(Colinus Virginianus) by Drew G. Arnold, BS a Thesis In

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The Immunocompetence and Immunomodulation of Northern Bobwhite Quail (Colinus virginianus)

Drew G. Arnold, B.S.

A Thesis

Wildlife, Aquatic, and Wildlands Science and Management

Submitted to the Graduate Faculty of Texas Tech University in Partial Fulfillment of the Requirements for the Degree of

MASTER OF SCIENCES

Approved

Dr. C. Brad Dabbert Chair of Committee

Dr. Michael A. Ballou

Dr. Steven Presley

Mark Sheridan Dean of the Graduate School

May, 2019

Texas Tech University, Drew G. Arnold, May 2019

ACKNOWLEDGMENTS I dedicate this thesis to my late father Richard L. Arnold and grandfather Noel G. Harvison, both of whom instilled in me a love of the outdoors, natural resources and conservation from an early age. Without their nurturing and encouragement in these areas I certainly would not have pursued a career in this field, and I am indebted to them for the foundation they built for me. I miss you both dearly.

I would like to acknowledge the financial support of the Burnett Foundation for making this project possible. The contributions the Burnett Foundation has made in quail conservation is of great value and critical importance to this corner of the world and is tremendously appreciated. Thank you to Dr. Charles B. Dabbert, who took a chance on me when I showed up on a January day with no particular project and funding available at the time. Thank you for the opportunity to conduct this research and contribute to the understanding of quail ecology. I apologize for the delay in finishing this research but greatly appreciate the support and flexibility from Dr. Dabbert to return home to share the remaining time I had with my father and grandfather.

Thank you to Charles Starkey for the formulation of the feed study diets and fielding questions regarding feed study feed composition. Thank you to Joel McAtee, Feed Mill Manager of the Kansas State University O.H. Kruse Feed Technology Innovation Center and his staff for their expertise in manufacturing feeds for the feed study component of this research. Many thanks to Dr. William Cannon, Director of Nutrition and Quality Systems at Nurtablend, LLC for your assistance in determining the necessary rates of nutrients and vitamins to include in the feed study diets. Additional thanks to Dr. Michael Ballou for your contributions of minerals, nutrients, and vitamins from the Texas Tech Research Farm and access to your lab and lab equipment to complete this research.

A special thanks to Byron Buckley, John McLaughlin, Dakota Neel, Matthew McEwen, and Cody Griffin. I have a deep appreciation for the friendship you all provided. You all made graduate school infinitely more interesting and fun. A special thank you to my fellow Mississippian, Byron Buckley, for all of your early tutelage and your mentorship

Texas Tech University, Drew G. Arnold, May 2019 when I first entered graduate school. I had no idea what I was doing (sometimes I still don’t) and I could not have made the transition into graduate school without your aid.

I want to thank my mother, Polly Arnold, for your tough love that day in the field. You gave me the gumption to apply to graduate school and follow my dreams no matter the circumstances. Sometimes a little tough love is what I need. I am forever indebted to you and dad’s unceasing love and support.

Last of all, to my wife, Candace Arnold, I owe many lifetimes of thanks for your unending support and patience throughout grad school. It was because of you that I ended up at Texas Tech and I am grateful for the life we established in Lubbock and many great memories and friendships formed there. I love you.

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Texas Tech University, Drew G. Arnold, May 2019

TABLE OF CONTENTS ACKNOWLEDGMENTS ...... ii

ABSTRACT ...... v

LIST OF TABLES ...... viii

LIST OF FIGURES ...... ix

CHAPTER I ...... 1

IMMUNE FUNCTION PARAMETERS AND SURVIVAL IN BOBWHITES ...... 1

METHODS ...... 12

RESULTS ...... 19

DISCUSSION ...... 21

LITERATURE CITED ...... 31

CHAPTER II ...... 40

NUTRITIONAL MODULATION OF THE BOBWHITE’S IMMUNE SYSTEM .....40

METHODS ...... 59

RESULTS ...... 66

DISCUSSION ...... 67

LITERATURE CITED ...... 87

CHAPTER III ...... 99

NUTRITIONAL MODULATION OF MUSCLE DAMAGE ...... 99

METHODS ...... 109

RESULTS ...... 112

DISCUSSION ...... 114

LITERATURE CITED ...... 127 iv

Texas Tech University, Drew G. Arnold, May 2019

ABSTRACT Bobwhite populations have been declining across their historic range for several decades. A number of hypotheses have been posited regarding factors contributing the observed decline. One such factor that that has recently gained attention from wildlife professionals is the contribution of disease and parasitism to bobwhite population regulation. Despite limited evidence implicating infectious agents in bobwhite population declines, researchers appear to largely disregard the role of the immune system in controlling said infectious agents. The immune system is the most important component of any organism’s defense against infectious agents. Although the immune system is well studied in a number of gallinaceous species (e.g. chickens and other poultry) there is a paucity of information regarding the immune system of bobwhites. Furthermore, the lack of information also pertains to the effects of varying factors encountered in the natural environment that may impact the immune system, susceptibility to infectious agents, and potentially mortality. Nutrition plays an important role in supporting the immune system with evidence of tradeoffs between other physiologically important processes and the immune system resulting from nutritional or resource limitation commonly encountered in natural systems. Other factors inherent to natural systems cannot typically be controlled by land managers (i.e. weather, disease processes, predation, etc.) with the exception of habitat management. Even then, responses of vegetative communities must yield to abiotic factors such as the weather (e.g. drought). However, nutritional pathways may be altered through the provision of supplemental food resources by land managers. Hence, nutritional pathways may be utilized to alter the immune function of bobwhites and consequently may buffer bobwhite populations against the effects of infectious agents if these processes are limiting bobwhite survival. The goals of my study were to assess whether measures of immune function from wild captured bobwhites were important parameters in bobwhite survival. To assess the effects of immune function on the survival of bobwhites, I utilized two immune assays; a bacteria killing assay (BKA) and an enzyme linked immunosorbent assay (ELISA) to assess the innate and adaptive immune function. I used the Nest Survival data type in Program Mark to asses survival of bobwhites captured over the years of 2010 – 2011, v

Texas Tech University, Drew G. Arnold, May 2019

2011 – 2012, and 2013 – 2014. Immune function measures (BKA and ELISA – denoted as IgY in survival models) as well as covariates related to year of monitoring, age (juvenile/adult), initial capture mass and feed supplementation status (supplemented/control) were utilized as covariates within the analysis to determine their relative importance in explaining survival. Only bobwhites with relevant immune data (BKA/IgY) were included to achieve a sample size of 124 individuals. The top models for survival through the analysis interval included the models containing the parameters, Year, Feed_Stat, and IgY. The top model (Year+Feed_Stat+IgY) contained the additive effects of all three aforementioned parameters (Wi = 0.501). The parameter Year was determined to be the most important parameter and appeared to “wash-out” the effects of Feed_Stat and IgY. A post-hoc analysis excluding Year indicated that Feed_Stat and IgY were the most important parameters. The model averaged probability of a bobwhite collected over a 3-year period surviving during the study was ~22%

The role of nutrition on the bobwhite immune system was evaluated by providing a captive population of all male bobwhites with feeds of varying nutrient concentrations and compositions (i.e. vitamin, trace mineral, and antioxidant content.). The adaptive and innate immune systems were assessed via a battery of laboratory assays as well as in vivo assays. Diet did not influence innate immune function as measured by bactericidal killing capacity of serum, adaptive immune function measured as circulating immunoglobulin Y (IgY), and cell-mediated immune function as measured via subcutaneous swelling following phytohemagglutinin injection into the patagium. A significant time effect was observed for the adaptive and innate immune function parameters, circulating IgY and bactericidal killing capacity between time points (Day #1, Day #14, and Day #28). Through what nutritional pathways (e.g. nutritional component) the immune parameters were elevated is unknown. Whether nutritional quality is of consequence to the immune system of wild birds is yet to be seen and studies implicating nutritional profiles, available resources, and condition of bobwhites during immune compromising events warrants further study.

Texas Tech University, Drew G. Arnold, May 2019

The role of nutritionally supplemented diets consisting of varying nutritional qualities and components on muscle damage following extreme exertion was evaluated by providing a captive population of all male bobwhites with feeds of varying nutrient concentrations and compositions (i.e. vitamin, trace mineral, and antioxidant content.). Bobwhites were then exposed to a trial of muscular exertion for 15 minutes. Muscle damage resultant from exertion was assessed via determination of the concentration of enzymes indicative of muscle damage, creatine kinase (CK) and aspartate aminotransferase (AST) at 4 hours and 24 hours post exertion. Pathological examination of muscle tissues from the breast and thigh muscles were also utilized to determine effect of diet on incidence of muscle damage. A difference in CK concentrations between the NRC diet and NATURAL diet at 4 hours was observed. Four hours post exertion, CK concentrations in the NRC diet were an average of 8,690 +/- 3,572 U/L. No other differences between diets on CK and AST activity were observed at either 4 hours or 24 hours post exertion. I found that there was a significant difference in incidence of muscle damage as indicated by muscle damage lesion identification. It appears that nutritional quality did indeed affect muscle damage in bobwhites following strenuous muscular exertion, with bobwhites on the HVTM+A diet having the highest incidence of muscle damage as indicated by muscle damage lesions. The specific pathways regulating the occurrence of muscle damage in bobwhites fed diets with elevated nutrients is not known. However, the potential for reduction of muscle damage through increased nutritional supplementation does not appear in this specific study to be beneficial. A better understanding of the implications of specific nutrients, vitamins, antioxidants, etc. is necessary and additional research warranted.

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Texas Tech University, Drew G. Arnold, May 2019

LIST OF TABLES 1.1 Akaike’s Information Criterion, adjusted for small same size (n =124), ranking logistic regression models (Nest survival data type) relating to the annual survival of Northern bobwhites (Colinus virginianus) on the 6666 Ranch on the Rolling Plains of Texas, USA...... 29

2.1 Composition of complete diet base mix for feed trials...... 77

2.2 Composition of premix added to base mix to achieve experimental feed nutrient concentrations...... 78

2.3 Concentrations of nutrient additives in grams and milligrams added to base mix (see table 2.2) ...... 80

2.4. Analysis of nutritional components performed by A & L Plains Analytic Laboratory (302 34th St, Lubbock, TX 79404)...... 83

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Texas Tech University, Drew G. Arnold, May 2019

LIST OF FIGURES 2.1 Bactericidal killing capacity of diets for Trial #2 and Trial #3 combined in percentage bacterium, Escherichia coli ATCC 8739, killed compared to controls over the duration of the feed trials (28 days) ...... 84

2.2 Circulating immunoglobulin Y (IgY) of diets for Trial #2 and Trial #3 combined observed as the optical density (OD) at wavelength λ450 over the duration of the feed trials (1, 14, and 28 days)...... 85

2.3. Wing web swelling in response to phytohemagglutinin (PHA) injections of Trial #2 and Trial #3 combined at Day 21. Values presented as a wing web swelling index calculated as the difference between wing web thicknesses at PHA injection site versus thicknesses at un-injected sites...... 86

3.1 Response of liver enzyme, creatine kinase (CK), among diet treatments for both Trial #2 and Trial #3 combined at 4 hours and 24 hours post muscular exertion..... 121

3.2 Response of creatine kinase (CK), among diet treatments for Trial #2 and Trial #3 combined at 4 hours post muscular exertion...... 122

3.3 Response of liver enzyme, creatine kinase (CK), among diet treatments for Trial #2 and Trial #3 combined at 24 hours post muscular exertion...... 123

3.4 Response of liver enzyme, aspartate aminotransferase (AST), among diet treatments for Trial #2 and Trial #3 combined at 4 hours and 24 hours post muscular exertion...... 124

3.5 Response of liver enzyme, aspartate aminotransferase (AST), among diet treatments for Trial #2 and Trial #3 combined at 4 hours post muscular exertion. .. 125

