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Salmonella vaccination is a widely accepted practice in the United States poultry industry. These vaccines stimulate a strong humoral response to Salmonella serovars and significantly reduce Salmonella load in the bird. In general, a Salmonella vaccination program for an egg-type pullet flock consists of two administrations of a live attenuated Salmonella vaccine at 1- and 6-wk-of-age followed by one administration of a Salmonella bacterin at 12-wk-of-age. Although these vaccines are regarded as effective against paratyphoid Salmonella infections, there are incidences of breakthrough Salmonella infections and severe injection site reactions after bacterin administration. Therefore, the main objective of this dissertation was to conduct longitudinal studies to assess local (growing feather, a dermal test) and systemic (blood, spleen, liver, cecal tonsil) cellular- and humoral-immune responses to live attenuated- and killed-Salmonella vaccines in egg-type chickens. In Chapter 1, intradermal (i.d.) injection of commercial Salmonella vaccines or vaccine components (lipopolysaccharide or water-oil-water emulsion) into the pulp of growing feathers (GF) of non-vaccinated (primary response) and Salmonella-vaccinated (secondary response) Light-brown Leghorn pullets revealed that a secondary administration of Salmonella vaccines prolongs heterophil recruitment and stimulates local T helper (Th)-17 cell-mediated responses at the site of injection. In Chapter 2, specific pathogen-free White Leghorn pullets were administered a multiphase Salmonella vaccination program (two administrations of a live attenuated Salmonella vaccine, POULVAC® ST, followed by an intramuscular administration of killed S. Typhimurium, or ST, bacterin) and found that CD4+ T cell levels decreased in peripheral blood and liver while γδ T cell levels increased in the spleen and cecal tonsils after live vaccine administration and booster administration of the bacterin. At the end of the Salmonella vaccination program, GF pulps of Salmonella-vaccinated (sensitized)- and non-vaccinated (non-sensitized)-chickens were i.d. injected with ST. Sensitized chickens that were i.d. injected with ST had greater heterophil-, macrophage-, and B cell-localization at the site of injection when compared to those of non-sensitized birds, while non-sensitized birds i.d. injected with ST had prolonged γδ T cell localization. When assessing Salmonella-specific antibody responses in peripheral blood, there was evidence of a T cell-mediated IgM-to-IgG isotype switch in sensitized birds. In Chapter 3, cecal pouch microbiota was assessed prior to vaccination and at 10 d post-vaccination during the multiphase Salmonella vaccination. There was a compounding effect with repeated vaccinations on the cecal pouch microbiota alpha diversity in Salmonella-vaccinated chickens, where indices were higher in Salmonella-vaccinated chickens at 12-wk-old when compared to those of 12-wk-old non-vaccinated chickens. Additionally, the compositions of cecal pouch microbiota in Salmonella-vaccinated- and non-vaccinated-chickens had distinct clustering in Jaccard and Bray-Curtis PCoA plots during the entire vaccination program, where Salmonella-vaccinated birds had lower relative abundances of Lactobacillus with higher abundance of Lachnospiraceae when compared to that of non-vaccinated chickens. The results of these studies, when assessed together, provide qualitative descriptions of leukocyte profiles and ceca microbiota status during a longitudinal assessment of multiple Salmonella vaccine administrations. Future studies investigating interactions between vaccine responses and gut microbiota status in poultry can provide valuable insights into improving vaccine regimens in the future.

Salmonella infection in chickens is a significant and complex challenge to manage, affecting poultry health and food safety. Vaccination against Salmonella is a promising preventative approach to control flock infection and decrease the loads of this pathogen. Vaccination strategies against Salmonella often comprise the application of live attenuated and killed (inactivated) vaccines, including inactivated autogenous vaccines. Live attenuated Salmonella vaccines stimulate both cellular and humoral immunity but can potentially return to a virulent state; meanwhile, inactivated Salmonella vaccines trigger more of a humoral response and are safer since they are entirely rendered inactive. However, there is a lack of information on the cellular immune mechanisms occurring at the site of administration of inactivated Salmonella vaccines, limiting our understanding of how these vaccines can trigger chickens' immune defenses and how they could be improved. The dual-window approach, which comprises the growing feather (GF)-pulp test system and concurrent blood sampling, has been established as a minimally invasive comprehensive tool for monitoring in vivo local tissue/cellular and systemic immune responses to i.d. GF-pulp administration of test materials. In this dissertation project, the dual-window approach was used to monitor and assess the local- and systemic-cellular and humoral immune responses following different inactivated Salmonella vaccines and their components to gain insights into the impact of formulation, method of inactivation, route of administration, and antigen re-exposure on innate and adaptive immune responses in egg-type pullets. Administration of inactivated Salmonella vaccines or their components into GF-pulp stimulated tissue/cellular inflammatory responses, distinguished by the recruitment of different leukocytes from the blood into the GF-pulp, influencing circulating blood leukocyte profiles and leading to robust T-dependent Salmonella-specific antibody production in the plasma characterized by isotype switching from IgM to IgG and IgA. This investigation provides comprehensive knowledge about the temporal, quantitative, and qualitative local- and systemic-cellular and humoral immune responses to different inactivated Salmonella vaccines and their components in chickens. Overall, these findings not only provide practical insights for the development of inactivated Salmonella vaccines that can effectively stimulate protective levels of immunity against Salmonella in chickens but also serve as a basis for future investigations on the understanding of local and systemic immune responses to other types of immune modulators that can enhance poultry health.

Enterococcus cecorum (EC) has primarily been associated with osteomyelitis and lameness in older chickens. The recent emergence of pathogenic EC causing morbidity and mortality in young broiler chicken flocks requires further investigation. The purpose of this thesis was to develop a model to simulate horizontal transmission of virulent EC during the hatching phase in small scale hatchet cabinets. The model will be used to investigate novel pre- and post-hatch strategies to mitigate EC infections in young broiler chickens. The current thesis contains four chapters. Chapter I includes the thesis introduction. A literature review focusing on EC-associated disease in chickens, with a focus on transmission and virulence attributes, has been provided in Chapter II. Chapter III provides a summary of the EC horizontal transmission model development and effect of exposure to virulent EC during the hatching phase on colonization, macroscopic lesions, and early performance in broiler chickens. Lastly, Chapter IV contains the thesis conclusion.

The agricultural sector is experiencing a profound shift driven by technological advancements and the imperative for sustainable food production. Within this context, Precision Livestock Farming (PLF) has emerged as a transformative approach to animal management (Berckmans, 2017), leveraging data-driven decision-making, automation, and monitoring systems to enhance livestock production efficiency while minimizing resource usage and environmental impact. This thesis delves into a comprehensive examination of PLF as applied to broiler farming, particularly focusing on current usage perceptions, needs, and its potential to revolutionize the poultry industry. Research in Precision Livestock Farming has steadily increased since 2000, with a growing percentage of publications focusing on poultry. However, a significant portion of this literature centers on prototype products, with limited exploration of PLF perceptions. The primary objective of this thesis is to gauge the current perceptions, usage, and needs of PLF systems among academia. An online survey was conducted, targeting Animal Scientists, Engineers, and Veterinarians. Results indicated a consensus among poultry researchers regarding the future potential of PLF to enhance poultry production. While most respondents were familiar with PLF terminologies (92%), only about half reported current utilization of PLF technologies (55%). Cost and usability emerged as the primary barriers to implementation, cited by 93% and 88% of respondents, respectively. Notably, opinions varied based on experience with PLF technologies, with current users expressing greater confidence in PLF's potential to expand research opportunities. The thesis also includes a field implementation project, testing the Scout® PLF system in a commercial broiler house. Scout® is a rail-mounted robot system designed for mortality detection and environmental monitoring. Regression analysis revealed strong linear relationships between Scout® measurements and reference sensors for temperature (R2 = 0.88) and relative humidity (R2 = 0.86), albeit with discrepancies outside expected sensor accuracy. Mortality detection accuracy was modest (15%), with unexpected clustering between feed and water lines. Bird occlusion was identified as a contributing factor, supported by experiments showing higher detection rates for birds within pens. In summary, addressing barriers such as cost, usability, and improving environmental monitoring systems are critical for the widespread adoption and success of PLF in the industry.

King Rails (Rallus elegans) are a secretive marsh bird species of conservation concern in Arkansas. Freddie Black Choctaw West WMA in southeast Arkansas has multiple confirmed pairs of breeding King Rails and offered an opportunity to investigate King Rail migration ecology, as well as nesting ecology and nest site selection. In Chapter 1, we determined migratory status (resident vs. migratory) and described seasonal movement. To do this, we captured and outfitted 23 birds with Argos GPS tags to track migration patterns throughout the season. We found that most of the population did not make long distance migrations but instead were residents year-round. We examined monthly movements on the landscape to determine how their movement changed seasonally to help understand how management impacts their use of habitat. In Chapter 2, we investigated nest site selection relative to the habitat available across the Wildlife Management Area, in a used versus available habitat framework. To do this, we performed habitat surveys at confirmed nest sites and random points throughout the WMA to assess what characteristics are significant to King Rails. We ran generalized linear models to see what rails select for in a used versus available habitat framework. King Rails selected for nest sites that had higher average vegetation density and percent of rush cover. From this research, we corroborated that heterogeneous habitat is important for both the breeding and movement ecology of this species and recommend management that promotes diverse habitats that support these birds year-round.

