobial infection comparable to humans must therefore be developed. IAV has been studied extensively in small animals, including mice, rats and ferrets. However, these species frequently require IAV adaptation, reducing the capacity of these models to adequately represent human infection. Furthermore, species commonly used lack likeness to humans in both the presentation of symptoms and in lethality of infection. However, pigs are naturally susceptible to unadapted IAV and are considered to be the 'mixing vessel' for the recent pandemic IAV virus. Pigs are also susceptible to infection with Staphylococcus aureus, the most commonly isolated bacteria from IAV-infected human adults. Therefore, the use of pigs in the study of polymicrobial respiratory infections would be ideal for characterizing a host immune response comparable to humans, as well as for the development of diagnostics and therapeutics. Using this novel model, we determined that pigs are susceptible to Staphylococcus aureus, swine IAV, and polymicrobial infection. Furthermore, we showed that IAV infection predisposes pigs to Staphylococcus aureus pneumonia, and this susceptibility is dependent on day post-IAV infection. Master of Science

Pronuclear stage murine embryos received electrical stimulation in 5, 10, or 20 µs pulse lengths, and 0, 100, 200, 250, 300 or 400 voltages. Minimal embryo development occurred with 400 V. Irreversible electroporation occurred in embryos electroporated for 5 µs pulse length at 100 and 400 V, 10 µs pulse length at 400 V, and 20 µs pulse length at 100, 250, 300 and 400 V. Electroporated embryos that underwent reversible electroporation received 100 V for 5 µs, 400 V for 10 µs, and 250 for 20 µs and had similar development (P > 0.05) between the best and worst developed groups. Enhanced green fluorescent protein on a cytomegalovirus promoter (CMV-EGFP) was condensed with MgCl2 and injected into the cytoplasm of murine zygotes at three concentrations (100, 425 and 625 µg/ml). Zygotes injected with the highest concentration had the highest percentages of fluorescing embryos (44%), fluorescing morula and blastocysts (16.7%), and the lowest percentage of mosaicism after 4 d in culture. Five PCR analyses of tail DNA gave conflicting results between 33.3% positive in two or more analyses to 2.8% positive in all five analyses. Southern Analysis detected 2.8% transgenesis. Cytoplasmic injection of linear CMV-EGFP (625 µg/ml in water) was 3.7% transgenic. Pronuclear injections produced 7.9% transgenesis. This research identified a range of reversible electroporation that could easily be verified in vitro with a selectable dye or marker protein and applied in transgenic as well as preclinical treatment models of research. Furthermore this research identifies the benefits and disadvantages of using Mg2+ in DNA condensation and injection buffers. Master of Science

Dairy cows inefficiently convert dietary protein to milk protein causing economic and environmental costs. Amino acids (AA), insulin, and glucose significantly enhance muscle protein synthesis efficiencies. The objectives of this research project were 1) to investigate the regulatory effects of essential AA (EAA) and their interactions with insulin, glucose and acetate on mammary protein synthesis rates, 2) to investigate whether branched chain amino acids (BCAA): leucine , isoleucine , and valine , become limiting for milk protein synthesis when Met and Lys supply were not limiting, and 3) to develop a mathematical representation for the EAA and insulin effects on cellular signals for protein synthesis. MAC-T cells were treated with EAA, insulin, glucose, and acetate to observe their individual and interactive effects on phosphorylation of mTOR, rpS6, S6K1, 4EBP1, eEF2, eIF2α, Akt, and AMPK. These signaling effects on protein synthesis rates were examined with mammary tissue slices. A mathematical representation of the insulin and EAA effects was developed. The effects of supplementing BCAA on milk protein synthesis were investigated using nine Holstein cows, assigned to 7 d continuous jugular infusions of saline, Met and Lys, and Met and Lys plus BCAA. Multiple essential amino acids, Leu, Ile, Met, and Thr were able to substantially regulate protein synthesis rates in bovine mammary cells by increasing (P < 0.05) phosphorylation of mTOR, S6k1, 4EBP1, and decreasing (P < 0.10) eEF2 phosphorylation. Insulin considerably (P < 0.10) exerted similar signaling effects in MAC-T cells, independent of EAA. Supplementation of only acetate increased (P = 0.09) mammary cell energy status as indicated by reduced AMPK phosphorylation in MAC-T cells. Neither acetate nor glucose had substantial regulatory effects on mammary protein synthesis rates. Although Met and Lys supplementation increased (P < 0.01) milk protein yields and protein efficiencies, there were no apparent benefits of BCAA supplementation under the feeding circumstances of our study. The developed mathematical model adequately represented the regulatory effects of EAA and insulin. Such mathematical representations of regulatory effects of EAA and their interaction with other nutrients may improve our current AA requirement models to predict AA requirements of dairy cows with increased accuracy. Ph. D.