3.6 Response of liver enzyme, aspartate aminotransferase (AST), among diet treatments for Trial #2 and Trial #3 combined at 24 hours post muscular exertion. 126

Texas Tech University, Drew G. Arnold, May 2019

CHAPTER I

IMMUNE FUNCTION PARAMETERS AND SURVIVAL IN BOBWHITES

Introduction The northern bobwhite quail (Colinus virginianus), hereafter bobwhite(s), were once found thriving in a number of early successional habitat types and ecoregions across their historic range (Brennan 1991, Williams et al. 2004, Hernandez et al. 2013). However, bobwhite populations have experienced marked range-wide declines the past five decades with population indices indicating population declines in excess of 3% annually across the bobwhite’s historic range from 1966 to 2015 (Sauer et al. 2017). In the state of Texas, where bobwhites are of high cultural, economic, and ecological significance (Guthery and Hernandez 2012, Johnson et al. 2012), the North American Breeding Survey indicates bobwhite populations have declined 1.8% annually (Sauer et al. 2017). The decline of bobwhite abundance over the past 50 years has drawn the concern of researchers across their range and has prompted investigations into the cause(s) of bobwhite population decline. The extent of factors hypothesized to contribute to bobwhite population declines range from widespread landscape changes such as expansion of industrial agricultural agriculture (Peterson et al. 2002), high density silvicultural practices (Burger et al. 2002, Jones et al. 2010), and urbanization (Veech 2006), to more localized phenomena such as predation (Rollins and Carroll 2001), exotic species (Sands et al. 2011, Allen et al. 1995), releases of pen raised bobwhites (Hutchins and Hernandez 2003, Evans et al. 2006), pesticide use (Brewer et al. 1996), and disease (Hernandez et al. 2013) among others. Despite the numerous hypothesized contributing factors, the general consensus among bobwhite researchers is that the ultimate cause for the decline of bobwhites is a consequence of large-scale habitat loss and fragmentation due to expansion of land use changes (i.e. agriculture, forestry, urbanization, etc.) that alter the successional state beyond use for bobwhites (Brennan 1991, Burger 2002, Hernandez et al. 2013). The combined effect of more specific factors may contribute to 1

Texas Tech University, Drew G. Arnold, May 2019 habitat loss and fragmentation and subsequently affect bobwhite populations at a regional scale (Guthery 1997) but are not regarded as the ultimate cause of decline. However, the complete mechanisms regulating bobwhite populations at regional or local scales vary and many relationships remain unclear.

At a regional/eco-region level drastic fluctuations of bobwhite populations are known to naturally occur among years and are particularly observable in arid and semi- arid portions of their range (e.g. Texas and Oklahoma) (Hernandez et al. 2005). Such drastic fluctuations have largely been attributed to bobwhite response to weather variables (e.g. precipitation, heat loads, etc.) and the subsequent response of vegetative factors necessary to meet critical life-history events (i.e. nesting and rood rearing) and physiological requirements (thermal cover) (Hernandez et al. 2005, Parent et al. 2015). Within the arid and semi-arid regions bobwhite fluctuations may vary considerably between years so much so that the phenomenon is often described as “boom and bust” (Jackson 1962, Bridges et al. 2001, Hernandez et al. 2005). An example of the boom and bust phenomenon was evident during 2011 and 2012 when much of the state of Texas experienced one of the most severe droughts on record for the state, effecting many regions of the state known for exceptional quail populations (e.g. the Rolling Plains and Rio Grande Plains ecoregions) (Rollins 2007, Buckley et al. 2015, Parent et al. 2016). Observed bobwhite abundances within the Rolling Plains ecoregion during 2011 and 2012 indicated depressed populations below the 15 year mean (Texas Parks and Wildlife). As adequate precipitation returned and drought conditions receded in successive years, bobwhite abundances rebounded and exceeded the 15 year mean by 2015 (Texas Parks and Wildlife). Within the Rolling Plains ecoregion of Texas, where the boom and bust phenomena is commonly observed, Jackson (1962) hypothesized that in addition to weather variable (i.e. drought and precipitation events), grazing and plant succession were factors potentially contributing to population fluctuations among years. Bobwhite abundance has been positively associated with areas dominated by rangeland (Brady et al. 1998, Peterson et al. 2002, Guthery et al. 2001). The Rolling Plains ecoregion of Texas is an area characterized by an abundance of rangeland and largely

Texas Tech University, Drew G. Arnold, May 2019 lacks many of the landscape factors currently exerting pressure on bobwhite populations (Rollins 2007). Within the Rolling Plains of Texas and Oklahoma, a region where, despite adequate precipitation and favorable habitat conditions during 2010 bobwhites inexplicably declined (Dunham et al. 2016). This unusual localized decline sparked an abundance of regional research interest in the role that disease and parasites (hereafter infectious agents) might play in the regulation of bobwhite populations, a topic that has largely been unexplored in the bobwhite’s life history (Peterson 2007, Hernandez et al. 2013, Hernandez and Guthery 2012).

Infectious Agents The role that infectious agents play in regulating wildlife populations has historically been viewed as irrelevant in free roaming populations except as a consequence of poor habitat conditions or natural disasters (Herman 1969, Peterson 2004, Peterson 2007). Consequently, the effects of infectious agents on wildlife population regulation has not been well studied at an appropriate scale to draw conclusions for many wildlife species, including bobwhites (Peterson 1996, Tompkins et al. 2002). Nonetheless, bobwhite researchers have performed numerous studies and continue to study infectious agents in bobwhites; many of these studies occurring at the eco-regional or local scale in Texas (Bruno et al. 2015, Dunham et al. 2014, Henry et al. 2017, Brym et al. 2018). A lion’s share of the research related to bobwhite infectious agents has largely focused on surveillance for disease causing and parasitic agents (hereafter infectious agents) and their prevalence among wild populations (e.g. Jackson and Green 1965, Jackson 1969, Bruno et al. 2015, Dunham et al. 2017). Only recently, have researchers begun to investigate the roles infectious agents play in bobwhite physiological status and the effects infectious agents might have on bobwhite survival and subsequently population trends (Bruno et al. 2015, Henry et al. 2017, Brym et al. 2018). For example, Henry et al. (2017), observed declining abundances of bobwhites in Mitchell County, TX (located within the Rolling Plains ecoregion) concomitant with elevated helminthic parasite levels over a 5-year monitoring period. Henry et al. (2017) also noted lower bobwhite abundances in 2017 following high infection rates in 2016

Texas Tech University, Drew G. Arnold, May 2019 suggesting that high rates of infection may have reduced bobwhite survival resulting in low abundances. The brunt of recent research has heavily favored the potential effects of parasitic genera, specifically nematodes (caecal worms – Aulonocephalus pennula) and eyeworms (Oxyspira spp.) (Villareal et al. 2012, Dunham et al. 2014, Henry et al. 2017, Brym et al. 2018) on the bobwhite’s physiological system. These studies have hypothesized that the bobwhite’s daily life activities (e.g. flight, foraging, etc.) may be impaired by infectious agents and lead to higher risk of mortality from predation, trauma, and subsequent co-infections. Likewise, high profile wildlife diseases (e.g. West Nile virus, high pathogenic avian influenza, etc.) with the potential to inflict high mortality in avian species (Brown et al. 2007) and their recent emergence in the U.S. has spurred additional surveillance studies to determine presence and prevalence of emerging diseases among bobwhites (Urban et al. 2013). While past and current research is certainly is useful in determining the status of infectious agents among bobwhites, it fails to identify and quantify a key aspect of a bobwhite’s defense against infectious agents, the immune system. The functional aspects of the avian immune system and its potential to effect individual fitness (e.g. fecundity, mortality, etc.) has recently gained the attention of biologists and ecologists (Lochmiller and Deerenberg 2000, Sheldon and Verhulst 1996, Norris and Evans 2000). However, even with improved insights into the function aspects of the immune system, the influence of the immune system on wild avian species appears to be largely context dependent and as such is still poorly understood especially in regard to survival for many species.

To my knowledge, the relationship between immune system function and the survival rates of wild bobwhites has not been previously described. Estimation of survival rates for wildlife species are necessary to assist researchers in monitoring species population trends (Sandercock et al, 2008). Likewise, identification of mortality risks, both biotic or abiotic, provides vital information about factors driving survival and aids in identification and implementation of appropriate management activities by managers. Those existing studies describing bobwhite immune function have limited their scope to laboratory-based studies with captive bobwhites under artificial conditions, identifying

Texas Tech University, Drew G. Arnold, May 2019 effects of external stimuli (i.e. thermal extremes) (Dabbert et al. 1997) or nutritional requirements on the immune function of bobwhites (Lochmiller et al. 1993, Dabbert et al. 1996). Due to the lack of research available on the relationship between immune function and survival rates of bobwhites, an opportunity to expand knowledge of factors potentially driving bobwhite survival exists and seems prudent to explore.

The Avian Immune System Sandercock et al. (2008) identified survival of bobwhite chicks to independence, juvenile survival, and winter survival of bobwhite adults as having the greatest impact on population growth in declining bobwhite populations. It is clear that survival rates drive variation in bobwhite populations, a fact that stresses the need for a better understanding of factors influencing bobwhite survival. Further, insights into related factors would be advantageous for the future persistence of the species. A factor that has received limited research attention amongst bobwhite research is the immune system. Moreover, the limited research that has occurred on the bobwhite’s immune system have heavily relied on captive bobwhites (Lochmiller et al. 1993, Dabbert and Lochmiller 1994, Dabbert et al. 1997) which can be attributed to the difficulties in implementing meaningful immune techniques or assays in field studies (Millet et al. 2007) and the subsequent interpretation of immune responses under variable context dependent factors (e.g. nutritional status, current/prior infection status, reproductive state, etc.) is difficult to discern in natural settings (Demas et al. 2011). The immune system has been implicated as an important physiological function regulating survival in vertebrates (Sheldon and Verhulst 1996, Lochmiller and Deerenberg 2000). Likewise, Lochmiller and Deerenberg (2000) identify survival in conjunction with reproductive effort as an important factor contributing to an organism’s fitness. Consequently, investigating the role the immune system potentially plays in the survival of bobwhites is warranted. Within available scientific literature, outside of studies on captive bobwhites, there appears to be no such studies documenting the effects of immune function parameters on bobwhite survival.

The immune system is an exceedingly complex and multifaceted physiological system responsible for defending animals from parasitic and pathogenic infections and

Texas Tech University, Drew G. Arnold, May 2019 non-infectious molecules and organisms (Lochmiller and Deerenberg 2000, Roitt 2001, Korver 2012). The physiological costs of maintaining a competent immune system in the face of variable environmental and climatic conditions experienced by an organism throughout their lifetime and competing physiological functions (i.e. growth, locomotion, thermoregulation, reproduction, etc.) has been hypothesized to be nutritionally and energetically costly (Sheldon and Verhulst 1996, Lochmiller and Deerenberg 2000, Norris and Evans 2000, Martin et al. 2003). Although there is some uncertainty regarding costs in terms of other physiologically important processes (e.g. growth) (see Klasing 1998), the impression that the cost of mounting and maintaining a competent immune system is costly is prevalent within scientific literature. However, the underlying mechanisms regulating costs are poorly understood. The energy requirements of maintaining a competent immune system - which we will refer to as immunocompetence and functionally define as “the ability of an organism to prevent or control infection by pathogens and parasites” (Norris and Evans 2000) – has yielded data suggesting there are a number of tradeoffs between the immune system and other physiological functions (i.e. growth, thermoregulation, reproduction, etc.) (See Sheldon and Verhulst 1996, Lochmiller and Deerenberg 2000, and Norris and Evans 2000 for reviews). Immune function has been tied to survival through measures of the immune system (e.g. circulating Immunoglobulins) or via applied immune challenges (e.g. PHA injections, antibody injections, etc.) to wild species (Moller and Saino 2004). It is assumed that an individual’s ability to resist infection or clear an infection should be associated with its immunocompetence and subsequently higher measures of immune response should indicate a strong immune system (Ardia and Schatt 2008, Wilcoxen et al. 2010). Additionally, an animal’s ability to defend itself from infectious agents and survive is assumed to be tied to its ability to maintain a competent immune system in the face of competing energetically costly physiological processes (i.e. growth, reproduction, thermoregulation, etc.) (Lochmiller and Deerenberg 2000). Thus, having an understanding of the physiological foundations of the immune system are necessary to draw conclusions regarding immune function in varying contexts.