Globally, poultry is one of the most versatile and cost-effective sources of animal-based protein. Due to its wide variety of uses and variability of growing styles, poultry production is applicable in nearly every country. With the increase of scale and concentration of production, pathogenic diseases have decreased the efficiency of production through worsened feed conversion, morbidity, and mortality. In addition to physiological health, climate largely impacts the suitable conditions for growing. With more extreme climate events, locations once ideal for poultry are less efficient due to increased resource cost. This analysis seeks to determine the heterogeneity of production and the impact of disease and climate factors from 2000 to 2020 as they affect productivity by region. Using time series panel modeling, we predict that disease has a negative impact on the value of poultry production with a larger effect in upper-middle income countries with the transition to higher stocking densities and less established resources when compared to high income countries. This analysis provides a better understanding of the drivers for global productivity and the impact of disease and climate as they continue to emerge and change.

The purpose of the present research was to evaluate the bactericidal activity of a microencapsulated Lippia origanoides essential oils (LOEOs) against Salmonella Enteritidis (SE) in an in vitro crop assay and in a prophylactic broiler chicken model using three low doses of the product: 75 ppm, 150 ppm and 300 ppm. Two trials of each assay were conducted. Day-of-hatch male Cobb-Vantress broiler chickens were randomly allotted to one of four groups: 1) positive control group; 2) 75 ppm LOEOs group; 3) 150 ppm LOEOs group; 4) 300 LOEOs group. Chicks were fed and watered ad libitum in heated brooder batteries in a controlled, age-appropriate environment. On day seven of age, all chickens were orally gavaged with 108 cfu of SE. Chickens were euthanized by CO2 exposure 24 hours after the SE challenge. Recovery of SE was evaluated in liver- spleen, and ceca (including cecal tonsils). None of the three microencapsulated LOEOs doses changed SE concentration (P > 0.05) in the in vitro crop assay. Similarly, no significant differences in SE were observed in the organ and intestinal colonization in broiler chicken trials. Therefore, the low concentrations of the microencapsulated LOEOs used do not reduce SE concentration in different poultry models. Further studies will be needed to optimize the formulation, delivery, models, and concentration of essential oils to control Salmonella infections in broiler chickens effectively.

Broiler breeder performance is based upon egg production. Breeders are reared from day of hatch to ovulation with the main goal of controlling body weight. Maintaining body weight in breeders is highly emphasized prior to first egg as to reach optimum peak production feeding increases dramatically to meet energy needs. Energy is supplied to the breeder in three forms carbohydrates (fast energy), protein (medium energy), and fat (slow energy). Carbohydrates are quickly used by the breeders, while protein is constantly being utilized by the breeder for growth and egg production, fat is stored and used lastly as the energy requirements exceed protein turnover’s ability to meet these requirements. Protein turnover (synthesis and degradation) works constantly to supply energy for the breeder to grow, produce eggs, and complete maintenance within the breeder. Protein turnover is not enough to supply energy to meet every metabolic energy need in the breeder consistently for extended periods of high energy strain, thus lipid turnover supports protein turnover during extended periods of energy strain. Lipid turnover is an evaluation of triglyceride synthesis and degradation in the breeder. Lipid turnover is extremely slow, synthesis of triglycerides occurs at very low rates and requires long period to build up reservoirs of energy. Degradation of fat is a last resort for the breeder to supply energy to perform, due to the slow nature of lipid mobilization it requires time for the breeder to begin utilization of fat. Protein and lipid turnover working in tandem with one another can generate increased energy to meet egg production. Energy is important for the breeder to be able to do the work required for egg production, but good egg quality is necessary for reproduction of chicks. To produce eggs of good quality multiple minerals are needed, of this calcium is one of these important minerals. Calcium is provided in the diet from young ages and is an essential mineral for skeletal development and maintenance of the pullet. As calcium is continuously supplied to the breeder over long periods of time prior to egg production the body has the ability to store it for utilization at a later time. Calcium mobilization concentrations can be altered to increase or decrease the available calcium for egg production. Along with multiple other minerals, tracing the calcium being given to the breeder from day of hatch to the output of calcium in eggshells evaluates when breeders are altering concentration levels of minerals to support increased egg production, growth, and maintenance of the body.

Understanding species’ distributions and how a species uses its environment is a vital aspect of ecology and important for the development and implementation of conservation planning. In migratory species, with distinct breeding and nonbreeding ranges, understanding distributions is a complex endeavor. Nevertheless, evaluating movement behavior by linking seasonal distributions and identifying source areas is vital for understanding a species’ full annual cycle ecology. Understanding distributions is particularly important for predators, like birds of prey, that can exert top-down effects on community structure and ecosystem function through their influence on prey populations. Among raptors, owls are severely understudied in large part because their cryptic habits prevent them from being easily detected using widespread, standardized study approaches—something that is especially apparent during the nonbreeding season. In recent decades, concerning trends have been observed in the movement dynamics of diurnal raptors and how this may translate to owls is poorly understood. The Northern Saw-whet Owl (Aegolius acadicus) is a small, migratory forest owl whose widespread distribution and abundance makes it an excellent model for highlighting research priorities in owls and other migratory raptors. In Chapter I, I explored sources of variability for d2Hf in feather samples obtained from juvenile saw-whet owls during the breeding season from 26 nest sites located in three regions: Quebec (CA), South Dakota and Nebraska (USA). I explored the relationship between d2Hf of juveniles and a set of covariates, as well as within-nest d2Hf variation of juveniles and a set of covariates. Results demonstrate total precipitation (year prior to feather growth), latitude, and number of siblings within a nest to be important in predicting d2Hf enrichment, but d2Hf values differed from predicted values commonly used for diurnal raptors. In samples from Quebec, I found unpredicted d2Hf enrichment, as well as temporal variation in d2Hf enrichment in juveniles and their parents. These results demonstrate the complex nature of using d2Hf in studies of migratory raptors and help inform the use of d2Hf in studies of cryptic migratory species. In Chapter II, I used acoustic monitoring to explore nonbreeding season occupancy and detection probabilities of saw-whet owls in the southern Interior Highlands of central North America, a region where they have only recently been recognized as occurring during the nonbreeding season. I used autonomous recording units to sample 56 sites across a large region, and results demonstrated overall high occupancy and low detection probabilities in the region’s pine forest. Occupancy probability was greater at more northerly study sites, where conifer cover was patchier on the landscape, as well as at study sites with greater Barred Owl activity. My study provides valuable information on the poorly-understood nonbreeding distribution of the saw-whet owl and highlights a successful effort to better understand the overwintering ecology of a cryptic, migratory raptor. In Chapter III, I conducted an experiment to better understand the thermal environment experienced by saw-whet owls at diurnal roost sites during the nonbreeding season. Using operative temperature models, I demonstrated that roost sites in conifer crowns—commonly used by saw-whet owls—were more thermally buffered and experienced lower wind speeds than other roost sites likely to be used by owls. My results suggested the species may choose roosting sites to avoid thermally stressful temperatures, particularly at the milder, southern fringe of their nonbreeding distribution. This study provides critical information on the thermal environment experienced by roosting saw-whet owls and could have implications in predicting other novel regions in which the secretive species may occur.

Concerns around the economic and environmental consequences of suboptimal or excess dietary P levels in broiler diets has led to increased interest in determining ileal P digestibility values for feed ingredients. Therefore, two experiments (EXP) were conducted to determine the apparent (AID) and standardized ileal digestibility (SID) of P in various inorganic phosphates and meat and bone meals (MBM) using the direct method. In both EXP 1 and 2, ileal samples were collected at 22 d post-hatch from birds fed semi-purified diets based on dextrose and corn starch containing test ingredients and one N and P free (NPF) diet for determination of ileal endogenous P losses (IEL). In EXP 1, diets containing monocalcium phosphate (MCP), dicalcium phosphate (DCP), monodicalcium phosphate (MDCP), defluorinated phosphate (DFP), bovine meat and bone meal (B-MBM), and porcine meat and bone meal (P-MBM) were formulated to 0.31% total P and balanced to 0.65% total Ca with limestone. Monosodium phosphate (MSP) was included at 3 levels to provide 0.13, 0.23, and 0.33% total P. Apparent ileal digestibility of P values were separated on two levels of statistical significance (P73%), no P source, including MSP, was totally available. Additional work is needed to establish the factors that influence ileal P digestibility among inorganic and animal protein sources.