The highly regulated maturation of the mammary gland is poorly understood. Our studies were designed to further characterize the role of ovarian hormones, growth hormone (GH)/IGF axis proteins and extracellular matrix (ECM) in the growth and development of prepubertal mammary glands. Prepubertal heifers were injected with either exogenous GH or subjected to ovariectomy (OVX). Mammary parenchyma (PAR) and mammary fat pad (MFP) were harvested for DNA, protein, lipid, and western blot analysis. Remaining tissues were preserved for histological staining or snap frozen for quantitative real-time PCR. We examined 13 genes that work in conjunction with the extracellular matrix to regulate mammary proliferation and morphogenesis. Administration of GH, while impacting composition of MFP, had no effect on expression of the selected genes; there was a decrease in expression of fibronectin in PAR. Ovariectomy had no effect on gene expression in MFP but decreased expression of epimorphin, a potent regulator of morphogenesis, in PAR. In both experiments, the presence of a 55 kDa band corresponding to androgen converting enzyme aromatase was detected but its expression was unaffected. In another study, we used in vitro cell culture to evaluate the role of ECM in mammary gland maturation and employed quantitative real-time PCR to evaluate gene expression profiles of select genes involved in proliferation and differentiation. Expression of Rac1 was decreased in response to bovine insulin (BI) but increased on collagen I (Col). Expression of aldehyde dehydrogenase was decreased in BI and serum on plastic and on Col in the presence of BI. Expression of IGF binding proteins (BP) 3, -4, and -6 were decreased in the presence of serum on laminin (LM). Also, IGF-BP2 expression was decreased on Col while IGF-BP6 was increased on LM with BI. Clusterin, a ubiquitous non-adhesive ECM protein was not affected by ECM substrate but did increase over time. In conclusion, we propose that the mammary gland is not able to respond to GH at this age and that while OVX did effect the expression of some genes, the presence of aromatase maintained local estrogen concentrations. Furthermore, ECM alone is insufficient to regulate mammary gland development and growth. Ph. D.

Bacterial mastitis is a significant problem for the dairy industry. A vaccine against mastitis pathogens could potentially target dendritic cells (DC). While there has been some research describing bovine DC populations in-vitro, little is known about DC in mammary tissue. In this study, immunohistofluorescence was used to identify and localize bovine mammary DC. DC were found in alveoli, in epithelia, and in interalveolar tissue. Fluorescence-activated cell sorting (FACS) was used to characterize mammary DC as expressing CD11c, MHC-II, CD205, CD11b, and CD8α. FACS allowed us to distinguish DC (CD14lo) from macrophages (CD14hi). Two DC subsets, CD11a-, CD11alo, were evident in the mammary gland while an additional CD11ahi population was identified in the supramammary lymph node. After phagocytosis of bacterial components such as lipopolysaccharide (LPS), DC undergo a maturation process, in which they upregulate homing receptors, such as CCR7, and antigen presentation markers, including MHCII and CD80. A primary cell culture model was used to evaluate changes in transcription of CD80 and CCR7 after LPS stimulation. Cell cultures contained digested and Ficoll separated mammary tissue or supramammary lymph node tissue. While the presence of CCR7 and CD80 was confirmed, CD80 and CCR7 transcripts were not upregulated after LPS stimulation. Further, CD11c, CD14, MHCII, CD11b, CD11a, and CD205 protein levels, as assessed by FACS, were similar in LPS stimulated cultures and unstimulated controls. Overall, these studies provide a better understanding of mammary gland immunology, while potentially aiding in the development of novel DC based vaccines. Master of Science