Texas Tech University, Drew G. Arnold, May 2019

The avian immune system is composed of two branches; the innate immune system and the acquired or adaptive immune system, hereafter referred to as innate immunity and adaptive immunity. These branches can be further broken into constitutive, humoral, and cell-mediated immunity. Constitutive immunity refers primarily to initial immune responses of the innate immune system following encounter of an infectious agent and its components are largely derived from epithelial defenses, various antimicrobial peptides and proteins, certain antimicrobial actions of important immune cells (heterophils, macrophages, and other phagocytic cells) (Millet et al. 2007). Constitutive immunity is also comprised of the activation of the acute phase inflammatory response which is highly effective at non-specific response to infection but is typically considered substantially costly to mount (Lochmiller and Deerenberg 2000, Klasing 2004). Humoral immunity refers in general to the enzymatic action of certain cellular pathways that eliminate infectious agents by promoting inflammatory responses, facilitate the lysis and/or phagocytosis of infectious agents, and signaling and activation of the adaptive immune system. Innate immunity relies on rapid non-specific responses to eliminate pathogens/parasites and non-self molecules when encountered. The innate immune system is composed of anatomical barriers (i.e. integumentary and mucosal membranes), resident flora (non-pathogenic microbes), humoral factors (e.g. complement and associated enzymes), and cellular responses (e.g. neutrophils, monocytes, macrophages, etc.) (Demas et al. 2011). Innate immunity is typically the first branch of immunity activated when challenged by pathogens and quickly responds to infection by release of chemical signals from receptor cells to recruit phagocytic cells (e.g. heterophils, macrophages, etc.). The release of phagocytic cells allows for elimination of pathogens via the release of cytotoxic chemicals such as reactive oxygen species and nitric oxide and degranulation of pathogen cells. In addition of release of toxic chemicals leading to pathogen death, innate immune cells activate plasma complement proteins and enzymes that further identify and target pathogens for removal by other immune cells. The innate immune response is again, non-specific, attacking non-self molecules/pathogens and does not display pathogen memory. This non-specific response and the induction of an acute phase response often results in a multitude of symptoms 7

Texas Tech University, Drew G. Arnold, May 2019 that outwardly present in avian species such as inflammation at the site of infection, fever, reduced feed intake, lethargy, and catabolism of skeletal muscle and energy reserves (Korver 2012).

The acquired or adaptive branch (hereafter acquired immunity) of the immune system involves a slower and pathogen specific targeted response following exposure to a pathogen (Demas et al. 2011, Korver 2012). Acquired immunity relies on the recognition of certain immune system activators and a resulting memory of pathogen exposure, facilitating a rapid and specific response after subsequent pathogen exposures. The acquired immune system works in accompaniment with the innate immune system relying on cues from the innate immune system in order to effectively clear an infection. The innate immune system aids in presenting antigen to the acquired immune system, allowing for future recognition of said pathogen. Additionally, the acquired immune system can become activated if the innate immune system reaches a threshold of antigen (Murphy et al 2007). Future exposure to or infection by a recognized pathogen antigen allows for a specific, targeted response to a pathogen and can limit energetic costs to avian species (Korver 2012). Alternatively, the innate immune system, provides a rapid and generic response to an assortment of pathogens but this generic response has been demonstrated to frequently be more energetically and physiologically costly to avian species. For further insights into the avian immune system there are several valuable papers that provide characterization of aspects of the avian immune system (Fearon and Locksley 1996, Klasing 1998, Korver 2006, Demas et al. 2011, Korver 2012)

Measuring the Immune System/Immune Response The immune system is an exceptionally complex physiological system that has evolved as a means to defend an organism against infection by infectious agents (e.g. viruses, bacteria, parasites, etc.) (Moller and Saino 2004). The immune system has been hypothesized to have been shaped by selective pressures from environmental factors and associated density dependent processes within animal populations (e.g. nutrition, opportunistic pathogens, etc.) (Lochmiller 1996). Additionally, the maintenance and deployment of the immune system against infectious challenges are hypothesized to incur

Texas Tech University, Drew G. Arnold, May 2019 considerable costs to an organism (Lochmiller and Deerenberg 2000, Bonneaud 2003). Lochmiller and Deerenberg (2000) offer that immunity is a major physiological mechanism regulating an organism’s survival. There appear to be trade-offs among other physiologically important life-history components such as reproduction, growth, or thermoregulation associated with the maintenance and deployment of the immune system. As such, it seems logical to assume that allocation of resources towards other important life-history components and away from the immune system may result in a reduction in the fitness of an organism (Norris and Evans 2000). These trade-offs may have negative consequences to an organism in regard to fitness

In order to a trade-off to be evolutionarily significant, Norris and Evans (2000), propose the following three constraints: 1) immunocompetence must compete with life- history components for limited resources; 2) investment into one life-history component must reduce immunocompetence; 3) a reduction in immunocompetence must result in fitness. Such trade-offs and subsequent reductions in fitness in avian species have largely focused on reductions in reproduction and survival and examples are prevalent in the literature.

Animals encounter a host of infectious agents in their natural environment, which seek to exploit host resources for their own use (Sheldon and Verhulst 1996, Moller and Saino 2004). Because of the numerous infectious agents encountered, it is not surprising that animals have developed a variety of physiological and behavioral defenses against infectious agents. The immune system is one such defense and is an animal’s the primary defense mechanism to eliminate or control infectious agents (Zuk 1994, Lochmiller and Deerenberg 2000). In addition to defending an animal against infectious agents, the immune system is also a major component regulating the survival of an animal (Lochmiller and Deerenberg 2000). Likewise, an individual animal’s ability to appropriately mount an immune response upon exposure to infectious agents, known as “immunocompetence” is considered critical for resistance to infectious agents and consequently survival (Demas et al. 2011). The ability of an individual to survive infection by an infectious agent (i.e. disease or parasite) is presumed to be related to the

Texas Tech University, Drew G. Arnold, May 2019 strength of an individual’s immune system or “immunocompetence” (Wilcoxen et al. 2010). The process of maintaining a competent immune system and deploying an immune response has been speculated to be energetically costly and may incur physiological costs to other important physiological functions (e.g. growth) or life-history functions (e.g. reproduction which consequently may impose reductions in fitness (Sheldon and Verhulst 1996, Lochmiller and Deerenberg 2000, Norris and Evans 2000, Bonneaud 2003). Costs associated with immune response are most notably proposed to occur during energy limited conditions and mechanistically occur by allocation of energetic resources towards physiological functions that are most beneficial in regard to fitness (Norris and Evans 2000). Furthermore, it is hypothesized that this priority allocation of resources towards physiologically important life-history traits or physiological functions (e.g. immune function vs. growth) results in trade-offs that have the capacity to induce negative effects on fitness (Lochmiller and Deerenberg 2000, Moller and Saino 2004, Demas et al. 2011) In order for a trade-off associated with immunocompetence to be evolutionarily significant, Norris and Evans (2000), propose the following three constraints: 1) immunocompetence must compete with life-history components for limited resources; 2) investment into one life-history component must reduce immunocompetence; 3) a reduction in immunocompetence must result in fitness. Such trade-offs and subsequent reductions in fitness in avian species have largely focused on reductions in reproduction and survival and examples are well documented in the literature (Lochmiller et al. 1993, Saino et al. 1997, Hanssen et al. 2004, Moller and Saino 2004, Hanssen et al. 2005).

It follows that a functional measure of immunocompetence is necessary to evaluate and translate the effects of investment into competing physiological functions on fitness. According to Norris and Evans (2000) there are two different types of techniques commonly used to assess immunocompetence and life-history trade-offs; 1) Monitoring techniques, characterized by multiple sampling occasions and collection of biological specimens (e.g. blood sampling) which can then be assessed in a controlled environment to indicate the immune status of an individual over a time interval.; 2) Challenge

Texas Tech University, Drew G. Arnold, May 2019 techniques, which are characterized by exposure of a particular component of individual’s immune system to a challenge and a measurement of the strength of the response. Of the two techniques, challenge techniques are typically preferred by investigators due to their standardized application and quantification of immune response (Norris and Evans 2000). For example, the injection of phytohemagglutinin (PHA), a mild mitogen that elicits cellular proliferation of T-cells at the site of injection, into the patagium of avian species is commonly used as standardized measure of cell-mediated immune function (Smits et al. 1999). A criticism shared by many researchers is that those studies utilizing one technique or measurement of one specific component of the immune system assume that all components of the immune system respond in the same manner as that component being measured, such that the magnitude of investment into one component of the immune system likewise occurs in all components of immune system (Norris and Evans 2000, Demas et al. 2011). Because immune responses are not static but rather are highly variable and dependent upon a number of factors including but not limited to season, reproductive status, sex, energetic and nutritional state, stress, and past and current infections status, coupled with the complex nature and interrelated aspects of immune system components it seems logical to utilize multiple assessments of the immune system to generate an accurate representation of an individual’s immunocompetence (Demas et al. 2011).

For this study we utilized several measures of the immune system to evaluate immunocompetence. Among those used were the Bacteria Killing Assay (BKA) and Total Immunoglobulin (Ig) levels. Both of these assays fall under monitoring techniques (Norris and Evans 2000). BKA assesses the actual ability of the immune system to eliminate an infectious agent (E. coli ATCC #8739) in this case and provides a functionally relevant assessment of the bobwhite’s immune function versus challenging the immune system with novel antigens (Demas et al. 2011). In contrast to the protocol of Millet et al. (2007) which utilized whole blood, serum samples were utilized for this study. Fortuitously, E. coli ATCC #8739 is most effected by complement (i.e. serum) derived mechanisms of immune function (Millet et al. 2007). The pathways for

Texas Tech University, Drew G. Arnold, May 2019 infectious agent destruction in serum relies on the abilities of natural antibodies, typically Immunoglobulin M (IgM) or Immunoglobulin A (IgA) which are components of the innate immune system (Millet et al. 2007, Palacios et al. 2009, Demas et al. 2011) and according to Demas et al. (2011), allows for a more functionally relevant assessment of an individual’s immune system than challenge-based assessments.

Total Immunoglobulin (Ig) assessment allows for the assessment of circulating immunoglobulins (Ig), an important component of the adaptive immune system. Immunoglobulin Y, (IgY) was assessed in this study. IgY is an important and widely circulating component of the adaptive immune system that is responsible for the coating of infectious agents for recognition and destruction by neutrophils and macrophages (Bourgeon et al. 2010, Demas et al. 2011). The downside to this assessment is the lack of ability to assess whether circulating IgY is reflective of the individual’s immunological state or if the levels of circulating IgY reflect current infection status (Martin et al 2006). It should be noted that due to logistical difficulties in recapturing bobwhites, these techniques provide a “snap shot” of the captured bobwhite’s immune system. Techniques that can provide a baseline of immune function should be used in conjunction with infectious agent challenges and along with techniques that track immune function over time would yield a better understanding of how different components of the immune system responds within a particular context. However, studies are often constrained by the natural life histories of an organism being studied (Demas et al. 2011), and as such investigators should evaluate immune response assessment techniques based on the life history and context driven factors that may arise during their study.