The objective of this dissertation was to evaluate the impact of environmental and nutritional enrichments on the behavior, bone health, and welfare of Hy-line brown and Hy-line W-36 laying hens. Laying hens are prone to experiencing a progressive increase in bone fragility due to the ongoing mobilization of calcium from the bones for eggshell formation. Over time, this increases their susceptibility for bone fracture, which is a welfare concern. Prior research suggests that exercise, especially during the developmental stage, can aid in minimizing bone fractures by strengthening muscles and increasing bone mass. Furthermore, nutrition plays an integral role in laying hen skeletal health. We investigated two interventions to prevent or reduce the negative effects observed on laying hen skeletal health: 1) environmental enrichment, which included the provision of multi-tier perches at various time points within the lifespan of laying hens and 2) nutritional enrichment, which involved supplementing the diet with boron. The first aim of this dissertation (described in Chapter 2) was to determine the effect of perch provision during the rearing phase on the activity and musculoskeletal health of pullets. Pullets (n=810) were either housed with or without multi-tier perches from 0 to 17 weeks of age (15 pens/treatment, 29 birds/pen). We expected pullets with perches to show improved musculoskeletal characteristics compared to pullets reared without perches. At 5, 11, and 17 weeks, birds were individually monitored for activity level over 3 consecutive days. At 11 and 17 weeks, 60 birds were euthanized for computed tomography (CT) scans to quantify tibiotarsal bone mineral density (BMD) and cross-sectional area (CSA). After CT scanning, birds were dissected for measurement of muscle size, tibiotarsal breaking strength, and tibiotarsal ash percentage. Novel markers of bone mineralization (bone-specific alkaline phosphatase [BALP] and pro-collagen type 1 n-terminal propeptide [P1NP]) were measured from serum samples of 60 birds/week. Results indicated that pullets reared with perches from 0-17 weeks of age exhibited increased levels of vertical activity, with no significant effect on overall activity level. Pullets with perches had greater total and cortical BMD at week 11, with increased cortical bone CSA and higher total and cortical BMD at week 17 compared to pullets without perches. At week 11, pullets with perches had heavier leg muscles, with heavier triceps, biceps, pectoralis major and minor, and leg muscles at week 17 than pullets without perches. At both weeks, pullets with perches had greater tibiotarsal breaking strengths, higher ash percentages, and greater concentrations of BALP and P1NP than pullets without perches. These results indicate that activity resulting from perching elicits a beneficial impact on measures of pullet musculoskeletal health at both 11 and 17 weeks of age. The second objective (described in Chapter 3) was to determine whether there were enduring impacts of perch provision timing on the musculoskeletal health of laying hens. Pullets (n=812) were housed under different conditions (7 pens/treatment, 29 birds/pen) with either continuous access to multi-tier perches from 0 to 40 weeks of age (CP), no access to perches (NP), early access to perches during the rearing phase from 0 to 17 weeks of age (EP), or solely during the laying phase from 17 to 40 weeks of age (LP). We expected hens from the CP group to exhibit improved musculoskeletal health as a result of perch-related activity compared to hens from the NP group, with hens from the EP group having improved musculoskeletal health compared to hens from the LP group due to increased activity during the developmental stage. At weeks 24, 36, and 40 of age, birds were individually monitored for activity level over 3 consecutive days, and blood samples were collected from a separate set of 3 birds per pen to analyze serum concentrations of tartrate-resistant acid phosphatase 5b (TRACP-5b) and C-terminal telopeptide of type I collagen (CTX-I) as novel markers of bone demineralization. At 40 weeks of age, 3 birds per pen were euthanized for CT scans with further analysis including muscle weights, tibiotarsal breaking strength, and tibiotarsal ash percent. During week 24, hens from CP, EP, and LP pens had the highest overall activity compared to hens from NP pens, with no differences at week 36 or 40. During all weeks, hens from CP and LP pens had greater vertical and less horizontal activity compared to those from EP and NP pens. TRACP-5b and CTX-I concentrations did not differ at week 24 of age, with hens from CP pens having the lowest TRACP-5b and CTX-I concentrations compared to NP pens at 40 weeks of age. Total bone CSA did not differ between treatments, but CP had greater total BMD than NP with no differences between EP and LP pens. CP and LP hens had heavier biceps, pectoralis major, and leg muscle groups, as well as greater tibiotarsal breaking strengths than EP and NP pens. CP hens had higher tibiotarsal ash percentages compared to all other treatment groups. The results from this chapter indicate that the continuous provision of perches throughout the rearing and lay phase beneficially impacts activity level and measures of hen musculoskeletal health at 40 weeks of age, contributing to an overall improvement in laying hen welfare compared to no access to perches. Perch access during the early lay phase (17-40 weeks of age) had a positive impact on activity, muscle weight, and bone strength, but these benefits were not as great as those observed with continuous perch access. Perch access during the rearing phase (0-17 weeks of age) was associated with a decline in measures of bone demineralization, but did not have an overarching beneficial impact on other measures of hen musculoskeletal health or activity at 40 weeks of age, suggesting there was not a long-term benefit of perch access during the developmental stage. The third objective (within Chapter 4) was to determine the influence of perch provision timing on laying hen behavior, specifically anxiety and fearfulness. While providing perches may enhance biological functioning and animal welfare, their effectiveness could be age-dependent. This chapter investigated the effects of early and late perch access on anxiety and fear in hens through attention bias (AB) and tonic immobility tests (TI). Pullets (n=728) were raised with or without multi-tier perches either continuously (CP; 0-37 weeks), during only the rearing phase (EP; 0-17 weeks), during only the laying phase (LP; 17-37 weeks), or not at all (NP; no perch access). We expected hens from the CP group to show responses consistent with reduced anxiety and fear compared to hens from the NP group, with intermediate responses from hens in the EP and LP groups. AB was conducted in weeks 21 and 37 (n=84 birds/week) and TI was performed in weeks 20, 25, and 37 (n=112/week). CP hens fed quicker in the AB test than EP, LP, and NP hens at weeks 21 and 37. CP and NP feeding latencies were stable, while EP and LP fed faster at week 37 compared to 21. CP had the shortest TI duration at week 20, while both CP and LP had the shortest TI durations in weeks 25 and 37. Hens from LP pens showed increased anxiety levels at week 21 of age, which they adapted to by week 37, indicating that adaptation to a new adult environment requires at least 16 weeks. Also, LP hens exhibited reduced fearfulness by 20 weeks of age compared to hens that lost their perch access (EP) or never had perch access (NP). At 25 and 37 weeks of age, LP hens showed similar fear levels as hens from CP pens, indicating that current perch access reduces fearfulness. Removing perches at 17 weeks of age (EP) increased fear at weeks 20 and 25 and anxiety at week 21, effects that disappeared by week 37 of age. Furthermore, birds from EP pens showed decreased anxiety at 37 weeks of age compared to NP birds, suggesting that perch access, even when removed at 17 weeks of age, is more beneficial to anxiousness at 37 weeks of age than not having access to perches at all. Our findings indicated that providing hens with multi-tier perches throughout their lifetime improved affective state by reducing anxiety and fearfulness, while no access to perches negatively impacted measures of emotion and affective state. The last objective (described in Chapter 5) was to determine the effects of boron supplementation on pullet musculoskeletal health and performance parameters. Boron plays a role in the metabolism of calcium, which may help improve bone strength and prevent fracture. A total of 529 Hy-Line W-36 pullets were distributed across 24 pens and fed basal diets containing varying amounts of boron (C: (C: 0mg/kg; L: 50mg/kg; M: 100mg/kg; H: 150mg/kg) for 17 weeks. We expected pullets from the M group to show improved musculoskeletal health compared to the other treatment groups. Performance parameters (body weight, average daily weight gain/bird, and average daily feed intake/bird) were measured at weeks 4, 7, 10, 13, and 16, while all other measures were recorded at 11 and 17 weeks of age. Performance measures did not differ between treatment groups. Pectoralis major weights were higher in H pullets at 11 weeks of age, and we also observed higher pectoralis major, minor, and leg muscle weights in H pullets at 17 weeks of age. Pullets fed the H diet had larger cortical CSA than the other treatment groups at 11 weeks of age. At 17 weeks of age, both H and M groups had larger cortical CSA than L and C groups, but the M group had slightly smaller cortical CSA. Pullets fed the H diet had higher BMD values than other treatment groups at 11 weeks of age. At 17 weeks of age, pullets fed the H diet had the highest total BMD values compared to the other treatment groups, and cortical BMD increased with increasing boron inclusion. Pullets fed the H diet had the highest tibia ash percentages and concentrations of BALP and P1NP. Pullets fed the M and H diets had greater failure load and maximum bending moment than pullets fed the L or C diet at 11 weeks of age, with H pullets having greater stiffness values than other groups. At 17 weeks of age, pullets fed the H diet had greater failure load and maximum bending moment compared to all other treatment groups. Our results imply that providing boron within the diet at 150mg/kg improves musculoskeletal characteristics of Hy-Line W-36 pullets up to 17 weeks of age, without impacting performance parameters.