Klebsiella spp. are coliform bacteria that cause mastitis in dairy cattle and account for high mortality rates in infected cows leading to a significant financial loss. Recent outbreaks indicate that within farms a single strain can be responsible for clinical signs in multiple animals. Identification of the virulence of factors enabling Klebsiella spp. survival in the mammary glands of multiple animals may provide insight into host adaptation. In this study, Klebsiella spp. strains were evaluated for their ability to evade neutrophil killing, the primary immune defense in the bovine mammary gland. Our research focused on capsule and biofilm production by Klebsiella spp. when strains were grown in Luria Broth or skim milk to examine the effects on evasion of neutrophil killing. Biofilm production was not significantly related to the ability to resist neutrophil killing nor was capsule (P = 0.29). Farm (P < 0.001), media type (P < 0.005), and strain type by cow (P < 0.001) were found to play significant roles in neutrophil evasion. This suggests farm of origin, media type used, and cow all may play a role in evasion of neutrophils by Klebsiella spp. Further evaluation of virulence factor expression in different media types and the role of individual cow immune responses may provide insight into ability of Klebsiella spp. to cause outbreaks of mastitis in multiple animals. Master of Science

Two studies were conducted to assess factors affecting estrogen excretion in dairy heifers. The objective of the first study was to quantify estrogenic activity in feces and urine during the estrous cycle. Ten non-pregnant Holstein heifers were fed the same diet for 28 d. Plasma, feces, and urine samples were collected daily. Plasma 17-β estradiol (17-β E2) was quantified with RIA and used to confirm day of estrous. Feces and urine samples from days -12, -6, -2, -1, 0, 1, 2, 6, 12 of the estrous cycle were analyzed with RIA and Yeast Estrogen Screen (YES) bioassay. Plasma 17-β E2 concentrations peaked on day of estrus, with feces and urine estrogenic excretion peaking a day after. The objective of the second study was to quantify variation in estrogenic activity in feces and urine due to increased dietary phytoestrogen content. Ten Holstein heifers were randomly assigned to treatment sequence in a two-period crossover design. Dietary treatments consisted of grass or red clover hay, and necessary supplements. Feces and urine samples were collected and pooled for analysis. Estrogenic activities of pooled samples were quantified using the YES bioassay. Estrogenic excretion in feces and urine was higher for heifers fed red clover hay. Fecal and urine samples from five heifers were analyzed using LC/MS/MS to quantify excretion of phytoestrogenic compounds. Heifers fed red clover hay excreted more equol than heifers fed grass. Identifying sources of variation in estrogenic activity of manure will aid in the development of practices to reduce environmental estrogen accumulation. Master of Science