METHODS

Study Area I conducted my research using blood samples and subsequent serum samples collected as a by-product of the studies of Buckley (2013), McLaughlin (2016), and

Texas Tech University, Drew G. Arnold, May 2019

Wiley (2017). In short, these studies evaluated the effect of broadcast supplemental feed on survival, reproduction, and recruitment of bobwhites on the Rolling Plains of Texas, USA. All of these studies were conducted on the historic 6666 Ranch (also locally known as the “4 Sixes Ranch”) located in Guthrie, King County, TX. The location of the 4 Sixes is located within the Rolling Plains Ecoregion of Texas. Refer to Buckley (2013), McLaughlin (2016), and Wiley (2017) for a complete description of the study area, management activities, and experimental procedures during respective studies. Pertinent to my research, bobwhites were trapped using walk-in funnel traps (Stoddard 1931) baited with milo. Trapping for most studies began in early to mid-October (~October 1st – October15th) and ended just prior to breeding season on approximately March 31st or until nesting activity was confirmed (McLaughlin 2017). Bobwhites weighing ≥ 150 grams were fitted with a necklace style radio transmitter weighing approximately 7 grams (American Wildlife Enterprises, Monticello, FL). Individuals were added to the sample size through the duration study in accordance with the staggered entry design as outlined in Pollock et al. (1989). Bobwhites were allowed an acclimation period of seven days following capture, fitting with a radio-collar, and release before inclusion into the survival study. A period of seven days is commonly used in radio-telemetry studies to minimize any potential effects capture, handling, and tagging may have on survival (White and Garrott 1990, Burger et al. 1995). Bobwhites failing to survive past this seven-day period were subsequently censored from my analysis. Bobwhites were tracked and homed in on at least once a week for the duration of each study. See the respective study manuscripts of Buckley (2013) and McLaughlin (2016) for a more detailed description of tracking efforts. Bobwhites that were unaccounted for, could not be assigned a mortality cause, experienced radio-failure or simply disappeared were right censored from the last known location and date (Buckley et al. 2018) for my survival analysis. Due to inconsistencies among the studies and collection of data of Buckley (2013), McLaughlin (2016), and Wiley (2017) some bobwhites may have been censored that possibly could have contributed to the analysis.

Texas Tech University, Drew G. Arnold, May 2019

Blood collection occurred as a by-product of trapping efforts. Not all bobwhites trapped and fitted with radio-collars had blood samples collected. Collection of blood samples was an opportunistic practice and was largely dictated by logistical constraints facing the collecting researcher (i.e. time, supplies, stress on bobwhites, etc.) (Personal Communication Byron Buckley and John McLaughlin). In short, bobwhites trapped in walk-in funnel traps were collected from traps and placed in cotton bags and placed in a research vehicle for the collection of demographic (i.e. age and sex), morphometric (i.e. mass), and biological (blood) data/samples and radio-tagging when appropriate (i.e. mass ≥ 150 grams). Blood was collected via venipuncture of the brachial vein with a 25-gauge hypodermic needle and collected in 200 µl Saf-T-Fill capillary collections tubes (RAM scientific) and stored on ice packs in a cooler until centrifuging could occur. Blood samples were centrifuged at 1500 RPM for 15 minutes in portable centrifuges (product information here…) and the resulting separated serum component was removed via sterile pipette and placed into Nalgene® cryo-tubes (Nalgene) and frozen in a commercially available freezer at the 6666 Ranch until samples could be transferred to a - 20°C or -80°C freezer for later analysis.

Bacteria Killing Assay (BKA) We utilized a bactericidal killing capacity immune assay that takes advantage of the natural antibodies and lysozymes present in the humoral fluid to destroy bacteria as a measure of the functional aspect of the constitutive innate immune (Millet et al. 2007). We chose to use a modified protocol developed by Millet et al. (2007). Modifications pertained to reductions in the amount of serum used in the assay through altered dilutions. Modifications were necessary due to the limited and variable amounts of serum collected from wild birds and the need to measure other functional aspects of immunity (i.e. multiple assays requiring serum). The bacterium species E. coli (Escherichia coli) ATCC 8739 (3.1 x 107 CFUs per pellet, Epower Microorganisms no. 0483E7, MicroBioLogics, St. Cloud, MN) was reconstituted in 25 ml of tryptic soy broth (TSB) and incubated for 24 hours at 37°C. After 24 hours the stock solution was centrifuged at 1200 relative centrifugal force (rcf) for 10 minutes and tryptic soy broth

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(TSB) removed via sterile pipette. Tryptic soy broth was immediately replaced with 25 ml sterile phosphate buffered saline (PBS). Twenty-five microliters (µl) of previously collected and frozen serum (wild caught and captive was diluted 1:4 with CO2 independent media plus 4 mM L-glutamine (catalog no. 18045; Gibco-Invitrogen,

Carlsbad, CA) yielding a final volume of 100 µl (25µl serum:75 CO2 independent media). A 20µl aliquot of E. coli ATCC 8739 serially diluted to contain approximately 200 colony forming units (CFU) was then added to the diluted serum samples. The day prior to assaying samples, serial dilutions were performed and plated on agar plates at varying concentration to assess stock solution concentrations. Diluted serum samples were incubated for 40 minutes at 37°C and then 50 µl aliquots promptly spread on plates in duplicate. Control samples were included for every 10 samples run. Control samples consisted of 100µl of CO2 independent media with the addition of 20 µl of E. coli ATCC 8739 working solution. Duplicate control samples of 50 µl aliquots were plated samples and incubated at 37°C overnight. Viable colonies (i.e. colony forming units – CFU’s) were enumerated per plate and the average number of colony forming units (CFU’s) per duplicate calculated ((Plate #1 + Plate #2)/2). Bactericidal capacity of bobwhite serum was measured as the proportion of bacteria (CFUs) “destroyed” between the control sample average CFU count and the serum sample plate average CFU count, ((Control CFU average – Serum Sample CFU average)/Control CFU average). The chosen dilution of 1:4 and time stop point of 40 minutes were used based on a prior optimization experiment to identify the time stop point and dilution factor.

Enzyme Linked Immunosorbent Assay (ELISA) To assess the acquired or adaptive branch of the immune system we utilized the protocol of Killpack et al. (2013) (Originally Fassbinder-Orth (2013)) to determine total IgY levels via an enzyme linked immunosorbent assay (ELISA). The ELISA assay is used to assess humoral immunity, a component of the acquired or adaptive immune system, by quantifying total IgY, the most important circulating plasma protein within the humoral immune system of avian species (Roitt et al. 2001), levels within the serum of bobwhites. Flat bottomed 96 well microplates were coated with 100 µL of bobwhite

Texas Tech University, Drew G. Arnold, May 2019 serum diluted to 1:100 in coating buffer (0.015 M Na2CO3, 0.035 M NaHCO3, pH 9.6) in duplicate. Negative controls consisting of coating buffer and positive controls of a composite serum sample (i.e. a mixture of 10 randomly selected pooled serum samples) were also included on every plate. Plates were then incubated overnight at 37°C after which the coating buffer was removed and 200 µL of blocking buffer (PBS with 5% non- fat dry milk, 0.05% Tween) was added to each well of the plate. The plate was incubated at room temperature for 30 minutes and then washed with wash buffer (PBS with 0.05%Tween) four times. Fifty µL of detecting conjugate goat anti-bird IgY – horse radish peroxidase (Bethyl Laboratories, Inc., Montgomery, TX) was then added at a dilution of 1:1000 in blocking buffer, incubated for 1 hour at 37°C and then washed away with wash buffer. One hundred µL Tetramethylbenzidine (TMB)-peroxidase substrate (catalog no. 34028; ThermoFischer Scientific, Waltham, MA) was then added to each well and incubated for 5 minutes, and the reaction was stopped with 100 µl 1 M H2SO4. Optical density (OD) was then read with an automated microplate reader SpectraMax

340PC (Molecular Devices, Sunnyvale, CA at A405. Total IgY was reported as (OD of sample) – (OD of negative control sample).

Experimental Design and Statistical Analysis I developed 12 a priori models using biotic and abiotic variables that I deemed biologically relevant to bobwhite survival. No grouping variable was utilized for model development, rather due to highly variable group size among years (2010 – 2013), year was utilized as a covariate resulting in one cohesive group. There were 61 bobwhites, 19 bobwhites, and 44 bobwhites for the intervals of 2010 – 2011, 2011 – 2012, and 2013 – 2014 respectively. Bobwhites with only complete records for both BKA and ELISA assays were included in the survival analysis. Six covariates were included in model development. Parameters or covariates for model development included: 1. Year: Due to stochastic events such as drought, inclement weather conditions, and other unpredictable factors wild bobwhites experience in conjunction with different experimental designs over the course of prior studies I predicted Year would significantly contribute to overall survival estimates; Age: Although age has not been shown to contribute to survival

Texas Tech University, Drew G. Arnold, May 2019 among bobwhites (Pollock et al. 1989, McLaughlin et al. 2018), there is evidence that the immune system may not be well formed in juvenile cohorts of avian species (Hausmann et al 2005, Lavoie et al. 2007) and as such might contribute to survival if individuals with immature immune systems are more susceptible to mortality; 3. Feed_Status: Bobwhites receiving supplemental feed have displayed increased survival versus their unfed counterparts (Buckley et al. 2015, McLaughlin et al. 2018), although some studies note neutral or limited benefit to bobwhites (Guthery et al. 2004, Henson et al. 2012). The goal of such supplemental feeding programs centers of buffering bobwhites against stressors (drought, harsh cold, resource limitations, etc.) and subsequently may allow bobwhites to adequately meet increased energetic and nutritional demands. Moreover, there is evidence that immune function is affected via nutritional pathways in bobwhites (Lochmiller et al. 1993). I predict that the feeding status (i.e. receiving supplemental feed) will be an important contributing parameter to the best approximating model for survival.; 4. Mass: Mass at time of capture can be used as a body condition index, which may indicate a bobwhite’s fitness and overall ability to withstand stressors (Hernandez and Guthery 2012). Additionally, body mass might be suggestive of nutritional state and serve as an indicator of ability to reallocate resources towards immune function (Bourgeon et al. 2010, Wilcoxen et al. 2010). I predict that bobwhites with higher capture masses (mass) will be in better physiological and immunological condition and will be an important contributing parameter to the best approximating model for survival; 5. IgY: The immune system is a dynamic system whose mechanisms of regulation often varies seasonally (Nelson 2004) and a single measure of immune function is a “snapshot” of the individual’s immune status at time of capture. However, circulating IgY is a reliable indicator of adaptive immune function (Hegemann et al. 2017) and measures of immune status at time of status might indicate the overall physiological condition of the individual captured (i.e. poor immune function measures = poor condition) which subsequently may translate into susceptibility to infectious agents ultimately poor survival (Hanssen et al. 2004, Moller et al. 1998). I predict that due to the importance of Immunoglobulin Y (part of the adaptive branch of the immune system) in the avian immune system and its ability to be affected via nutritional pathways (Bourgeon et al. 17

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2010) that IgY will be an important contributing parameter to the best approximating model for survival.; 6. BKA: Represents the bactericidal killing capacity primarily of natural antibodies (IgM or IgA) of the innate immune system (Millet et al. 2007, Palacios et al. 2009). The innate branch of the immune system is susceptible to the effects of nutritional pathways and may be used as a representation of the physiological status of an individual which may subsequently translate into survival (Bowers et al. 2014). I predict that the immune function measure “BKA” will be an important contributing parameter to the best approximating model for survival.

Program MARK I used the nest survival model in Program MARK (Dinsmore et al. 2002) to assess the annual survival of radio-tagged bobwhites against 12 a priori models. A priori models were developed based on biologically relevant hypotheses. The nest survival model was utilized due to its flexibility in managing “ragged data”, that is data where logistical constraints (i.e. radio failures, weather conditions, time constraints, etc.) resulted in uneven time intervals or missing records (Buckley et al. 2015). Use of nest survival was deemed appropriate for my study assessing adult bobwhite survival based on a number of other studies likewise assessing adult or juvenile survival via nest survival models (Rotella et al. 2004, Ruthrauff and McCaffery 2005, Mong and Sandercock 2007). The nest survival model also seemed obvious to use due to the variation in data collection from different sources (i.e. Buckley 2013, McLaughlin 2016, Wiley 2017) and inconsistencies among data collection and time intervals. Bobwhites were recorded as either “Successful” or “Unsuccessful” and denoted using binary notation as “0” for “successful” and “1” for “unsuccessful”. Bobwhites that experienced radio failure or were unaccounted for during the monitoring period were right censored and subsequently marked as “successful” (Dinsmore et al. 2002).