Three experiments were conducted to examine the effects of different feed additives in mitigating stress in broiler chickens. Experiment 1 used 504-day-old male by-product Cobb breeder chicks with 8 treatments replicated 9 times with 7 birds per replicate cage, structured as a 2x4 factorial arrangement of treatments. All birds were on the experimental diets for 20 days. There were 2 levels of lipopolysaccharide (LPS) challenge [no challenge vs. LPS challenge at 1 mg/kg BW) and 4 types of feed additives [no additive, 0.05% PostB, 0.1% PostB, and 0.1% TynagenTM]. In experiment 2, 672-day-old male by-product Cobb breeder chicks were randomly assigned to 8 treatments replicated 12 times with 7 birds per replicate, structured as a 2x4 factorial arrangement of treatments. Birds were on the experimental diets from day 0 to day 29. The treatments consisted of 2 HS levels [no HS (averaging 80oF from days 10-29) vs. HS (averaging 94oF for 6 hours per day from days 10-29)] and 4 feed additive types/levels [no additive, 0.04% ActigenTM, 0.08% ActigenTM, and Stepdown (0.08% ActigenTM in starter diet, 0.04% in grower diet)]. Each temperature group was replicated in two rooms (n=2) to check for the room effect. In experiment 3, 672-day-old male by-product Cobb breeder chicks were randomly assigned to 6 treatments replicated 16 times with 7 birds per replicate for 29 days. The treatments consisted of 2 HS levels [no HS (averaging 80oF from days 10-29) vs. HS (averaging 94oF for 6 hours per day from days 10-29)] and 3 supplemental feed additive types [no additive, 0.1% TynagenTM, and 0.1% PostB]. Each heat group was replicated in two rooms (n=2) to check for the room effect (8 replicates/room). In experiment 1, LPS challenge reduced (P < 0.05) bodyweight gain (BWG) and feed intake (FI) over days 0-20, while the supplemental feed additives improved (P < 0.05) feed efficiency (FE), especially in LPS-challenged birds. In addition, the LPS challenge reduced (P < 0.05) the levels of immunoglobulins A and M, but no significant effect of supplemental feed additives was observed. In experiment 2, HS reduced (P < 0.05) FI but increased (P < 0.05) FE between days 0-28. Across days 0-10, 0.08% ActigenTM improved the LBW and BWG compared to the basal diet. Furthermore, HS altered blood chemistry by decreasing (P < 0.05) the partial pressure of CO2, bicarbonate levels, base excess, total CO2, sodium, ionized calcium, hematocrit, and hemoglobin, but ActigenTM did not impact these response variables. In Experiment 3, HS reduced (P < 0.05 the LBW, BWG, and FI. However, birds that received 0.1% TynagenTM had higher (P < 0.05) FE than those placed on the basal diet across both d 11-28 and d 0-28. Furthermore, HS altered blood chemistry levels as it increased (P < 0.05) oxygen saturation and the partial pressure of oxygen while decreasing (P < 0.05) blood bicarbonate, total carbon dioxide, and ionized calcium levels. In addition, the HS-birds had a reduced (P < 0.05) jejunal villi width (VW) and crypt depth (CD), while 0.1% PostB improved the villi height (VH), VW and VH:CD in non-HS birds. In conclusion, both LPS challenge and heat stress had a detrimental impact on the performance, blood chemistry, and gut health of the birds, while the supplemented feed additives had a moderately ameliorative effect overall.

The availability of P in feedstuffs and the effect of feedstuffs, feed additives, and anti-nutritional factors on secretory IgA (sIgA) production in chickens were evaluated. A rapid method was developed using the precision-fed rooster assay to evaluate phytic acid disappearance, P digestibility, and phytase efficacy. Further, exogenous phytase was found to be effective at degrading myo-inositol 1,2,3,4,5,6-hexakis, however, the enzyme was less efficient at P release from lower inositol phosphates. The availability of P in feedstuffs was found to be affected by dietary Ca content, where increasing Ca linearly reduced (P < 0.05) both phytic acid hydrolysis and P digestibility. For example, the standardized ileal digestibility of P was reduced from 73 to 47% in soybean meal when the dietary Ca content was increased from 0.30 to 0.75%. The digestibility of P in distillers dried grains with solubles (DDGS) and corn fermented protein (CFP), however, was high across all Ca levels (71 to 80%). Relative P bioavailability based on tibia bone ash was also found to be high for DDGS and CFP, being 81 and 78%, respectively. Upon further evaluation, it was observed that utilizing low dietary Ca levels (0.30%) when P digestibility in individual plant-based feedstuffs was determined overestimated the P digestibility in practical grower diets by up to 16 percentage units. Utilization of higher Ca levels (0.75%) when P digestibility in individual plant-based feedstuffs was determined, however, resulted in calcualted P digestibility values that were within 1 to 3 percentage units of the measured digestibility in practical grower diets. Aside from P, feedstuffs were also evaluated to determine their effect on sIgA production in the intestine. Roosters excreted approximately 6 to 30 mg of sIgA / 24 h. Further, the proportional contribution of sIgA to total endogenous amino acid (AA) losses ranged from 1 to 13%, with the greatest contribution being observed for Trp. The addition of casein to a protein-free diet increased (P < 0.05) sIgA production in both roosters and broiler chickens. There was no change (P > 0.05) in sIgA production compared with the protein-free diet when the same amino acids (AA) were added in purified form; therefore, this effect was unique to the intact protein source. A variety of feedstuffs, feed additives, and anti-nutritional factors were also evaluated to determine their effect on sIgA production. Raw soybeans reduced (P < 0.05) ileal and excreta sIgA concentrations and total sIgA excretion (mg / 24 h). Feeding DDGS and CFP were also found to result in lower (P < 0.05) sIgA concentrations in excreta. Results from these studies demonstrate that 1) phytic acid disappearance in precision-fed roosters can provide preliminary estimates of P digestibility in plant-based feedstuffs, 2) utilization of low dietary Ca levels in when determining P digestibility in individual plant-based feedstuffs overestimates P digestibility in commercially relevant diets due to reduced hydrolysis of phytic acid as Ca content increases, and 3) feedstuffs and other dietary components can regulate sIgA production in the gastrointestinal tract through host-diet interactions.

As a result of the advancement of genetics, understanding broiler requirements for dietary energy and amino acids is essential for improving live performance and carcass characteristics. The energy system used to formulate diets in poultry is based on metabolizable energy (ME); this equation can be described as the total amount of energy in the feed minus gases and feces. However, the net energy (NE) system may be the best depiction of the energy utilized by birds for production and maintenance purposes once the heat increment (HI) is measured. Thus, a series of studies were conducted to establish the responsiveness of broilers (Cobb 500 and Ross 708) fed varying dietary energy, amino acid (AA) levels, and energy systems. The experiment in chapter II analyzed the response of Cobb 500 broilers (males) fed varying levels of digestible lysine (dLys) and ME during the starter phase (1 to 18 d) on live performance as well as the carryover effects on carcass characteristics. Birds that were fed increased ME obtained an improved live performance. Additionally, it appears safe to utilize 1.18% dLys to provide the standard results regarding live performance and carcass traits. Experiment 1 (male) and 2 (female) in chapter III served to assess the responsiveness of Ross 708 broiler performance and carcass characteristics to different energy systems and crude protein (CP) formulations. It was determined that male birds fed increased CP levels had decreased feed conversion ratio (FCR) and both sexes decreased fat pads with high CP diets; additionally, male broilers fed decreased CP were more sensitive to dietary energy variations regarding abdominal fat pad deposition. In addition, in chapter III, experiment 3 aimed to evaluate the performance and carcass characteristics of broilers (Cobb 500) at different NE levels and CP during the 32 to 41d finisher period, in addition to comprehending possible interactions between NE formulation and CP. It was established that increasing the levels of NE from 2,250 to 2,400 kcal/kg improved live performance and did not affect the abdominal fat pad. Furthermore, birds fed increased CP had greater live performance and breast meat yield, and decreased fat pad yield.

Eimeria species oocysts cause coccidiosis, one of the costliest diseases in the poultry industry. Coccidiosis infections cause significant economic losses due to reduced performance in chickens and turkeys. The objective of this thesis was to determine fecal bacterial recovery from chlorhexidine (CHX) salt suspensions containing Eimeria maxima M6 oocysts and evaluate the effect of CHX salts on oocysts during long-term cold storage. For bacterial recovery experiments, CHX digluconate and CHX gluconate were tested at different concentrations on poultry fecal slurries during oocyst shedding. Eimeria maxima M6 oocysts were evaluated in CHX salt suspensions to test the effect on oocyst degradation and sporulation. Chickens were challenged with oocysts stored in CHX salts for five months at 4°C in order to test the effect of CHX salts on Eimeria viability during storage as an alternative to potassium dichromate (PDC). PDC is a chemical commonly used to store oocysts. PDC can effectively control aerobic and facultative anaerobic microbes that compete with oocysts for oxygen in the storage suspension. PDC, however, is dangerous for personnel utilizing it and the environment. A safer alternative, therefore, should be sought. Chapter 1 consists of a brief summary of this thesis. Chapter 2 consists of a literature review about PDC, CHX salts, and Eimeria, including the physiology, life cycle, anticoccidials and vaccination methods. Chapter 3 consists of research conducted, including bacterial recovery, oocyst degradation, oocyst sporulation, and oocyst infectivity from CHX salt assays.

The employment of ozone in livestock production as a means of mitigating ammonia is used for enhancing air quality within animal confinement facilities, such as swine or poultry barns. Ammonia is a byproduct of animal excrement. Elevated ammonia concentrations can adversely affect the well-being of both animals and humans. The aim of this thesis was to investigate the effects of Ozone Technology Machine Unit (OTMU) utilization in poultry barns regarding ammonia concentration and its potential benefit in terms of broilers performance and welfare. In recent years, there has been a significant boom in technological advances. New devices that help mitigate the concentration of ammonia in poultry production must be developed to help improve production in a sustainable way, protecting the workers who are dedicated to this field and improving the welfare of birds. Chapter I provides the whole thesis introduction. Chapter II provides a literature review on ammonia concentration effect on broilers performance and a brief overview of the ozone utilization in livestock. Chapter III comprises a study conducted to assess if the OTMU under research improves air quality, reduces ammonia concentration within the poultry barn, ameliorates bird welfare, and boosts broilers performance.