Dairy herds are major contributors to N pollution because 70% of the N intake is lost to the environment and 30% or less is retained in milk protein. Plasma amino acids (AA) that are not used for protein synthesis in mammary glands (MG) are catabolized in post splanchnic tissues (liver plus gastrointestinal tract, pancreas, spleen, portal system, and associated adipose tissue) and two thirds of the net supply of essential AA (EAA) are cleared in splanchnic tissues. Thus, increasing AA capture in MG would be expected to reduce AA catabolism and thereby increase efficiency of AA utilization. The objectives of the work presented in this dissertation were to test the effect of energy and N intake on cell regulatory mechanisms, nutrient kinetics, milk, milk protein yield, and N efficiency in dairy cows. The aim of the first study was to test whether metabolizable protein (MP) and dietary energy exerted independent effects on milk protein synthesis and postabsorptive N efficiency. Forty mid-lactation cows (32 multiparous Holstein and 8 primiparous Holstein x Jersey cross-breds) were used in a complete randomized design with a 2 x 2 factorial arrangement of diets. Cows were assigned to one of four dietary treatments: high-energy, high-protein (HE/HP); high-energy, low-protein (HE/LP); low-energy, high-protein (LE/HP); and low-energy, low-protein (LE/LP). Energy concentrations were 1.55 (HE/HP and HE/LP) or 1.44 (LE/HP and LE/LP) Mcal NEL/kg DM according to the NRC model. Changes in predicted MP were achieved by feeding diets with 6.6 (HE/HP and LE/HP) or 4.6% (HE/LP and LE/LP) ruminally undegradable protein (DM basis). Ruminally degradable protein was held constant at 10.1% of DM. All cows were fed HE/HP diet from day 1 to 21 followed by the respective treatments from day 22 to 43 (n=10). Milk protein yield was reduced as dietary energy was reduced. There were no interactions between dietary energy and protein for either milk or protein yield. Milk urea N was significantly affected by energy and protein with an interaction (HE/HP=17.2, HE/LP=12.2, LE/HP=21.0, LE/LP=12.2 mg/dl). Nitrogen efficiency was affected by energy and protein supplies with no interaction and ranged from a low of 31% (LE/HP) to a high of 43% (HE/LP). Although energy and protein independently affected milk and protein yield the tissue and cellular mechanisms that regulate milk production were not studied. The second experiment studied cellular mechanisms in MG that contributed to the regulation of protein synthesis in the presence of energy or protein supply. We hypothesized that metabolism of AA in the MG is controlled by systemic and local tissue adaptations and when combined with altered mammary cell function controlled milk protein yield. Six primiparous mid-lactation Holstein cows with rumen cannulas were randomly assigned to abomasal infusions of casein and starch using a 2 x 2 factorial arrangement. The design was a replicated incomplete 4 x 4 Latin-square. All animals received the same basal diet (17.6% CP and 1.58 Mcal NEL/kg DM) throughout the study. Cows were restricted to 70% of ad libitum intake and infused abomasally for 36 h with water, starch (2 kg/d), casein (0.86 kg/d), or the combination (2 kg/d starch + 0.86 kg/d casein) using peristaltic pumps. Milk weights, milk samples, and arterial and venous blood samples were collected during the last 8 h of infusions. Mammary biopsy samples were collected and tissue protein prepared to evaluate cell signaling. Animals infused with casein had increased arterial concentrations of NEAA and EAA, as well as net uptake and clearance; however, milk protein yield did not increase. Animals infused with starch however, exhibited reduced arterial concentrations of NEAA and EAA but increased clearance and net uptake of most AA. Additionally, infusions of starch increased circulating concentration of insulin, IGF-I, and glucose as well as the rate of mammary plasma flow. Abomasal infusions of starch activated mammary activity of ribosomal protein S6 irrespective of other treatments. However, mammary tissue mTOR increased activity in response to casein only when starch was present during the infusions. These results suggest that cell signaling activation responded to different nutritional stimuli. Milk and protein yield increased in animals infused with starch. Therefore, MG positively responded to energy supply and engaged local and intracellular regulatory mechanisms to achieve that response. Understanding these adaptations could be beneficial in the development of mathematical representations for nutrients utilization in lactating animals. These two studies supported our hypotheses that regulatory mechanism are activated during limiting supply of AA to sustain protein synthesis in MG. The accuracy of mathematical models for lactating animals would increase if effects of energy on AA metabolism and cell signaling related to protein synthesis were included in the representation of milk protein synthesis. Ph. D.

Ovarian secretions and growth hormone (GH) are major endocrine regulators of mammary growth and development in bovine mammary gland; but information on endocrine regulation during early prepubertal period is limited. Our overall objective was to study the regulation of mammary growth and development as well as transcript abundance in early prepubertal bovine mammary gland by ovarian secretions and exogenous bovine somatotropin (bST). In the first study, we determined the effect of staged ovariectomy on mammary growth and development in two to four month old Holstein heifers. In the second study, effects of bST on mammary growth and development, and also on putative stem cell population were evaluated by beginning bST treatment in one month old Holstein heifers. Mammary growth and development was determined by mass of mammary tissue, biochemical analyses, histological examination, transcript abundance and protein expression in mammary parenchyma (PAR) and fat pad (MFP). Ovariectomy reduced mass and lipid content of PAR without affecting the histological characteristics or rate of epithelial cell proliferation. There was a marked reduction in progesterone receptor expression both at the mRNA and protein level. Ovariectomy also reduced transcript abundance in GH receptor (R), insulin-like growth factor (IGF)-1, IGF binding protein -6, estrogen responsive and proliferation marker genes, but increased the mRNA abundance of IGF-1R in PAR. On the other hand, administration of bST did not have an impact on PAR growth, epithelial proliferation, putative stem cell population or transcript abundance of IGF-axis genes. However, bST reduced the relative abundance of GHR, signal transducers and activators of transcription-5b and suppressors of cytokine signaling-2 in mammary PAR. Transcript abundance of IGF-axis molecules, estrogen responsive genes and proliferation markers in MFP was not affected by ovariectomy or bST. Overall, our data suggest that ovary is a predominant regulator of mammary growth and development in prepubertal heifers and that exogenous bST is not effective as a mammary specific mitogen in very young prepubertal heifers. Ph. D.