Model performance was assessed, and the best approximating model selected by using Akaike’s information criterion for small sample sizes (AICc), ∆AICc values, Akaike weights (AICω), and model likelihoods (Burnham and Anderson 2002). Goodness of fit test (i.e. χ2) was not performed because the models are saturated (i.e. one

Texas Tech University, Drew G. Arnold, May 2019 parameter per each encounter history) fits the data exactly (Dinsmore et al. 2002, Cooch and White 2014, Buckley et al. 2015). Models with the lowest AICc values were considered to be the best approximating model given the data (Anderson 2008). Akaike weights (AICω) were then used to narrow the model selection process, where the model containing the highest AICω value was considered the best approximating model given the data (Burnham and Anderson 2002). In addition to AICc and AICω models with a ΔAICc value ≤2 were considered to have substantial support in explaining survival, although ΔAICc values of up to 6 may significantly contribute meaningful support to model selection (Cooch and White 2014, McLaughlin et al. 2018). If support for any one model was deemed uncertain (i.e. no model with AICω ≥ .90), unconditional variances of estimated parameters were calculated, and model averaging employed to assess contributions of parameters (Cooch and White 2014). Nomenclature and syntax of the model representations follows Lebreton et al. (1992).

RESULTS

The survival analysis consisted of 124 individual bobwhites with 61 bobwhites, 19 bobwhites, and 44 bobwhites corresponding to the intervals of 2010 – 2011, 2011 – 2012, and 2013 – 2014 respectively. Bobwhites trapped during Buckley (2013) yielded 196 bobwhites. From those bobwhites 80 had the necessary immune function measures to assess effect of immune function on survival. McLaughlin (2016) captured 261 bobwhites during 2013 – 2014. A total of 44 bobwhites from this study interval had associated immune function measures present. All of the bobwhites included in the study had corresponding measures for different components of immune function (i.e. innate vs. adaptive immunity).

Statistical Analysis Program MARK was employed to assess whether immune function or a combination of biologically relevant variables might affect bobwhite survival. Model uncertainty was prevalent in the survival analysis with no model containing more than 50% of the model set weight. Additionally, only two models presented a ΔAICc value 19

Texas Tech University, Drew G. Arnold, May 2019 less than 2, yielding a combined model set weight of ~68% for the top two models. The top best approximating model (Year + Feed_Stat + IgY) held 50% of the model set weight. The next best model (Year) held ~18% of the model set weight and a ΔAICc of 2.02, just above the threshold for models deemed to receive substantial support (i.e. ΔAICc ≤ 2). Because of the high level of uncertainty in model selection and the potential for models with ΔAICc values up to a value of 6 to significantly contribute meaningful support to model selection (Cooch and White 2014), the next best approximating model (Feed_status+ IgY) was considered to have substantial support with a ΔAICc value of 2.8 and holding ~12% of the model set weight. The parameter “Year” appeared to be the most important and was found within the top two models, which when combined held ~68% of the model set weight. The parameters “Feed_Stat” and “IgY” were likewise found in two of the top three models considered to have high levels of support yielding a combined value of ~62% of the model set weight for models with either parameter.

Model selection uncertainty was prevalent across all models and necessitated model averaging across all beta parameters in models containing support to account for such uncertainty. Model averaging indicated survival rates of bobwhites over the duration of the three-year interval was .2197 or ~22% (SE = .0392, 95% CIs: .1524, .3060). The unconditional standard error derived from model averaging allows for the assessment of both model specific variation in conjunction with variation from model selection uncertainty. An unconditional standard error value of 0.24% indicates there was negligible variation in parameter estimation within the model sets.

Due to the existence of the parameters “Feed_Stat” and “IgY” in two of the top three models in the analysis I investigated the role of these parameters to determine whether or not the parameters “uninformative” as detailed by Anderson (2008). Uninformative variables are those that “ride the coat tails” of the best approximating model (Cooch and White 2014). The parameter “Year” which was found within the top two of the three models with substantial support. “Year”, however, was not considered to be a biologically relevant parameter and a post hoc analysis was performed excluding the variable “Year” to determine if “Year” was “washing out” the influence of the parameters

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“Feed_Stat” and “IgY”. The beta estimates for Feed_Stat in two of the top three models indicated that the parameter was indeed important to the survival analysis and not a superfluous variable in models (Year + Feed_Stat + IgY) and (Feed_Stat + IgY)(β = - .5467, SE =.2327 , 95% CIs = -1.003, -.0906 ) and (β = -.5031, SE =.2317 , 95% CIs = - .9573, -.0489 ) respectively. Based on beta estimates for “IgY” in the model (Year + Feed_Stat + IgY) (β = -.0.3243, SE = .4349, 95% CIs = -1.1767, 0.5282) the parameter “IgY” appears superfluous due to confidence intervals overlapping zero. However, the parameter “IgY” in the third best approximating model (Feed_Stat + IgY) were deemed important based on confidence intervals not overlapping zero (β = -.5031, SE =.2317, 95% CIs = -.9573, -.0489) and IgY (β = -.0.3243, SE = .3390, 95% CIs = -1.5783, - .2494). Likewise, in the post hoc analysis without the parameter “Year”, beta estimates yielded support for IgY in the top models.

A post-hoc analysis was performed to assess whether the parameters Feed_Stat and IgY were capable of maintaining their status within the top-models without the effect of the parameter “Year”. Analysis results indicate “Feed_stat” and “IgY” are significant variables effecting the survival of bobwhites over the three-year duration in the absence of the parameter “Year”.

DISCUSSION

The objective of this study was not to determine whether the magnitude of immune function measures resulted in differences in survival between groups but rather was an exploratory analysis to identify whether immune function measures were important variables in the survival of bobwhites overall. Additionally, it is important to recognize that directionality (negative/positive) within parameter estimation may not be truly representative of the parameter’s perceived effect on survival. This analysis was simply an exploratory analysis to determine the importance of immune function measures on bobwhite survival. I acknowledge that this study is imperfect and propose additional structured analyses are needed to tease out the finer details of immune function and survival in bobwhites. 21

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My survival analysis for the duration of a three years, yields support that certain aspects of immune function may indeed affect bobwhite survival. The parameter “IgY”, which provides a functional measure of the adaptive immune system appears in conjunction with parameter “Feed_Stat”, indicating that adaptive immune function and supplementation of food resources, were important determinant variables for bobwhite survival. Model averaged results indicated that feed status (βfeed_stat) and circulating IgY levels (βIgY) had a negative effect on bobwhite survival (βfeed_stat = -0.4076, SE = 2369;

95% CI: -.5142, -.2948) and (βIgY = -0.4037, SE = .4493; 95% CI: -.6170, -.2168). The parameter “Year” was also deemed an important determinant for bobwhite survival but was not considered biologically relevant. While “Year” was found to influence on bobwhite survival, I do not believe that this was a particular useful variable due to naturally occurring fluctuations in bobwhite populations observed on the Rolling Plains of Texas. Furthermore, without the effect of “Year” in a post-hoc analysis, “IgY” and “Feed_Stat” still maintained positions in the top models indicating substantial importance.

The effect of the immune system on survival is well documented, with many examples occurring within avian species (review by Moller and Saino 2004). Hanssen et al. (2004) observed that the upregulation of the adaptive immune system via injection of novel antigens into common eiders (Somateria mollissima) reduced subsequent return rates the following year, suggesting a direct effect of the immune system and its deployment reduced survival. Similarly, Wilcoxen et al. (2010) observed that a functional measure of the innate immune system (BKA) prior to and following an epidemic in a population of Florida scrub jays (Aphelocoma coerulescens) indicated increased survival in those exhibiting higher function and in better body condition. While my study is not directly comparable to these studies, these examples help illustrate the direct impact that immune function might have on survival.

Immune function is also known to be mediated via nutritional pathways which may indirectly effect survival (Lochmiller et al. 1993, Klasing 1998, Korver 2012). The role that nutrition exerts on survival is evident in a number of studies examining the

Texas Tech University, Drew G. Arnold, May 2019 effects of supplemental feeding on bobwhite survival (Buckley et al. 2015, McLaughlin et al. 2018). Within these studies bobwhites supplemented with an artificial food source were observed to exhibit higher survival than non-supplemented counter parts. The link between nutrition and the avian immune system is well established (Klasing 1998, Kidd 2004, Korver 2012) but the species-specific pathways and use of nutrients to support differential immune components in avian species is largely context dependent and poorly understood in wild populations. As such, it is entirely possible that the immune system is bolstered by the provision of adequate resources such that it provides enhanced protection against infectious agents. This would explain the status of the immune function indicator “IgY” and the variable related to food resources, Feed_Stat, in the top models. Interestingly, the analysis indicated that the parameter “Feed_Status” had a negative effect on survival which contrasts with the results of Buckley (2013) and McLaughlin (2016); Studies that used the same individuals for survival analysis as this study and observed positive effects of supplemental feed. The negative effect observed within this study likely arises from methodological differences in analysis. In this analysis bobwhites were grouped together in order to increase statistical power. In Buckley (2013) and McLaughlin (2016) bobwhites were separated into groups (e.g. Year, Supplemental Feed, Control, etc.) and survival rates compared between groups. Within this analysis bobwhites, both provided supplemental feed and control birds were included together in the analysis rather than placed in individual groups and separate anaylsis. Additionally, bobwhites used in this analysis were those with adequate serum volumes to perform immune function assays representing only a subset of those utilized in Buckley (2013) and McLaughlin (2016). Likewise, many bobwhites included in Buckley (2013) and McLaughlin (2016) did not have blood samples and subsequent serum samples collected. These factors may have constrained my analysis to utilizing bobwhites that may not be truly representative of bobwhites used in Buckley (2013) and McLaughlin (2016) and thus explain the observed negative effect of “Feed_status”. Nonetheless, the analysis does indicate that nutritional pathways are important to bobwhites survival. Because of the combined nature of the parameter “Feed_status” where birds were assigned as being provided supplemental food or not, I propose that future analyses run 23

Texas Tech University, Drew G. Arnold, May 2019 individual analyses comparing groups rather than grouping and assignment of parameter values to provide better insight into the effects of feed status and immune function measure.

Models including the functional measure of the innate immune system “BKA”, that is the bacteria killing capacity of blood serum, interestingly did not yield much support in survival models with models including “BKA” as a parameter. BKA was not associated with top models. Within lower models, the beta parameter estimates and associated confidence intervals overlapped zero indicating that “BKA” was an unimportant parameter.