The emissions of ammonia (NH3), particulate matter (PM2.5), and carbon dioxide (CO2) are major concerns in poultry facilities. They can pose environmental concerns and nuances. Robust and affordable measurement systems are needed to accurately measure in-barn concentrations and quantify the emissions. The Intelligent Portable Monitoring Unit (iPMU or PMU3) developed in 2016 was reconstructed into PMU4 to include upgraded NH3 and PM2.5 sensors and wireless connectivity for a low-cost, robust, and accurate air quality monitoring device with contactless data transfer using the concept of Internet of Things (IoT). In addition, a user-friendly web-based interface was developed for 1) real-time and historical data visualization, and 2) estimation of NH3 and PM2.5 emissions with suitable ventilation measurement. The PMU4 device developed in this project features back-up data stores and leverages cloud computing for data analysis and visualization. It was designed to collect and store sensor data (temperature, relative humidity, NH3, PM2.5, and CO2) on an SD card, and simultaneously send the data to a secure server. The PMU4 device uses Wi-Fi for internet access, but it was programmed to tolerate internet outages by queueing data and automatically transmitting the data queue to the secure server when the internet restores. The PMU4 device was deployed in the Robert T. Hamilton Poultry Teaching & Research Facility (Iowa State University, Ames, IA) for 13 days (September 15 – September 27, 2023) for field evaluation. The results showed that the mean temperature and relative humidity were 23.2 ± 1.8 ˚C and 62.1 ± 11.8% respectively. The mean concentrations of NH3, PM2.5, and CO2 were 0.47 ± 0.27 ppm, 12.0 ± 12.6 µg/m3, and 581.3 ± 134.2 ppm. The NH3 and PM2.5 per bird emission rates for the monitoring period were estimated using facility temperature, NH3 and PM2.5 concentrations, atmospheric barometric pressure obtained from the nearest airport, and ventilation and chicken inventory obtained from the facility manger. The mean per bird emission rates for NH3 and PM2.5 were 0.23 g/d/hen and 7.44 mg/d/hen, respectively. Advisor: Yijie Xiong

Bone quality is an important measure of welfare in laying hens for researchers, veterinarians, and commercial producers who bear the responsibility of providing them with care. Breeding practices that have resulted in high egg productivity of laying hen strains have resulted in an increased susceptibility to developing osteoporosis. Osteoporosis, a decrease in mineralized bone, is accompanied by increased bone fragility that can lead to potentially painful bone fractures that can inhibit some aspects of the birds natural living, respiration and flight being two examples. Current methods of examining laying hen bone quality involve the implementation of diagnostic imaging and biomechanical assessments, most commonly conventional/digital radiography, computed tomography (CT), and the 3-point bending test. Current studies utilizing radiographic examinations to assess poultry bone health have largely relied on the use of scoring systems that are unique to the respective lab group conducting the research. This is apparent from reviews of the literature that have highlighted the discordance amongst reported scoring systems that use the presence or absence of fractures, fracture gaps, callus formation, bone deviations, and abnormal angulations of bone morphology to assign a qualitative score to a bone to characterize its damage status. The result of utilizing these varying scoring systems is that there is a lack of generalizability of the results and a subsequent inability to make comparisons between studies that would be beneficial in making efforts towards addressing the welfare concern. Additionally, scores do not allow the use of stronger statistical tests that can be performed with quantitative data based on discrete and continuous measures. Computed tomographic (CT) and biomechanical analyses are established, quantitative methods for characterizing the bone quality of laying hens. Reported analyses have assessed the 3 bone mineral density (BMD) of the keel, tibiotarsus, femur, and humerus; longitudinal morphology changes of the keel; cross sectional area of the keel and tibiotarsus; cortical thickness; and the angulation of the ventral margin of the keel using image reformatting. Current published methods of the assessment of keel and tibiotarsal bone quality include variations of assessing the BMD of different bone types. Total bone; the midpoint of the tibiotarsal diaphysis; and defined proximal, middle, and distal locations of the keel and tibiotarsus served as points for CT assessment of BMD/Bone mineral content (BMC) in the published literature. Less frequently however is CT muscle quality evaluated despite its potential to be correlated with bone health as is reported in human literature. Additionally, the 3-point bending test has been used as the primary method of assessing bone quality of laying hens biomechanically with differing testing parameters (fulcrum width and crosshead speed) used between studies. Given the knowledge gaps present in the literature pertaining to radiographic and CT assessments of poultry bone quality, the objective of the retrospective study detailed in Chapter II was to develop and assess the intra-observer repeatability of a standardized radiographic protocol for quantifying three types of KBD (keel bone fractures, ventral margin deviation, and dorsal margin angulation) using Horos open-source image analysis software and discrete and continuous variables. Objectives of the prospective study detailed in Chapter III were the following: 1) develop standardized protocols for quantifying keel bone, tibiotarsal, and muscle quality using CT and open-source image analysis software (Horos, https://horosproject.org); 2) test associations between CT cross sectional area of the muscle group surrounding the tibiotarsus and tibiotarsal BMD; 3) test associations between CT cross sectional area of the muscle group surrounding the keel and keel BMD; 4) develop a standardized protocol for quantifying biomechanical properties of tibiotarsal bones; and 5) test associations between biomechanical 4 measures and CT measures of bone quality. Our findings indicated that the radiographic protocol was repeatable and had the potential the increase the generalizability of results if applied to other studies. Implementation of the developed radiographic protocol that utilizes quantitative, discrete and continuous measurement variables would also allow for the use of stronger statistical tests. It was also found that associations exist between CT measures of tibiotarsal bone and muscle quality at the proximal location of the bone. Computed tomographic measures of bone and muscle quality were also predictive of biomechanical measures of bone quality of the tibiotarsus. These findings justify the use of the measure of the transverse, cross sectional muscle area of the tibiotarsus in future studies. This measure is not currently reported in the poultry literature, although current literature suggests there is a relationship between measures of bone quality and body weight. This measure, if utilized in studies aiming to address welfare concerns, has the possibility of identifying the effects of interventions on tibiotarsal musculature and can indirectly provide a measure of bone quality, further reducing the need for destructive testing. Additionally, findings further justify the exploration of the suitability of laying hens for use as translational research models for osteoporosis in humans.

Black seed oil concentrations of 0%, 0.1%, 1% and 5% were added to milk inoculated with kefir grains and incubated at 25°C for 22 h. The pH and microbial count indicated 1% black seed oil caused low inhibition (P > 0.05) of fermentation, but 5% black seed oil caused significant inhibition of the kefir microorganisms (P < 0.05). Cobb 500 male chicks (n = 256) were distributed in a randomized block design and received one of four treatments: CTRL1 (Non-medicated, no kefir, no Clostridium perfringens), CTRL2 (Non-medicated, no kefir, C. perfringens inoculated), CTRL3 (BMD medicated, no kefir, C. perfringens inoculated), KTRT (Non-medicated, kefir treated, C. perfringens inoculated). Kefir was supplied via a 1:10 kefir to water ratio in the chicken water supply. Birds were orally inoculated with Eimeria maxima and C. perfringens on Day 11 and Days 16 through 18, respectively. KTRT (kefir) weight gain was comparable to the medicated CTRL3. CTRL3 and KTRT feed conversion ratios were significantly lower than CTRL2 (untreated). There was no significant difference (P > 0.05) between CTRL3 and KTRT lesion scores. CTRL3 (antibiotic) mortality rate was significantly lower than other treatments. KTRL (kefir) reduced mortality but not as effectively as CTRL3. Cobb 500 male chicks (n = 320) were placed in randomized block design and received one of five treatments: CTRL1 (nonmedicated, uninoculated, no kefir, no black seed), CTRL2 (nonmedicated, inoculated, no kefir, no black seed), CTRL3 (medicated, inoculated, no kefir, no black seed), KTRT1 (nonmedicated, inoculated, 2:10 kefir to water supplied in drinking water, no black seed), KTRT2 (nonmedicated, inoculated, kefir, black seed), KH2O (sample of 2:10 kefir-water mixture from water trough). Birds were orally inoculated with E. maxima and C. perfringens on Day 14 and on Days 19 and 20, respectively. Weight gain of CTRL3 (antibiotic) and KTRT2 (kefir + black seed) increased during D14-D28. Feed intake of KTRT2 was significantly (P < 0.05) higher than CTRL3 and KTRT1 (kefir). CTRL3 and KTRT2 lesion scores were the lowest of all treatments. KTRT1 was as effective as CTRL3 in the prevention of mortalities. KTRT2 mortality rates were the lowest of all treatments. Results suggest KTRT2 was effective against necrotic enteritis in broiler chickens.

The genetic selection for high growth rate, breast muscle yield, and feed efficiency in modern broilers has been a double-edged sword. While it has resulted in the desired traits, it has also led to broiler myopathies. Recent omics studies have shown that hypoxia may be a potential factor that damages the myofiber niche and compromises muscle repair processes. The objective of this study was to optimize isolation protocols for primary broiler satellite cells (SCs) and fibroadipogenic progenitors (FAPs) and to characterize SCs proliferation and differentiation under hypoxia. The study first focused on the optimization of isolation procedures and culture conditions for primary broiler SCs. Simple and cost-effective preplating techniques were used to purify SCs, which may improve the fidelity of follow-on experiments. In addition, myogenic differentiation potential was maximized under optimal conditions with different types and concentrations of serum and chicken embryo extract. This dissertation further demonstrated the effects of hypoxia on the differentiation and metabolism of SCs. Satellite cells were exposed to hypoxia conditions for 48 h and normoxia recovery. Hypoxia improved SCs proliferation by maintaining Pax7 and reducing Myod1 and myogenin while reducing myogenic differentiation. The cell proliferation was not rescued by 24 h normoxia recovery, whereas myogenic potential was recovered. In addition, hypoxia-activated AMPK and upregulated proteins involved in lipid metabolism and cellular stress, with decreased mitochondrial activity and increased lipid accumulation in myotubes, which indicates that the balance between fatty acid uptake and oxidation was disrupted. Several differentially expressed genes were also identified across myogenic differentiation under hypoxia with targeted RNA sequencing. Lastly, we established an isolation protocol for broiler FAPs for the first time. Through the comparative analysis between FAPs and preadipocytes, the induction of adipogenic differentiation and morphology of adipocytes derived from muscle and adipose tissues are similar. However, fatty acid methyl ester and targeted RNA sequencing data revealed a distinct separation between FAPs and preadipocytes throughout adipogenic differentiation. In conclusion, these data provide valuable insight into the biology of SCs and FAPs and the effects of hypoxia on SC proliferation and differentiation to better understand broiler myopathies.