The Virginia Tech crossbreeding program began in the fall of 2002 by mating Holstein and Jersey foundation females to Holstein and Jersey bulls to create HH, HJ, JH, and JJ breed groups (sire breed listed first). Collection of daily dry matter intakes (DMI) began in the fall of 2005 and continued through spring of 2008. DMI were measured for two weeks out of every six week period in first lactation on 44, 32, 29, and 13 HH, HJ, JH, and JJ cows. Daily milk and body weights and monthly milk components were collected. The National Research Council (NRC) DMI prediction equation was used across breed groups to investigate breed differences. DMI prediction equations were developed across breeds. Random regression model were used to predict energy consumed (TEC), energy needed for production, maintenance, and growth at every week of lactation (WOL). Energy balance (EB) was calculated at every WOL by subtracting energy for production, maintenance, growth, and pregnancy from TEC. Common health events were recorded if they occurred in the first 100 days of lactation. Logistic regression was used to analyze health disorder. The HJ and JH were not different from each other in any analyses. The NRC under-predicted DMI for HH and over-predicted DMI for HJ, JH, and JJ. There were significant breed differences in the prediction equations developed. Results indicated that breeds differ in DMI after accounting for production and body weights. The HH cows consumed more energy than HJ and JJ cows. The HH, HJ, and JJ were not different from each other for production but were different from JJ. The JH allocated less energy to maintenance than the HH. There were no breed differences for weeks required to reach positive EB, return to positive cumulative EB, or EB at week 21 of lactation. The HJ, JH, and JJ were more likely to have an incidence of mastitis than HH. The HJ and JH were less likely to have an incidence of metritis than HH. The results indicate breeds differ in DMI, and health diseases and evidence suggests differences in characterization of EB and warrants further investigation. Ph. D.

This study was to evaluate effects of milk replacer (MR) composition on: mass and composition of mammary parenchyma (PAR) and fat pad (MFP), growth hormone (GH)/insulin-like growth factor-I (IGF-I) axis gene expression, and putative mammary epithelial stem cells. The hypothesis was that diet during the pre-weaning period alters the development, persistence, or activity of populations of putative mammary epithelial stem cells, possibly through involvement of GH/IGF-I axis molecules. Twenty-four newborn heifers were fed one of four MR diets: CON (20% CP, 21% fat MR fed at 441 g DM/d), HPLF (28% CP, 20% fat MR fed at 951 g DM/d), HPHF (27% CP, 28% fat MR fed at 951 g DM/d), and HPHF+ (27% CP, 28% fat MR fed at 1431 g DM/d). Animals were harvested on d 65 of life and mammary tissue was subjected to biochemical, molecular, and histological examination. By design, the effects of diet were evaluated at a common chronological age, but not necessarily at the same physiological age (body weight). Results from heifers reared on CON were compared to the average results from heifers reared on the other 3 diets. The second comparison evaluated the effect of increased fat in MR when protein content and intake were the same. The final comparison evaluated the effect of increased intake of a high-fat, high-protein MR. Neither diet composition nor nutrient intake in pre-weaned heifers affected PAR weight, PAR composition, GH/IGF-I axis gene expression, or putative mammary epithelial stem cell abundance when assessed at a common chronological age. Changes in MFP size and composition were observed, but no diet effect on GH/IGF-I axis gene expression in MFP was observed. This suggests nutrition is not critical for regulating the expression of local GH/IGF-I axis components or stem cell populations in the developing heifer mammary gland. Ph. D.