Specific components of the immune system are documented to respond differentially depending on context (Norris and Evans 2000). For example, Bourgeon et al. (2010) noted that the innate immune system of mallards responded more rapidly, returning to baseline conditions to the reintroduction of resources (i.e. food) than the adaptive immune system, suggesting allocation of resources towards the innate immune system was more beneficial in regard to immediate life-history needs (i.e. potentially survival). Red crossbills (Loxia curvirostra) experiencing experimentally induced food shortages did not display reductions in immune parameters but rather maintained immune parameters consistent with controls, suggesting that crossbills did so at the expense of body mass (Schultz et al. 2017). The lack of contribution of the measure for innate immune function, BKA, may be the result of life-history traits that are most beneficial to bobwhites. Perhaps the maintenance and/or deployment of the adaptive immune system is more advantageous to bobwhites, considering the wide range of infectious agents they may encounter in the wild. Induced innate immune responses and acute phase responses induced via pathogen encounter are considered costly to initiate and maintain (Klasing 2004) while the adaptive immune system is considered less costly to initiate. It is conceivable that the energetic costs associated with mounting an innate immune response and acute phase response might impart negative consequences in future fitness and survival, favoring allocation of resources towards maintaining an immune system of lesser cost and specific targeting of pathogens (adaptive). Moreover, these life history

Texas Tech University, Drew G. Arnold, May 2019 traits could be the result of behavioral traits such as bobwhite’s covey forming behaviors in the fall and winter could expose bobwhites to infectious agents not encountered when in proximity to conspecifics. Seasonal variation of the immune system is observed in many species, including many avian species. Changes in cues (e.g. photoperiod) that signify seasonal changes have been implicated in upregulation of the immune system during certain seasons. During the winter, many species enhance physiological processes that should ensure survival (i.e. thermoregulation, immune function, other cellular processes) (Nelson 2004). Bobwhites have been observed to follow a similar trend when exposed to thermal extremes analogous to winter conditions, with elevated immune function in response to cold temperatures (Dabbert et al. 1997). Because a majority of the bobwhites in the study were collected during the months leading up to winter, the duration of winter, and transitioning to spring, it is plausible that the negative effect of the adaptive immune system (IgY) may be the result bobwhites exhibiting poor immune function succumbing to infectious agents or increased susceptibility to predation as a result of current infection. Increased circulation and/or abundance of immune cells and increases in lymphoid organ and tissue size also occurs during the winter (Nelson et al. 2002). Despite IgY’s ability to represent adaptive immunity, there are difficulties in interpretation of these changes in immune supporting processes and may indicate either current infection status or alternatively enhanced resistance to infectious agents (Norris and Evans 2000). Organisms with current infections likewise display similar patterns in re-distribution and abundance of immune cells coincidental with enlarged lymphoid organs and tissues. With regard to this study, it is suggestive that bobwhites displaying higher immune measures (i.e. high circulating IgY) may have upregulated the immune system to combat a current infection. Those with current infection may be more susceptible to succumbing to said infection or the immune response may incur physiological costs in the form allocation of resources away from other costly processes (i.e. thermoregulation, maintaining body condition, locomotion) and subsequently result in reduced survival. However, we were not able to determine whether the magnitude of immune function status (e.g. high or low) was responsible for the negative effect on survival. It can be presumed that subsequent studies utilizing groupings of bobwhites by 25

Texas Tech University, Drew G. Arnold, May 2019 immune status might yield insight into the effect of different immune status has on survival. However, it will likely be difficult to discern immune function status based on magnitude due to difficulty of interpretation of status, especially since standardized and baseline measures of immune function are not well documented in most species and the variability of conditions and variables that might influence the immune system. As such, I recommend that future investigations should focus efforts on describing immunological parameters, specifically IgY baseline levels with bobwhites with known infection status (i.e. no infection) under sterile conditions, and under similar nutritional status and environmental conditions as bobwhites in the wild to provide adequate comparisons of immunological condition.

Related to difficulties associated with functional measures of the immune system, evaluation of the adaptive immune system via Enzyme Linked Immunosorbent Assay (ELISA) may not truly reflect an individual’s immune status (Demas et al. 2011). Furthermore, drawing conclusions about the significance of the results are difficult to discern whether immune function is reflective of past or current infection. Because we did not directly assess whether data indicating higher circulation of Immunoglobulins positively or negatively affected survival, we can only infer that this measure of the adaptive immune system is important for bobwhite survival. It is conceivable that higher levels of circulating IgY may indicate a bobwhite’s current or past infection state (Martin et al. 2006) and those with higher IgY may have lower survival due to incurred costs (i.e. reduced fitness and susceptibility to infection). Alternatively, those with higher IgY levels may simply reflect the nutritional or energetic state of a bobwhite and the ability to allocate resources to maintaining a strong immune system.

The lack of evidence for the bacteria killing capacity (innate immune function) could also be the result of inherent faults within assay performance. For example, there is evidence that the freezing and thawing of samples such as those used in the bacteria killing assays (BKA) may significantly affect the results of the assay, providing a poor indication of the bacteria killing capacity (Liebl and Martin 2009). However, Hegemann et al. (2017) observed no effect of the freeze/thaw processes on the bacteria killing

Texas Tech University, Drew G. Arnold, May 2019 capacity of the BKA when plasma samples were exposed to up to ten freeze/thaw cycles. However, Hegemann et al. (2017) did note that for two commonly applied immune assays, length of storage (i.e. 1 year) decreased efficacy of assays but generally observed a proportional change across times, lending values comparable in magnitude to values from one year prior. Although neither assay was the BKA, we propose that there should not have been an effect from the freeze/thaw cycle since samples were likely only handled 2 two to three times in the course of performing assays. Hegemann et al. (2017), comment that because individual samples used in their study were collected at a single time point, the proportional declines in measurement over time are directly comparable but warns against comparing samples collected at different time points and with differing factors (e.g. different populations) may prove problematic in interpretation and should likely be avoided. Therefore, the lack of relevance of “BKA” in survival might have resulted from inconsistencies of collection and storage. That is to say that because bobwhites serum samples were collected and stored over the course of 3 years, results of the BKA may not truly represent the immune capacity of the innate immune system and an effect on survival missed. Although the evidence on age of samples is relevant to the BKA, it appears that the IgY concentration as determined via ELISA is not affected by similar effects. Hegemann et al. (2017) did not observe the effects of aging on an agglutination assays, which utilizes antibodies mechanisms of the innate immune system, albeit not directly related to circulating IgY concentrations, provides evidence of the well-known durability of circulating proteins and antibodies (Argentieri et al. 2013). Consequently, the relevance of circulating IgY on bobwhite survival might be apparent due to the lack of confounding properties associated with the assay itself. We cannot discount the usefulness of BKA as an assessment of the innate immune system owing to its ease of interpretability based on higher in vitro killing of bacteria equates to greater capacity of a host to limit infection by a non-species-specific infectious agent (e.g. E. coli) (Matson et al. 2006). Likewise, we cannot discount BKA and its relevance to survival based on this study due to the possibility of confounding factors that may have limited interpretation (i.e. varying ages of samples). I, therefore, recommend that future investigations attempt to process serum samples in immune assays in an expedient 27

Texas Tech University, Drew G. Arnold, May 2019 manner, performing assays at a standardized time point post collection or integrate age variables into their analysis to determine the effects of sample storage on results.

Texas Tech University, Drew G. Arnold, May 2019

Table 1.1 Akaike’s Information Criterion, adjusted for small same size (n =124), ranking logistic regression models (Nest survival data type) relating to the annual survival of Northern bobwhites (Colinus virginianus) on the 6666 Ranch on the Rolling Plains of Texas, USA.

Model N AICc Δ AICc ωi L K Deviance

S(Year+Feed_Stat+IgY) 124 762.78 0.00 0.50 1.00 4.00 754.78

S(Year) 124 764.81 2.02 0.18 0.36 2.00 760.80

S(Feed_Stat+IgY) 124 765.63 2.85 0.12 0.24 3.00 759.63

S(Feed_Stat+IgY+BKA) 124 766.91 4.13 0.06 0.13 4.00 758.91

S(Year+Age+Mass+Feed_Stat+IgY+BKA) 124 767.70 4.91 0.04 0.09 7.00 753.69

S(IgY) 124 768.31 5.52 0.03 0.06 2.00 764.31

S(Mass+Feed_Stat+IgY+BKA) 124 768.85 6.07 0.02 0.05 5.00 758.85

S(Feed_Stat) 124 770.34 7.56 0.01 0.02 2.00 766.34

S(Age+Mass+Feed_Stat+IgY+BKA) 124 770.37 7.59 0.01 0.02 6.00 758.37

S(Feed_Stat+BKA) 124 771.68 8.90 0.01 0.01 3.00 765.68

Texas Tech University, Drew G. Arnold, May 2019

Table 1.1 Continued

Model N AICc Δ AICc ωi L K Deviance

S(.) 124 772.35 9.57 0.00 0.01 1.00 770.35

S(BKA) 124 774.05 11.27 0.00 0.00 2.00 770.05

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CHAPTER II

NUTRITIONAL MODULATION OF THE BOBWHITE’S IMMUNE SYSTEM Introduction The northern bobwhite quail (Colinus virginianus) – hereafter bobwhite(s) – is an economically, culturally, and ecologically important gamebird that has experienced a range-wide decline across much of its historic range (Brennan 1991, Williams et al. 2004, Hernandez et al. 2013, Sauer et al. 2017). Declining populations have been largely attributed to habitat loss and fragmentation at a large scale (Brennan 1991, Hernandez et al. 2013, Hernandez and Guthery 2012). Even in areas considered strongholds of quality and productive habitat conditions such as the Rolling Plains of Texas, wild bobwhite populations have likewise experienced a decline (Rollins 2007). Bobwhite densities are known to fluctuate annually based on weather patterns especially in arid or semi-arid portions of their range where annual precipitation heavily influences bobwhite population demographics (Rollins 1999, Lusk et al. 2002, Herndandez et al. 2005). The underlying factors controlling these fluctuations have been largely debated by a number of researchers. However, Jackson (1962) hypothesized that within the Rolling Plains the observed “boom and bust” nature of bobwhite populations was the result of a combination of drought, grazing practices, vegetative succession, and periods of heavy precipitation. Although there have been many studies to determine the underlying processes governing the boom and bust phenomenon, the exact mechanisms and interactions of biotic and abiotic factors still elude researchers (Hernandez, Guthery, and Kuvlesky 2002, Hernandez and Peterson 2007). Regarding bobwhite population irruptions, during 2010 the Rolling Plains received an abundance of precipitation during the growing season, resulting in the prediction of a boom year for bobwhites (Dunham et al. 2016). However, bobwhite abundances did not respond positively as predicted, despite adequate precipitation and favorable habitat conditions, leading many concerned parties to launch investigation into the role diseases, parasites, contaminants, and other pathogens (e.g. viral infections) play in the observed bobwhite declines (Dunham et al.

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2016). The unexpected “bust” year and suspected involvement of infectious agents culminated in a concerted effort among multiple regional research entities to discern causative agents of the decline and was collectively known as Operation Idiopathic Decline (Brym et al. 2018). Research efforts from this collaborative initiative have observed a high prevalence of parasitic infections (helminthic worms) and have likewise noted evidence of negative physiological effects of infections (Bruno et al. 2015, Dunham et al. 2014, Brym et al. 2018). Although these recent investigations have observed high rates of infection in bobwhites on the Rolling Plains, providing valuable information; a wide knowledge base regarding disease, parasites, and other infectious agents (e.g. viral and bacterial infections) already exists in bobwhites throughout their range with numerous studies identifying potential pathogens their distributions, prevalence, and occurrence in bobwhite populations. For a detailed review of diseases and parasites found within bobwhite populations see Peterson (2007 89:115). Although pathogen occurrence in bobwhites is well documented, many researchers have noted the absence of research tying pathogens and disease processes to bobwhite population regulation and have stressed the need for additional research into said processes (Peterson 2007, Hernandez et al. 2013, Hernandez and Guthery 2012).