In the past, histidine warranted little concern from poultry nutritionists because it was seldom deficient in practical broiler diets. With most essential amino acids now commercially available as feed-grade forms to partially replace intact protein ingredients, histidine and its minimum in least-cost formulation are receiving fresh attention. Published histidine requirement data for broilers are sparce, and many of these studies have been conducted on males within the first three weeks post-hatch. Moreover, rudimentary dose-response experimental designs may not be applicable to estimate histidine requirements in later growth periods because histidine is uniquely stored within the body. In times of dietary histidine deficiency, endogenous histidine reserves can be catabolized to furnish free histidine and possibly confound requirement estimates. Therefore, two experiments were conducted to evaluate broiler responsiveness to dietary histidine concentrations with and without endogenous histidine reserve consideration. Chapter II utilized a 2 x 4 factorial design to determine whether female broilers responded differently to increasing digestible histidine to lysine ratios from 22 to 42 days when previously fed different digestible histidine to lysine ratios from 0 to 21 days. Live performance data were collected for both feeding phases and broilers were processed on day 43 to evaluate carcass characteristics. No interactions were found between the starter and grower diet digestible histidine to lysine ratios for any parameters assessed. Increasing the grower diet digestible histidine to lysine ratio elicited linear and quadratic responses for 42 day final body weight, 22 to 42 day body weight gain, 22 to 42 day feed conversion ratio, and 43 day total breast meat yield. The optimal digestible histidine to lysine ratio was calculated to be 34% for 43 day total breast meat yield and 35% for 22 to 42 body weight gain and feed conversion ratio. Chapter III utilized a dose-response design to assess male broiler responsiveness to digestible histidine to lysine ratios of 28 to 43% from 15 to 35 days. A positive control diet differing in the crude protein concentration was formulated to determine the efficacy of the test diet. Live performance and carcass characteristics were measured as response criterion. Increasing the digestible histidine to lysine ratio elicited linear responses for final body weight, body weight gain, feed conversion ratio, and carcass yield. Linear and quadratic responses were observed for both Pectoralis major and minor yield. The optimal digestible histidine to lysine ratios were calculated to be 37 and 39% for Pectoralis minor and major, respectively. Despite the fact that further reductions in dietary crude protein often lead to impaired broiler performance, several benefits could be achieved in doing so, especially during times when prices for intact protein ingredients are high. Broiler responsiveness to increasing dietary histidine concentrations suggests that a deficiency could occur during practical formulation. Thus, understanding requirements for histidine and other less limiting amino acids are some of the steppingstones in the quest towards the more sustainable production of chicken meat.

Poultry houses might have high levels of airborne pathogens, including Escherichia coli (E. coli) and avian influenza (AI), which may be transmitted through the air and pose risks of infection. The objective of the dissertation is to provide an insightful understanding the of airborne transmission of E. coli and AI which were attached to poultry dust particles. Chapter I and VI summarized background, gap in knowledge, and discussed the limitations and implications of the study. Chapter II compared the efficiency of Andersen six-stage impactor, all-glass impinger, and ACD-200 Bobcat, in collecting airborne E. coli carried by dust particles. The results showed that the Andersen six-stage impactor and the all-glass impinger outperformed the Bobcat sampler. Airborne E. coli were found to mainly aggregate on large particles (>7.0 µm). The survivability of E. coli in poultry litter under different environmental conditions was described in Chapter III. The survivability of airborne E. coli was found to have a half-life time of 5.7 ± 1.2 min, while the survivability of settled E. coli and E. coli in poultry litter were much longer with half-life times of 9.6 ± 1.6 hrs and 15.9 ± 1.3 hrs, respectively. The effect of ultraviolet (UV) light on the inactivation of airborne E. coli carried by poultry dust particles under laboratory conditions was explored in Chapter IV. The inactivation rates varied from over 99.87% and 99.95% at 5.62 s of contact time to 72.90% and 86.60% at 0.23 s of contact time, with 1,707 µW cm−2 and 3,422 µW cm−2 of UV irradiance, respectively. Utilizing computational modeling to assess the risk of airborne and deposited AI carried by poultry-litter dust particles was investigated in Chapter V. Results showed that concentrations of airborne AI transmitted to other farms in a day were lower than the minimal infective dose for poultry. The study suggests that factors such as infected location and type of poultry house may influence the risk of airborne transmission of HPAI. In conclusion, this dissertation explores sampling methods, survivability, mitigation technologies for airborne transmission of pathogens in poultry, and factors affecting infection probabilities in the poultry industry.

The poultry industry is integral to the global agricultural system as consumption rates continue to rise worldwide. With the global population projected to reach 10 billion by 2050, there is an increasing need for efficient and sustainable animal protein sources. Feed efficiency is a significant factor in poultry production costs, as feed accounts for 50 to 70% of total production costs. Given increasing concerns surrounding feed efficiency improvements, there is a focus on the genetic improvement of broiler digestibility to improve nutrient utilization and reduce production costs. Water availability is also of critical concern for the poultry industry, as estimates suggest that freshwater availability needs to increase by ~25 percent to meet the needs of increasing population and production. Although broilers have the lowest water footprint of any meat protein, there is still an opportunity to improve water efficiency in broilers. One study aimed to evaluate the effect of sex on the growth performance and feed efficiency of Japanese quail. Female quails consistently reported higher body weights and feed intake; however, no differences were reported in FCR among male and female quails, suggesting that male quails may have the potential for increased relative feed efficiency. Another study assessed the efficacy of conventional and novel water monitoring systems on commercial broiler strains. The study demonstrates that bell waterers lack accuracy and result in increased water intake. Furthermore, the novel low-flow water monitoring system accurately measures water-related production traits without negatively affecting growth performance. Lastly, a final study examined the influence of conventional and novel water monitoring methods on the processing performance of commercial broiler strains. Research results show that water monitoring methods have minimal impact on processing performance and meat quality. These results are encouraging in that the novel low-flow water monitoring system is an accurate and viable method of measuring water intake in poultry. The poultry industry has made significant strides in improving efficiency and sustainability; however, further research into resource sustainability is critical for ensuring food security and reduced environmental impacts in the face of an increasing global population and resource scarcity.

The objective of this thesis was to investigate the effects of Enterococcus cecorum (EC) administration into the amnion at day 18 of embryogenesis on performance and septicemia development in broiler chickens. Chapter I consists of a brief overview of the thesis. Chapter II provides a literature review on EC-related disease in broiler chickens and methods to isolate, detect, and differentiate between commensal and pathogenic strains of EC. Chapter III consists of a study conducted to determine if different EC strains isolated from field cases that exhibited septicemia and high mortality impacted hatchability, livability, organ colonization, and early performance in broiler chickens.

Streptococcus gallolyticus (SG) is a Gram-positive found as commensal gut flora in animals and humans. SG has been associated with acute mortality in poults 2 to 3 weeks of age, with lesions mainly consisting of splenomegaly, hepatomegaly, and pericarditis. The objective of this thesis was to reproduce disease in poults caused by SG recovered from clinical field isolates and evaluate different routes of administration. Three field isolates from geographically disparate areas in Arkansas and Missouri were used for the experiments contained in this thesis. Antibiotic sensitives of the three field isolates revealed sensitivity to penicillin as well as a wide panel of antibiotics. The three isolates had similar antibiotic sensitivities with the exception of SG2 and SG3 being intermediate for neomycin. All samples tested were intermediate for Sulfamethoxazole/trimethoprim. Penicillin is widely used to treat Gram-positive bacteria such as S. gallolyticus, which have a thick layer of peptidoglycan, by inhibiting the production of peptidoglycan. However, in antibiotic free flocks, growers do not have the luxury of treating with antibiotics, therefore higher mortality has been reported and managing outbreaks may be difficult. Learning more about this bacterium, as well as finding alternatives to antibiotics for SG may lead to clues on how to combat this novel pathogen. Chapter 1 consists of a brief summary of this thesis. Chapter 2 consists of a brief literature review about S. gallolyticus consisting of its relevance, pathogenicity, antibiotic resistance potential, and diagnostics involved in identification of SG. Chapter 3 consists of research conducted, which includes preliminary work, to recreate disease in poults due to challenge from different SG isolates.