The overall objective of this dissertation research was to understand the mechanisms by which serum insulin-like growth factor-I (IGF-I) is regulated by nutritional intake in cattle. Two studies were conducted to achieve this objective. In the first study, effects of feeding levels on basal and growth hormone (GH)-stimulated serum concentrations of IGF-I, IGF binding protein-3 (IGFBP-3) and acid-labile subunit (ALS), and their mRNA expression in the liver were determined in beef cows. It was found that increased nutritional intake did not alter basal concentrations of serum IGF-I, IGFBP-3 or ALS, or their mRNA expression in the liver. However, under increased nutritional intake, GH administration stimulated a greater increase in serum IGF-I concentration, and this greater increase was not due to reduced degradation of IGF-I in serum. Increased nutritional intake did not enhance GH-stimulated IGF-I mRNA expression in the liver, but it increased the amount of IGF-I mRNA associated with polysomes, suggesting that liver translation of IGF-I mRNA is enhanced under increased nutritional intake. Under increased nutritional intake, GH also stimulated greater increases in serum IGFBP-3 and ALS concentrations, but these greater increases were not due to greater expression or translation of their mRNAs in the liver. Taken together, these results suggest that translation of GH-stimulated IGF-I mRNA in the liver is enhanced under increased nutritional intake and this enhancement may be partially responsible for the greater GH-stimulated increase in serum IGF-I concentration. These results also suggest that the greater GH-stimulated increases in serum IGFBP-3 and ALS may be secondary to the greater increase in serum IGF-I because increased IGF-I may increase the formation of IGF-I/IGFBP-3/ALS complexes, thereby increasing the retention of IGFBP-3 and ALS in the blood. In the second study, the effects of food deprivation on serum IGF-I concentration in steers and the underlying mechanism were determined. It was found that food deprivation decreased serum IGF-I concentration and that this decrease was not due to increased IGF-I degradation in serum. Food deprivation decreased liver IGF-I mRNA expression, and this decrease was associated with decreased expression of GH receptor (GHR) mRNA and protein in the liver. Food deprivation was also associated with increased mRNA expression of two inhibitors of the GHR signaling pathway, suppressor of cytokine signaling-2 (SOCS2) and cytokine-inducible SH2 protein (CIS). These results suggest that decreased IGF-I gene expression in the liver may be at least partially responsible for the decrease in circulating IGF-I concentration during food deprivation, and that the former decrease may be due to increased expression of SOCS2 and CIS, and decreased expression of GHR in the liver. Overall, this dissertation research indicates that multiple mechanisms are involved in nutritional regulation of circulating IGF-I concentration in cattle. Ph. D.

Studies were conducted to investigate the tissue distribution of stearoyl-CoA desaturase-1 (SCD) and the regulation of SCD1 protein expression by dietary fat, insulin, polyunsaturated fatty acids (PUFA), and linoleic acid (cis-9, cis-12 18:2). The first study examined tissue distribution of SCD1 protein in Holstein calves (n=6/diet) fed one of four milk replacer diets for a nine wk period after which they were sacrificed. Milk replacer diets varied in fat content and were formulated and administered as follows: 0.4 kg/d 20% protein, 20% fat (20:20; CON), 0.97 kg/d (28:20; HPLF), 0.97 kg/d (28:28; HPHF), or 1.46 kg/d (28:28; HPHF+). Samples of subcutaneous adipose tissue (AT), perirenal AT, omental AT, duodenum, proximal jejunum, distal jejunum, ileum, and liver were collected from calves fed the HPHF+ diet to determine SCD1 tissue distribution. Tissue homogenates were prepared and used for Western blotting. Additionally, dietary effects were analyzed on tissues expressing SCD1 protein for all 24 calves. The second study investigated the regulation of SCD1 protein expression by insulin, fatty acids increasing in degree of unsaturation, and increasing concentrations of linoleic (18:2) acid. Subcutaneous AT was collected from Smith Valley Meats in Rich Creek, VA and used to prepare explants cultured in treatment media for 24 h. Treatments consisted of insulin at 0, 7, 14, and 21 nM; stearic (18:0), oleic (18:1), linoleic (18:2), and linolenic (18:3) acids at 100 μM; and linoleic (18:2) acid at concentrations of 0, 25, 50, 75, and 100 μM. Tissue explant homogenates were used for Western blotting to detect SCD1. In the first study, we found that SCD1 protein was detectable in subcutaneous AT, perirenal AT, and omental AT; however, it was not detectable in liver or small intestine samples. Also, the HPHF+ diet increased SCD1 protein expression in subcutaneous AT and perireanl AT. In the second study, SCD1 protein expression increased linearly with insulin concentration. There was no fatty acid treatment effect, but there was a negative linear effect with increase in degree of unsaturation. Finally, there was no effect on SCD1 protein expression with linoleic acid increasing in concentration. In conclusion, results indicate that SCD1 protein expression was detected in bovine AT depots, regulated by dietary fat, insulin, and by PUFA . Master of Science