Nutritional Modulation of the Immune System Disease processes have been suggested to contribute to population regulation in gallinaceous species, with the most notable example present in red grouse (Lagopus lagopus) populations. Cyclical fluctuations in red grouse populations in the moors of Scotland were discovered to be tied to parasitic helminthic infections (Hudson et al. 1997, Hudson et al. 1998). Such examples of infectious agents (e.g. bacterial, viral, parasitic infections) regulating populations are not well documented in bobwhite quail but have been speculated as a potential contributing factor regulating bobwhite populations (Hernandez et al. 2013, Peterson 2007 Dunham et al. 2014, Henry et al. 2017). Due to the propensity of bobwhite populations to unpredictably fluctuate across portions of their range, especially within the semi-arid, southwestern portions of their range (Brennan 1991, Hernandez et al. 2005), a number of other hypotheses have been

Texas Tech University, Drew G. Arnold, May 2019 posited regarding factors regulating bobwhite populations. Specifically, in relation to the observed boom and bust phenomenon, explanatory hypotheses can be described generally as non-specific stress, heat stress, and nutritional stress (Hernandez and Peterson 2007 p.58). Among those hypotheses, food limitation and nutrition have received considerable attention over the years (Stoddard 1931, Guthery 1997, Hernandez et al. 2005). Emphasis of research regarding nutrition has widely focused on the effects of supplemental feed on bobwhite survival. Although a number of studies have noted positive effects on the survival and productivity of bobwhites (Tall Timbers 2009, Buckley et al. 2015, Wiley 2017, McLaughlin et al. 2018) others have found a lack of or neutral evidence to support supplemental feeding as a beneficial management tool for bobwhites (Guthery 1997, Guthery 2004, Henson et al. 2012). Guthery (1997) hypothesized that food is generally not a limiting factor for bobwhites under most circumstances. However, supplemental feeding of bobwhites has been observed to improve survival and productivity of bobwhites through nutritional pathways, especially during times of resource shortages such as mid-winter when food resources may be depleted or during periods of heightened energetic requirements (i.e. thermoregulation, reproduction, locomotion, etc.) from environmental extremes (i.e. excessive snow coverage, drought, extreme cold (Giuliano et al. 1996, Buckley 2013, McLaughlin 2016). Limited food resources regardless of mechanisms driving limitations may have negative effects on bobwhites, resulting in the utilization of body reserves and depression of body mass (Robel 1969), indicating stressful conditions. Improvements in body condition have been noted in studies supplying supplemental food sources which assist bobwhites with accumulation of additional body fat reserves during the winter (Robel 1969; Robel et al. 1974; Larsen et al. 1994; Doerr and Silvy 2006). Animals in the wild are likely to experience seasonal fluctuations of resources, energy requirements, and stressors. Winter is an exceptionally demanding time for animals and energy expenditures to maintain physiological processes (e.g. thermoregulation, locomotion, immune function, etc.) may be costly. It is expected from a life history strategy that an animal must optimally invest resources among physiologically important processes that contribute to immediate survival (Norris and Evans 2000). Constrained by limitations of resources (i.e. food), 42

Texas Tech University, Drew G. Arnold, May 2019 high energetic demands, and nutritional deficits bobwhites may be unable to meet all physiological needs and consequently allocation of limited resources towards physiological processes deemed advantageous for survival seems likely.

The effect of food limitation and subsequent lack of nutrition on bobwhites is exemplified in Giuliano et al. (1996) where bobwhite hens fed diets deficient in energy and/or protein content exhibited reduced reproductive parameters (i.e. egg laying and ovary mass) and increased stress hormones (i.e. corticosterone). Because the net productivity of bobwhites is conditional on the hen’s reproductive success (i.e. nest success), and subsequent chick survival to recruitment into the fall population (Roseberry and Klimstra 1984, Cantu and Everett 1982, Sandercock et al. 2008), nutrition then has the capability to decrease reproduction and negatively affect bobwhite populations. In addition to productivity and recruitment of chicks into the fall population, the winter survival of adult bobwhites has also been identified as a contributing factor to positive population growth in bobwhites (Sandercock et al. 2008). Naturally occurring weather phenomena, specifically drought/drought-like conditions and excessive snow coverage that have the capability to limit availability to adequate food items have been suggested as important to the nutritional state of bobwhites (Giuliano et al.1996, Hernandez et al. 2005). These extreme environmental conditions may similarly exert additional pressure on physiologically important functions (e.g. reproduction, thermoregulation, locomotion, immune function). Among physiologically important functions, the immune system has been observed to directly affect survival of vertebrate species (Lochmiller and Deerenberg 2000, Moller and Saino 2004) but has not been well documented in bobwhites.

Nutritional Deficits Bobwhite populations are strongly influenced by climate patterns at a regional/ecoregional scale, especially in the semi-arid environments where annual precipitation heavily influences bobwhite population demographics (Lusk et al. 2002, Herndandez et al. 2005.) Food availability and limitations within the order Galliformes vary spatially (e.g. landscape, local level), temporally (e.g. within seasons or years), and

Texas Tech University, Drew G. Arnold, May 2019 along environmental gradients (e.g. elevations, latitudinally (Balasubramaniam and Rotenberry 2016) (McLaughlin et al. 2018). Food availability for bobwhites can be attributed to a number of natural and anthropogenic agents such as drought, extended snowfall cover, precipitation, plant succession and seral stage, agricultural practices and grazing practices of livestock among others (Jackson 1962, Wood et al. 1986, Rollins 1999, McLaughlin 2016). Within the Rolling Plains of Texas, Jackson (1962) hypothesized that drought, periods of heavy rain, livestock grazing practices, and plant succession principally influenced the classic boom and bust population pattern of bobwhites within the ecoregion. These factors working independently or in concert with each other can affect food resource availability, quantity, and accessibility (Errington 1939, Miller 2011, McLaughlin et al. 2018). Reduction of food resources, resultant from environmental or anthropogenic circumstances and/or the subsequent intraspecific or interspecific competition for resources that may occur could adversely affect bobwhites to the effect of altering survival. This deficit is especially relevant during times of high metabolic demands such as during extended periods of heat or cold, reproduction, infection, etc. Bobwhites display heightened thermoregulatory requirements during the winter (Swanson and Weinacht 1997). Subsequently, thermal stress from extended periods of cold temperatures and food resource limitations that commonly occur during the winter (Nelson et al. 2002) may have the capacity to affect the bobwhites immune system. For example, Dabbert et al. (1997) observed an upregulation of the immune system in bobwhites in response to stressful cold temperatures, a commonly observed physiological response in avian species (Nelson et al. 2002) Although Dabbert et al. (1997) failed to negatively affect cell-mediated and humoral immune function of the bobwhite immune system through prolonged exposure to heat or cold, the observed improvement of immune function in cold-stressed bobwhites were suggested to indicate higher susceptibility to viral infection. Associated with stressful cold temperatures, Dabbert et al. (1997) also observed mass loss in response to increased basal metabolic rates and proposed fat reserves were mobilized to meet metabolic demands. Body condition has been noted to influence immune function in other avian species. For example, Florida scrub jays (Aphelocoma coerulescens) with higher body condition 44

Texas Tech University, Drew G. Arnold, May 2019 scores exhibited enhanced innate immunity and were more likely to survive a naturally occurring disease epidemic (Wilcoxen et al. 2010). Related to body mass, the acute phase response mounted following infection and the subsequent mobilization of immune cells is the heightened need for amino acids to support protein synthesis, the consequences of which are significant reductions in lean body (Lochmiller and Deerenberg 2000). Body mass losses exceeding > 20% in bobwhites are typically fatal (Robel et al. 1979). It follows that reduction in body mass, whether derived from loss of lean protein or fat stores following infection or stressful environmental conditions, may affect survival. It also highlights the possibility that maintaining a competent immune system to prevent infection may be important to ensure additional physiological consequences (i.e. body mass loss), are not incurred.

A number of recent studies have documented trade-offs between the avian immune system and associated branches when experimentally challenged with resource limitations (food) or nutritionally inadequate food stuffs. For example, Bourgeon et al. (2010) fasted female mallards to the point of protein catabolism of muscular tissue (i.e. starvation), reducing the function of both innate and acquired immune system branches. Notably, Bourgeon et al. (2010) observed that the innate system recovered more rapidly than the acquired system to pre-experimental levels when mallards were reintroduced to food resources. This differential response by alternate immune system arms suggests that there are possible physiological priorities associated with maintaining a competent immune system. It might be that allocation of resources (i.e. food) to maintaining the innate immune system may be more beneficial from a life-history standpoint. That is, the benefits of investment in the innate immune system might be more beneficial than investment in the acquired branch of the immune system. Similarly, Lochmiller et al. (1993) compromised the immune system of bobwhite chicks through nutritional limitation of adequate protein levels resulting in reduced immune function and growth. These induced consequences further support the argument that allocation of vital resources towards a costly physiological process such as growth and maintaining a competent immune system are substantial.

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Recently, supplemental feeding has increased in popularity following research from Texas and the Southeastern U.S. noting its positive effect on survival and subsequently increased productivity (Sisson et al. 2000, Whitelaw et al. 2009, Buckley et al. 2015, McLaughlin et al. 2018). It has been hypothesized that the positive effect on survival is due to improved physical condition of bobwhites receiving supplemental feed, especially during times when food might be limited (e.g. droughts, extended snow coverage, etc.) (Porter 1980, Whitelaw et al. 2009, Buckley et al. 2015, Janke et al. 2015). Increases in body mass have been observed in bobwhites exposed to supplemental food sources (Robel 1969, Robel et al. 1974, Whitelaw et al. 2009). Increases in bobwhite body condition has been hypothesized to buffer bobwhites against environmental stressors (Doerr et al. 1988, Buckley et al. 2015). Body condition may be an important contributing factor in the maintenance of a competent immune system (Alonso-Alverez and Tella 2001, Bourgeon et al. 2010) especially during times of resource limitations. Resource limitation, specifically food, has not been hypothesized as a limiting factor in bobwhite populations (Guthery 1997). While not considered a limiting factor for bobwhites, food resources can certainly be critical for buffering bobwhites against stressors such as extreme cold or drought as evidenced in Buckley et al. (2015) and Janke et al. (2015). It follows that bobwhites in improved physical condition might maintain a stronger immune system due to adequate resources to maintain and deploy an immune response if facing an infectious agent.

The nutritional and energetic qualities of supplemental feeding regimens and commonly consumed naturally occurring food items have been explored in a number of studies (Fraps 1947, Robel et al. 1979, Wood et al. 1986, Peoples et al. 1994, Guthery 1999). A common theme among such studies is the emphasis on composition of food items in a diet or the energetics derived from said food items. A critical factor not wholly addressed in such studies is how these foods influence physiological systems outside of those that address thermoregulation and reproduction (Leif and Smith 1993, Giuliano et al. 1996, Guthery 1999, Swanson and Weinacht 1997). While a majority of studies emphasizing the positive effects of supplemental feeding programs and nutrition have

Texas Tech University, Drew G. Arnold, May 2019 conferred benefits primarily to improved nutrition and body condition, very few have investigated the effects of nutrition on other important physiological functions with the potential to affect bobwhite survival. The immune system is one such physiological system that is vital for defense against infectious agents whose maintenance and deployment is hypothesized to be energetically costly (Lochmiller and Deerenberg 2000) and has been demonstrated to impact survival in a number of avian species (see review by Moller and Saino 2004). Inherent to this hypothesis is that under limited resources differential allocation of resources to physiologically costly and important processes may result in consequences affecting individual fitness (Sheldon and Verhulst 1996, Lochmiller and Deerenberg 2000, Norris and Evans 2000). The lack of research on the implications of nutritional quality on the immune system of bobwhites along with the potential for the immune system to effect fitness (i.e. survival) provides an opportunity to investigate the link between nutritional quality and immune function. Moreover, the extent of knowledge about bobwhite immune response when exposed to improved nutritional supplementation is largely absent, with few studies examining the role of nutritional quality on bobwhite immune function such as the studies of Lochmiller et al (1993), Dabbert et al. (1996), and Dabbert et al. (1997). What is largely missing from current research is information on immune function of bobwhites in response to varying planes of nutritional quality and immune system enhancing nutrients that are commonly studied in the poultry science arena (e.g. antioxidants).