Breast myopathies have caused significant economic losses to the poultry industry over the last decade. The induction of woody breast (WB), white striping (WS), and spaghetti meat (SM) have caused textural issues, functional issues, and a decline in consumer acceptance. Of these three myopathies, WB causes the most pronounced effects on product quality and reduces functional use. Woody breast is also commonly associated with expansive breast meat growth/high performing broilers. However, progress in reducing these myopathies has been limited without hindering other aspects of poultry rearing. Generally, production performance reduces as myopathy occurrence or incidence reduces. This response is primarily driven by a reduction in final body weight rather than in a net myopathy reduction. For this reason, novel strategies for reducing myopathies are still being investigated. Therefore, this dissertation aimed to assess new technologies in these sectors to reduce or prevent the occurrence of breast myopathies in commercial broilers. Chapter 2 assessed the supplementation of guanidinoacetic acid (GAA) in poultry diets for reducing the occurrence of WB in commercial broilers. Supplementation of GAA was achieved through on-top additions of 0.06% and 0.12% of the diet. Results suggest that GAA can be used as a dietary source of breast myopathy reduction without suppressing body weight or broiler growth. The use of GAA in the diet also improved FCR in broilers at both supplied levels compared to the control diet. Data indicate that supplementation levels may be beneficial above those utilized in this experiment and optimizing levels should be investigated. Chapter 3 also assessed the use of GAA, but in formulation as a potential energy sparing ingredient while maintaining a reduction in breast myopathies. Supplementation was formulated as a 55 kcal/kg and a 110 kcal/kg reduction in energy. Results suggest that a 110 kcal/kg reduction of energy produced no differences in final body weight or body weight gain over the 49 day rearing period. Broilers also expressed no differences in carcass or part yields with or without the presence of GAA. In addition, meat quality differences were not present among treatments. However, GAA inclusion provided an improvement in feed conversion ratio and had a numeric shift in WB occurrence in broilers. The need to assess ingredient pricing and availability should be considered for GAA use as an energy sparing ingredient. Chapter 4 evaluated the use of a novel 0.9% in ovo feeding of nicotinamide riboside (NR), a vitamin B3 analog, to reduce the occurrence of WB in broilers. Three concentrations of NR were utilized in this experiment including: 250 mmol, 500 mmol, and 1000 mmol of the vitamin B3 analog. Results suggest that NR had little effect on performance or meat quality parameters yet improved final bird weight compared to the control. Optimizing the dosing level for WB reduction may not have been met in this study and should be further investigated for conformation. Changing to other forms of WB reduction, Chapter 5 evaluated the use of broiler stocking density as a combative measure. Three research accepted stocking densities were assessed to determine effects on meat quality. Results indicate that WB is reduced when stocking density is increased, but performance characteristics are reduced alongside a reduction in final body weight. Results also suggest that investigation into welfare limits and financial gain may be necessary to determine the most beneficial stocking density. Finally, Chapter 6 was conducted to understand statistical sampling needs to assess myopathies (specifically WB) for common broiler rearing experiments. There is a growing trend to include meat quality analysis in studies designed for nutritional, management, or physiological aspects. These types of studies typically use a low sample selection size per pen which have typically been adequate for analysis, such as yield. In opposition, myopathy occurrence in populations can vary and may require a larger sample size. Some studies conducted on performance characteristics have started assessing meat quality traits with limited samples and drawing conclusions that do not seem to match common practice. Meat quality based studies typically utilize a substantially larger sample size than those of performance-based experiments as variability is generally higher. Results indicated that sampling a larger proportion of the pen (up to 75%) would greatly reduce the variance present between samples and lead to a more accurate description of myopathies among treatments. Overall, this dissertation provided three new avenues for assessing WB in commercial broilers and potential ways to reduce occurrences. Limited results were present in WB reduction, but results indicate that differences in body weight were not as apparent as seen in previous studies. Suppressed growth comes at a significant price to the industry so finding nutritional or managerial strategies to reduce myopathies is very important. The need to better understand the relationship between each of the assessed strategies and WB are still necessary to improve breast product quality.

In Chapter 2, an experiment was conducted to evaluate the effect of different mixer types (MTY) and mix times (MTI) on mix uniformity (MU). A 2 (mix time) x 3 (mixer type) factorial was utilized in this experiment to create six treatments, each mixed in three randomized complete blocks. Mix times were designated adequate or inadequate, were based on the manufacturers’ recommendation, and varied based on the mixer utilized. The mixer types utilized included a vertical mixer, horizontal mixer, and transfer mixing system. The transfer mixing system can be described as a batch-to-mix system that simulates the process of conveying batched feed to a mixing location. Chloride ions (Cl-) via salt, crystalline DL-Methionine (Free Met), and crystalline L-Lysine-HCL (Free Lys) were utilized as coefficient of variation (CV) markers, with Cl- being used in a corn-salt (CS) ration and the Free Met and Free Lys being used in a complete diet (CD). An interaction between MTY and MTI affected Free Met CV (P = 0.005). The horizontal mixer and transfer mixing system did not demonstrate changes in the Free Met CV compared to the vertical mixer when MTI was manipulated. The authors hypothesize that additional mixing during load-out and added conveyance caused this interaction. The transfer mixing system resulted in the lowest Free Lys CV (P = 0.017); however, no treatment provided an industry-acceptable MU. In conclusion, Free Lys may not be an appropriate marker for MU, Free Met may be an appropriate marker for MU in complete diets, and the transfer mixing system may contribute to an acceptable MU. In Chapter 3, an experiment was conducted to evaluate the influence of pellet die thickness (PDT) and Azomite® (AZM) on feed manufacture. A 2 (AZM) x 3 (PDT) factorial was utilized in this experiment to create a total of six treatments, each manufactured in every run order position in a Latin Square Design. Azomite inclusion rate was either 0.0% (control) or 0.25%, and 32mm, 38mm, and 45mm PDTs were utilized in their appropriate treatments. The pellet diameter was held to a constant of 4.5mm. The objective of this study was to evaluate the effect of AZM and PDT on pellet production rate (PPR), pellet durability index (PDI), and hot pellet temperature (HPT) in corn, soybean meal, and dried distillers grains with solubles-based poultry diets, manufactured under a constant motor load. The inclusion of AZM increased overall PPR by 7.9% (P < 0.001) and decreased overall PDI by 1.6 percentage points (P < 0.001). As PDT increased from 32mm to the average of the 38 and 45mm treatments, PPR decreased by 28.5% (P < 0.001) and increased PDI by 10.1 percentage points (P < 0.001). A main effect interaction of AZM and PDT was recognized to influence HPT (P < 0.007), however, no difference was observed for the 45mm PDT when AZM was included. Linear contrasts showed that PPR increased by 5.0, 7.9, and 11.8% when comparing AZM treatments to their respective controls (All PDT; P < 0.001). The authors of this experiment concluded that AZM increased PPR across PDT and decreased PDI, albeit to the extent that would likely not affect broiler performance. In Chapter 4, a follow-up experiment to the previous was conducted to evaluate the influence of PDT and AZM inclusion rate on broiler performance and apparent ileal amino acid digestibility (AIAAD). A 2 (AZM) x 2 (PDT) factorial was utilized in this experiment to create a total of four treatments. The dietary treatments were sourced from the previous AZM experiment, and only the treatments utilizing the 32 and 45mm PDT were utilized. All feed was reground and slightly reformulated to reduce the confounding bias of PDI and to ensure an acceptable nutritional density. Ten randomized complete blocks of raised wire cages were utilized with each pen housing ten Ross 308 broilers. The objective of the study was to determine the effect of pelleted feed using AZM (0.0 or 0.25%) and PDT (32 or 45mm; using a constant die diameter of 4.5mm) on Ross 308 male broiler feed intake, live weight gain, feed conversion ratio, and AIAAD from 0 to 21 days of age. Live performance did not differ due to an interaction or independent main effects (P > 0.05). However, AIAAD was influenced by AZM and PDT interactions (P < 0.05). The amino acids Ala, Asp, Glu, Gly, Ile, Lys, Met, Pro, Thr, and Val demonstrated increased digestibility with the 45mm Control treatment. This treatment also produced the highest numerical HPT in the feed manufacture experiment. It was hypothesized that the digestibility resulted from increased frictional heat that deactivated trypsin and chymotrypsin inhibitors or degraded ingredient cell wall structure. While both inhibitors were determined to be present using SDS-PAGE and chemiluminescent detection, quantitative analysis showed no practically influential concentrations of either inhibitor or a decrease large enough to explain the increased AIAAD. The authors, therefore, speculate that the increase in AIAAD of various amino acids was due to the breakdown of the aleurone layer cell walls of the corn kernels caused by increased frictional heat exposure vis the 45mm PDT and the absence of AZM.

This dissertation explores the identity and role of immunoglobulin-like (Ig-like) receptors in chickens, with focus on their implications in disease and disease progression. These receptors, wisely expressed across immune cells, interact with the major histocompatibility complex (MHC) class I molecules to modulate immune responses in mammals. Due to the insufficient representation of chicken Ig-like receptors in online databases, this study systematically annotates the chicken Cluster Homolog of Immunoglobulin-like Receptors (CHIR) genes using advanced bioinformatic techniques, aligning with the release of the 7th edition of the chicken genome assembly that comprises builds for a broiler and layer chicken. The analysis identifies over 150 CHIR genes, refining functional classifications of activatory (CHIRA), inhibitory (CHIRB), bifunctional (CHIRAB), and CHIR-like (CHIRL) genes through InterProScan, phylogeny and motif searches. Variations in CHIR gene counts across different chicken lines (broiler, N = 124, layer, N = 70) suggest links to selective breeding demands, emphasizing their importance in poultry health and production. Phylogenetically, CHIRs show close relationships with other poultry Ig-like receptors, and structural comparisons indicate analogous roles to Ig-like receptors in the human and rat. As an outcome of the analysis, CHIR genes were renamed with the Chicken Genome Nomenclature Consortium from “chicken homolog of Ig-like receptors” to “cluster homolog of Ig-like receptors”. Reanalyzing next-generation sequencing data reveals CHIR genes are expressed across all tissues of a UCD001 line, with generally higher expression in blood-containing organs. Examination of CHIR gene single nucleotide polymorphisms across various in inbred lines (UCD001, UCD003, Line 0, Line 6, Line 7, Line 15, Line N, Line P, Line C, and Line W) indicates an overall variant rarity and slightly more occurrence in CHIRB genes. Over 1,000 protein-encoding variants are associated with differential resistance and susceptibility to Marek’s disease (P < 0.05). Two in vitro approaches assessed the roles of CHIR molecules in modulating immune responses or targeting pathogens. Re-examination of RNA-sequencing data of MHC-I types B2 and B19 macrophages, temporally stimulated with interferon-gamma, revealed dynamic and opposite CHIR expression trends, with B2s showing an increase and B19s displaying a decrease until returning to basal levels at 24 to 48 hours. These findings suggest nuanced and distinct regulatory patterns of CHIRs in different haplotypes during immune responses. Additionally, CHIR sequences were aligned for the design of small interfering RNA molecules targeting the CHIRB functional group on macrophages retrieved from birds of congenic (UCD331 and UCD335) and mixed (WVU1952) backgrounds. CHIRB silencing was observed to enhance cellular nitrate release and impact H2O2, particularly in specific MHC-I haplotypes and in different genetic backbones, in avian influenza virus infection. While this dissertation enhances our understanding of chicken Ig-like receptors and cellular involvement, it also acknowledges certain limitations, such as variations in gene annotations. Nevertheless, CHIRs merit a sizeable acknowledgment as pivotal contributors to the immune response, particularly in their intricate interactions with the MHC. Future studies integrating this understanding into breeding plans or other interventions becomes a strategic imperative for optimizing poultry health and immunity, ensuring wellbeing, and in turn, a more resilient and sustainable food supply.