Impact of precision feeding with feed management software was assessed for whole farm nutrient balance (WFNB) and feeding management from January through December 2006. Nine treatment and six control farms were selected in four regions of the Chesapeake Bay Watershed of Virginia. Herd sizes averaged 271 and 390 lactating cows for treatment and control farms while milk yield averaged 30 and 27 kg/d per lactating cow, respectively. Crop hectares grown averaged 309 and 310 ha for treatment and control farms, respectively. Treatment farms purchased and installed feed management software (TMR Tracker, Digi-Star LLC, Fort Atkinson WI) between May and October 2006 and received more frequent feed analysis and feedback. Data were collected for calendar year 2005 and 2006 to compute WFNB using software from the University of Nebraska. On treatment farms, up to five feed samples were obtained monthly from individual feedstuffs and each total mixed ration (TMR) fed to lactating cows. Control farms submitted TMR samples every 2 mo. Standard wet chemistry analysis of samples was performed. Data stored in the software were collected monthly from each treatment farm concurrent with feed sampling. Producers from each treatment farm participated in a 24-question personal interview in December 2006 addressing installation, operation, and satisfaction with the software. Daily feeding deviation of all ingredients across treatment farms averaged 173 ± 163 kg/d. This corresponded to average daily overfeeding of CP and P of 17.6 ± 17 and 0.4 ± 0.3 kg/d, respectively. Feeding deviation did not differ between feeders. Milk production was negatively associated with kg total deviation and kg CP deviation, but positively related to P deviation. Whole farm nutrient balance did not differ between treatment and control farms. All producers indicated TMR Tracker met expectations. Change made to the feeding program due to TMR Tracker was correlated (r=0.80) with perceived improvement in ration consistency. In conclusion, producers perceived feed management software as beneficial, but WFNB was not reduced after 3 to 6 mo of using feed management software; however, the large variation in daily over or under feeding indicates potential for future reductions in WFNB through reduced feeding variability. Master of Science

Lactation persistency is defined as the ability of a cow to maintain production at a higher level after peak yield. Hypothetically, more persistent cows are less susceptible to health and reproductive disorders. The objective of this research was to investigate the phenotypic relationships of common health disorders in dairy cows to lactation persistency. The relationships with peak yield and days in milk (DIM) at peak yield were also studied. Two separate investigations (Study 1 and Study 2) were performed. Study 1 used treatment incidence data and daily milk weights of 991 lactations from experimental dairy herds at Virginia Tech and Pennsylvania State University. Milk yield persistency (PM) was estimated for individual lactations using daily milk weights. In Study 2, producer recorded health data of 87555 lactations from 398 commercial herds were used. PM, fat (PF), and protein (PP) persistencies were estimated from TD yields. Mastitis only in the first 100 days, only after 100 DIM, and at any stage of lactation, and reproductive disorders including metritis, retained placenta, cystic ovaries, metabolic diseases including ketosis, milk fever and displaced abomasums, and lameness were considered in both studies. Mastitis both before and after 100 DIM was considered only in Study 1. Each disease was defined as a binary trait distinguishing between lactations with at least one incidence (1) and lactations with no incidences (0). Standardized measures of the persistencies, uncorrelated with yield, were calculated as a function of yield deviations from standard curves and DIM deviations around reference dates. Peak milk yield and DIM at peak of individual lactations were computed using Wood's function. Effects of persistency (PM, PF, and PP) on probability of the diseases in current and next lactations were examined through odds ratios from a logistic regression model. Conversely, the effects of diseases on persistencies, peak milk yield, and DIM at peak milk yield were also examined. Increasing PM, PF, and PP tend to reduce the incidence of mastitis, specifically in late stages of current and next lactation. PM and PP appear to have greater impact on mastitis than PF. No other likelihood of a disease was affected by the increasing persistencies. Post partum reproductive and metabolic diseases often had substantially positive effect on persistencies of both primiparous and multiparous cows (p