Two experiments were conducted to examine the effects of Allzyme® Spectrum supplementation to a reduced nutrient diet on the growth performance, energy and nutrient digestibility and utilization, bone quality, and jejunal digesta viscosity in broiler chickens. Each experiment used 300 one-day old Cobb by-product breeder chicks with 5 treatments, 10 replicates, and 6 birds per replicate cage in a randomized complete block design. The only differences between the two experiments were experiment 1 lasted for 21 days using a corn-soybean meal-based diet and experiment 2 lasted for 22 days using a wheat-soybean meal-based diet. The 5 treatments included a positive control (PC) diet that met or exceeded nutrient Ca (0.8%) and avP (0.4%) and energy (3,050 kcal/kg) requirements of birds of this age, a negative control (NC) diet that consisted of a reduction of 90 kcal/kg ME and 0.15%-point less Ca and P, and the last 3 treatments were varying inclusion levels of Allzyme® Spectrum (150, 200, 250 g/ton, respectively) added to the NC diet. Allzyme® Spectrum (Alltech, Inc., Nicholasville, KY, USA) is an enzyme complex containing xylanase and phytase. Data were analyzed using the GLM procedures of SAS (v 9.4). Simple contrasts were used to compare the PC vs. NC diet and PC vs. enzyme supplemented diets. A post-hoc test was used to generate the coefficients for the polynomial contrasts. Orthogonal polynomial contrasts were used to compare increasing levels of enzyme supplementation on the performance, jejunal digesta viscosity, nutrient digestibility and utilization, and bone mineralization of chickens. In experiment 1, increasing levels of enzyme supplementation resulted in a quadratic increase (P < 0.05) in feed intake (FI), body weight gain (BWG) and feed efficiency (FE) d 9-21 and a linear increase (P < 0.05) in FI d 9-21 and 0-21 and FE d 0-9, 9-21, and 0-21. In experiment 2, FI of chickens from d 9-22 and 0-22 and FE d 9-22 linearly increased (P < 0.05) with increasing level of supplemental Allzyme® Spectrum. There was a quadratic effect (P < 0.05) of enzyme supplementation level on BWG d 9-22 and 0-22 and FE d 9-22 and 0-22. In experiment 2, increasing levels of enzyme supplementation resulted in both a quadratic and linear decrease (P < 0.001) in jejunal digesta viscosity. In both studies, increasing levels of enzyme supplementation resulted in a quadratic increase (P < 0.05) in dry matter and N digestibility and retention as well as, digestible and metabolizable energy and a linear increase (P < 0.05) in P and Ca digestibility. Increasing enzyme supplementation resulted in both a quadratic and linear increase (P < 0.001) in P and Ca utilization in experiment 1, however it only resulted in a linear increase (P < 0.001) in Ca and P utilization in experiment 2. In experiment 1, there was a quadratic increase (P < 0.01) in ileal digestibility of all the amino acids with increasing level of enzyme supplementation. In experiment 2, there was a quadratic increase (P < 0.05) in ileal digestibility of Arg, His, and Val and nonessential amino acids (excluding Glu: P = 0.069 and Tyr: P = 0.088) and a linear increase (P = 0.001) in ileal digestibility of Thr when comparing the enzyme supplemented diets. There was a quadratic increase (P < 0.05) with increasing levels of enzyme supplementation in bone breaking strength and bone ash in both studies. In conclusion, Allzyme® Spectrum supplementation improved growth performance, energy and nutrient digestibility and utilization, and bone mineralization in broiler chickens and reduced jejunal digesta viscosity in 21- and 22-day-old broiler chickens fed wheat-based diets.

The purpose of this experiment is to determine the thermal kinetic parameters of Salmonella in moisture-enhanced, reconstructed ground chicken patties as affected by temperatures and salt concentrations. Salmonella is responsible for 35% of the foodborne illnesses associated with poultry products (Batz et al., 2011). Nonintact reconstructed chicken meat is mixed with brine solutions containing various salt and polyphosphate concentrations to increase water-holding capacity, decrease cooking losses, improve sensory tasting scores, and maintain the good quality of completed chicken products (Gill et al., 2004). Increasing salt concentrations within meats can increase the thermal inactivation of pathogens. In this study, Salmonella Typhimurium American Type Culture Collection (ATCC) 14028 was used, which is the same strain used in our previous validation studies of antimicrobials on broiler carcasses (Lemonakis et al., 2017). Survival curves for 62℃ of 0 and 1% salt samples indicate more heat susceptibility than 3 and 5% with roughly a 1.40 to 1.53 log CFU/g difference. For 66℃, survival curves of 1 and 3% salt samples indicate slightly more heat susceptibility than 0 and 5%, with a 1.21 to 1.66 log CFU/g difference. For 70℃, survival curves of 1 and 3% salt samples indicate more heat susceptibility than seen at other temperatures for 0 and 5%, with 1 and 5% differing from 1 and 3% by 1.20 to 1.23 log CFU/g. For 74℃, heat susceptibility for all salt concentrations (0,1.0,3.0,5.0%) is similar, with 1% and 3% being 0.64 to 0.99 log CFU/g different from 0% and 5%. Based on the results, it can be concluded that the thermal resistance of Salmonella is significantly affected by both temperature and salt concentration. There is evidence to suggest that 0% has the highest amount of resistance among the other concentrations, 1% is the next highest, while both 3 and 5% showed lower, but almost equal z-values. Suggesting that salt concentration above 3% will show a similar or less thermal resistance, and concentrations of 0 and 1% will have equal or more thermal resistance in future research.

The objective of this thesis was to determine the anticoccidial profile of Eimeria spp. derived from wild turkeys. These drug-sensitive Eimeria spp. have potential to be used as vaccine candidates to control coccidiosis in commercial turkeys. Chapter two consists of a brief literature review focused on coccidiosis in turkeys. Chapter three addresses the methodologies utilized to isolate, recover, and speciate Eimeria recovered from wild turkey feces in the eastern two-thirds of the United States. From this, we successfully obtained single oocyst-derived stocks for E. meleagrimitis, E. dispersa, E. meleagridis, E. gallopavonis, and E. adenoeides. Chapter four describes the experiment conducted to assess the protective efficacy of an E. meleagrimitis vaccine candidate isolated from wild turkeys with and without intermittent amprolium administration. Additionally, the impact of vaccination and/or challenge and a candidate bioshuttle program on gut permeability and the microbiome was evaluated. The E. meleagrimitis vaccine candidate induced mild disease without affecting performance. Future research must be conducted to elucidate the impact of amprolium with and without single or multi-species live coccidiosis vaccination on the microbiome and gut barrier function in turkey poults.

Histomonas meleagridis, is a parasitic protozoan, commonly found in the gallinaceous species of birds and is the causative agent of Blackhead disease. The cecal worm, known as Heteraskis gallinarum, acts as a vector in the spread of H. meleagridis in gallinaceous birds. H. meleagridis develops inside the cecal worm and in its eggs which allows H. meleagridis a method to survive in the environment. Blackhead disease causes high mortality rates in both chickens and turkeys; however, turkeys are noted to have a higher mortality rate compared to chickens. Molecular diagnostic testing such as PCR has been developed by creating primers for H. meleagridis allowing producers to identify disease reservoirs for the Blackhead disease. Also, ELISA testing for antibody production can be used as a tool to confirm outbreaks of histomoniasis in turkey flocks that may or may not be showing symptoms. Current options for producers to control the disease on their farms include following biosecurity protocols, applying litter acidification additives, and placing lime on the ground around the outside of production barns. These methods used by the farmers do not effectively stop the spread of H. meleagridis in the flocks. Therefore, determining other possible vectors that can spread the disease on the farms, could help the producers in controlling histomoniasis. For this study, insects were collected on three farms in Arkansas and one farm in Oklahoma. These insects were sorted out by species before PCR was performed. First, the insects were tested to see if DNA was present and then tested to see if H. meleagridis was present. Based on the study, no insect tested contained H. meleagridis.