Considerable changes have occurred in environmental regulations in recent years, only one of which is the requirement of stand alone heifer operations and feedlots to carry environmental permits. While growth of heifers is a widely researched topic, publications concerning nutrient utilization, partitioning, and excretion are scarce and are becoming necessary. That combined with the fact that feeding programs for heifers are highly variable from region to region and even from farm to farm indicated the need to examine the effects of diet on nutrient utilization. Therefore, the objective of this work was to evaluate how differences in levels of dietary protein and energy will affect growth and nutrient utilization in heifers from birth to calving. Two projects were conducted, one in 20 month old bred heifers where forage level in the diet was altered to provide the required nutrients in less feed and one in young calves where energy and protein content of the milk replacer was altered. Three diets were fed to 18 (n=6), 20 month old heifers who were in late gestation; the first was the high forage (HF) ration which was 90.7% forage and 9.3% concentrate, the second was a by-product (BP) ration which was designed to have the same level of fiber as the HF ration, however soybean hulls and cottonseed hulls were added at the sacrifice of the grain mix which increased the fiber content; the last ration was the low forage (LF) which was 54.7% concentrate and 45.3% forage and was fed at ~89% of the ad libitum intake of heifers fed the HF ration. Heifers fed the HF ration had greater fecal excretion compared to those fed LF, however heifers fed the LF ration exceeded the heifers fed HF and BP by 4.5 and 2.5 times, respectively, in urine volume excretion (40.2 vs. 8.9 and 16.9 kg/d, respectively). Although total N excretion (kg/d) was not different, heifers fed the LF ration tended to partition more N to urine than to feces. Phosphorus excretion in the feces was not different, however heifers fed HF and BP tended to have greater fecal P (P < 0.06). Urinary P excretion was less in heifers fed HF and BP compared to LF, however these heifers were excreting as much urinary P as a lactating cow. Calves were purchased from a commercial dairy at 3 d old (± 1d) and transported to the VT Dairy Center. They remained on study until 63 d when they were harvested for body composition. Four treatment diets were fed; a control milk replacer (24/17; 24% CP, 17% Fat), a high protein, low fat (32/17; 32% CP, 17% Fat), a high protein, high fat (31/24, 31% CP, 24% fat), and that same 31/24 milk replacer fed at 1460 g/d powder (31/24+). Calves were offered a 20% cottonseed hull starter at 1 d after arrival to the VT Dairy Center. Calves fed 24/17 consumed more starter than those fed the other milk replacers and therefore had a lower apparent digestibility and greater fecal excretion. Fecal N excretion was not different, although calves fed 24/17 tended to have greater fecal N excretion. Urinary N excretion was higher in calves fed 31/24+ compared to those fed 31/24. Total N excretion and N retention were not different. Empty body weight (EBW) gain was greater in calves fed 31/24+ compared to 31/24, however those same calves also had a higher percent of EBW as fat. Calves fed 32/17 had the most lean gain (in the form of N gain) compared to those fed extra energy (31/24) and also had a higher N as a percent of EBW. Limit feeding Holstein heifers late in gestation did not reduce nutrient excretion, however, more digestible nutrients were available to the heifer and fetus. Heifers in late gestation are likely over fed P and therefore excrete nearly everything they consume which has negative implications for nutrient management planning. Calves fed a low protein, low fat milk replacer did not grow as well as calves fed higher protein. Nitrogen retention and CP gain were higher when protein was at least 31% and fat was at least 17%. Feeding fat over 17% only increased fat gain and not CP gain. Overall, paying for extra nutrients in bred heifer diets seemed to be beneficial, however, feeding above 31% CP and 17% fat increased nutrient loss to the environment. Ph. D.