Reduction in food consumption is a negative consequence of infection. Suppressed food intake comes at a time when the body requires an increased intake of nutrients vital to maintaining an immune system (Lochmiller and Deerenberg 2000). The suppression of resource intake typically occurs as a consequence of the acute phase or inflammatory response which is characterized by fever, reduced appetite, hypermetabolism, protein malnutrition, and lethargy among other physiological and behavioral traits (Klasing 1998, Lochmiller and Deerenberg 2000) The acute phase response during infection, is considered to be a primary mechanism inflicting considerable metabolic costs on an organism during infection. Some argument exists

Texas Tech University, Drew G. Arnold, May 2019 among ecologists and immunologists of the costs associated with maintaining a competent immune system (Klasing 1998). However, the acute phase response has been observed to be energetically costly and as a response component associated with the immune system exemplifies energetic and metabolic requirements that may alter survival of an organism (Lochmiller and Deerenberg 2000). The immune system has high metabolic requirements and may become suppressed if energy requirements in the diet are not met, increasing risk of infection from opportunistic pathogens (Lochmiller and Dabbert 1993). It is of importance to note the metabolic costs of maintaining a competent immune system are not adequately able to be measured and according to Lochmiller and Deerenberg (2000) may be impossible to truly address.

Antioxidant Role in the Immune System The metabolic processes and functioning of the immune system of vertebrates can result in the production of compounds with high oxidizing potential that can damage biological molecules (Halliwell and Gutteridge 1999). One of the important consequences of physical activity is the acceleration of oxidative metabolism (Monaghan et al. 2009). The aforementioned compounds with high oxidizing potential are by- products of oxygen consumption (Finkel and Holbrook 2000). The build-up of oxidative damage may eventually lead to accelerated aging and development of degenerative diseases (Finkel and Holbrook 2000). The antioxidant machinery includes a series of endogenous and exogenous compounds that neutralizes oxidants and potentially enables individuals to avoid the harmful effects of oxidative damage (Halliwell and Gutteridge 1999). Because oxidative stress/damage is a ubiquitous phenomenon, these antioxidant) defenses are likely crucial for development and performance in vertebrates (Moller et al. 2000). If levels of free radicals in immune cells surpass normal levels, then these free radicals negatively affect the immune system (Szabo et al. 2010). Antioxidants boost the immune system by playing other important roles such as in cellular metabolism, signal transduction, gene activation, and transcription (Brambilla et al. 2008). However, there is evidence that the allocation of resources to antioxidant systems may incur costs, resulting in trade-offs with other fitness related investments such as reproduction, immune

Texas Tech University, Drew G. Arnold, May 2019 function, sexual signaling, and growth (Alonzo-Alverez et al. 2009, Bize et al. 2008, Monaghan et al. 2009, Nussey et al. 2009). Fruits, seeds, and invertebrates all provide sources of antioxidants in variable amounts and the levels of circulating antioxidants in wild birds are generally positively related with the levels of antioxidants in their diet (Cohen et al. 2009). Consequently, poor food resource availability can influence oxidative status by weakening dietary antioxidant defenses (Catoni et al. 2008). Supplementation of dietary antioxidants has been shown to increase total antioxidant capacity (Cohen et al. 2007). Szabo et al. (2010) found Japanese quail fed diets supplemented with different carotenoids (β-carotene, lutein, and lycopene), known antioxidants, had relatively similar oxidative prevention capacities, but certain carotenoids had a more immuno-modulatory effect than others. The supplementation of the carotenoids increased the effectiveness of antibody response in the adaptive immune system when challenged with an antigen and was efficient at oxidation prevention as compared to the control group that were fed a normal readily available quail ration. The enhanced host immunity/antioxidant defenses may translate into better health for the host (Szabo et al. 2010). This may have management implications when applied to a wild population by reducing oxidation when birds have to cope with the physiological trade- offs such as reproductive, developmental, and immunological costs associated with seasonal environmental constraints (Lochmiller and Deerenberg 2000). Availability of dietary antioxidants may be compensated for by the up-regulation of endogenously derived antioxidant defenses (Monaghan et al. 2009). It would seem if dietary needs of bobwhites were met and ample amounts of antioxidants were available that bobwhites could potentially limit the effects of reactive oxidative species and therefore improve the health and survival.

Trade-offs among biologically important physiological systems and the outcome of such trade-offs in regard to individual fitness is commonly investigated and is generally of ecological importance for understanding life-histories of animals (Nelson 2004). Trade-offs are frequently understood as the re-allocation of limited resources from one physiological system to another, leading to a lack of resources and negative

Texas Tech University, Drew G. Arnold, May 2019 consequences for the competing system (e.g. down-regulation of growth in response to immune system up-regulation (Lochmiller et al. 1993, Monaghan et al. 2009). However, less often considered is the trade-off that results as a consequence of the performance of one activity of a physiological system generating a negative consequence on another physiological system. It is this second type of trade-off and underlying processes that has brought into question the role of oxidative stress in such trade-offs (Monaghan et al. 2009). Organisms have evolved the capability to harness oxygen molecules through aerobic processes to generate energy. Generation of energy through aerobic pathways necessitates the need to prevent or offset the oxidizing reactive substances produced. Metabolic activity of an organism is often the production point for Reactive Oxygen Species (ROS) which includes free-radicals and non-free radical pro-oxidants (Constantini and Verhulst 2009). Oxidative damage from metabolic activities is an inescapable consequence of energy production (ATP genesis). The production of ROS must be countered by the production of endogenous anti-oxidants or incorporation of antioxidants from exogenous sources (i.e. diet) otherwise an imbalance of ROS may result in oxidative damage (Halliwell and Gutteridge 2007). The mechanisms between metabolic processes producing oxidative damage and the consequences on organisms is still poorly understood. However, the processes producing ROS are well understood. Free radical ROS are highly reactive and unstable molecules that trigger chain reactions among other damaging molecules leading to a cascade of damaging compounds and oxidative damage (Monaghan et al. 2009). However, not all ROS are damaging. In fact, around 10% of ROS in animal cells are produced by metabolic processes, primarily by certain enzymes for utilization in important metabolic processes such as cell signaling, cell transformation, regulation of smooth muscle relaxation, regulation of blood flow, and relevant to this research, immune defense (Dröge 2002). Pertinent to immune defense, ROS are utilized primarily during macrophage encapsulation of pathogens, where macrophages produce ROS upon encapsulation, leading to protease production and subsequent demise of the pathogen (Dröge 2002).

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The inability of the antioxidants to neutralize ROS produced by metabolic activities such that present ROS can continue to initiate additional reactions causing additional oxidative damage is known as oxidative stress (Monaghan et al. 2009). The implications of oxidative stress on an animal is relevant only if there are costs incurred upon the animal such that it effects fitness. While an imbalance of ROS can lead to oxidative stress, it does not necessarily occur unless the upregulation of antioxidant defenses is inadequate to combat the imbalance. No doubt, the omnipresence of ROS and oxidative damage, has led animals to development of effective means to combat ROS. Collectively known as the antioxidant system (Surai 2002), several defenses have developed within animals to combat oxidative damage. See Monaghan et al. (2009) for a complete review of antioxidant defenses and mechanisms. One pathway of antioxidant defense in particular utilizes the chain breaking antioxidant compounds made up of endogenously produced antioxidants, primarily enzyme derived, and exogenously derived compounds obtained via diet (Horak et al. 2007, Monaghan et al. 2009). Those antioxidants obtained through dietary sources are of particular interest to many researchers due to their ability to be supplemented via increasing dietary content of exogenous antioxidants to prevent the effects of ROS produced via metabolic pathways (Viña et al. 2000). Inclusion of a number of important antioxidants in experimental diets, especially those found naturally in dietary components or those derived from plants such as Vitamin E (tocopherols and tocotrienols), carotenoids (e.g. beta carotene and canthaxanthin), Vitamin C, etc. have been noted to contribute to reduced oxidative stress and damage. For example, Vitamin E, Vitamin C, biotin, folic acid and minerals like zinc and selenium, all which can be supplemented in diet have been shown to be active in buffering against oxidative damage (Cheng et al. 1990, Sahin et al. 2002, Citil et al. 2005)

Activation or upregulation of the immune system can result in the production of ROS via cellular and enzymatic activities of important immune components (i.e. heterophils, macrophages, T and B lymphocytes) during an inflammatory response (Constantini 2008). The primary production of ROS, namely free radicals, is the by-

Texas Tech University, Drew G. Arnold, May 2019 product of respiratory burst used to lyse infectious agents (McGraw and Ardia 2004). Consequently, oxidative damage and oxidative stress may be a result of immune response to infection. Because the effects of oxidative damage and stress may translate into physiological costs such as reduced immune function which accordingly may be buffered by provision of antioxidants (Horak et al. 2007), and the lack of information of antioxidants activity in bobwhites it seems logical to investigate if the provision of dietary antioxidants confer immunostimulatory effects on bobwhites which might translate into greater fitness and subsequently survival.

Justification for Antioxidant Components in Feed Study Antioxidants play an important role in the maintenance and deployment of the immune system in defense against pathogens. Several known exogenously derived antioxidant compounds such as Vitamin E, carotenoids (beta carotene, canthaxanthin, alpha tocopherol, etc.), Vitamin D and Vitamin C among others have been noted for their roles in immunomodulatory processes (Leshchinsky and Klasing 2001, Kidd 2004, Surai 2002). Within this study, feed development with the intention to positively affect immune function was the desired goal. Antioxidants with the capacity to reduce circulating reactive oxygen species resultant from metabolic processes or known to contribute to the physiological mechanisms were included in a diet denoted as High Vitamin + Antioxidants. Antioxidants (i.e. trace minerals and/or vitamins) that were present in this diet but not others or exceeded values in another nutritionally elevated diet

(HVTM) included Beta-carotene, Canthaxanthin, Vitamin D3 (Hy-D), Vitamin C, Docosahexaenoic Acid (DHA), Chromium, Carnitine, Selenium, Biotin, Vitamin E. The background for inclusion of these components follows:

Vitamin E is of particular importance for its role in immune function, serving as a critical antioxidant in membranes and active in disrupting lipid peroxidation (Monaghan 2009). Several examples exist displaying the beneficial effects of vitamin E on the immune system. For example, improved humoral and cell-mediated immune responses were observed in Vitamin E supplemented Coturnix quail (Coturnix coturnix) (Hooda et al. 2005). Similarly, a study by Leshchinsky and Klasing (2001) yielded positive effects

Texas Tech University, Drew G. Arnold, May 2019 on cell-mediated and humoral immune function in domestic chickens (Gallus gallus) supplemented with Vitamin E. However, it is important to note that Leshchinsky and Klasing (2001) observed a dose dependence effect, suggesting that Vitamin E beyond a certain concentration may not be effective as an immunostimulatory compound. Besides ineffectiveness of Vitamin E at levels above or below optimum doses, Vitamin E has also been shown to vary in effectiveness in relation to presence of other antioxidant compounds. One such relationship is that of Vitamin E, carotenoids, and Vitamin C (ascorbic acid). Carotenoids and Vitamin C have both been shown to be important in the recycling of Vitamin E which is often oxidized by ROS’s into a pro-oxidant, the recycling of which functionally alters Vitamin E back into an antioxidant form (Surai et al. 2001). It is therefore reasonable to expect that low levels of carotenoids or Vitamin C may limit effectiveness of Vitamin E. In addition to interactions with other known antioxidants, carotenoids have been thoroughly investigated for their antioxidant properties and relationship to immune function. Carotenoids are biologically active terpenic pigments, synthesized by plants, algae and some fungi and bacteria (Fenoglio 2002; Blount et al. 2000) and with respect to diet additives in this study include both beta-carotene and canthaxanthin. Carotenoids act as scavengers of reactive oxygen species, conferring protection from oxidative damage (Surai et al. 2001) and thus promote immune system function (Szabo et al. 2010). Manifestations of improved immune function were observed in Gray partridge (Perdix perdix) chicks and adult females fed diets with high concentrations of carotenoids (Fenoglio et al 2002, Cucco et al. 2006, Cucco et al. 2007).

Selenium is a trace mineral that is exogenously derived and must be consumed in a dietary form to receive beneficial effects. Selenium has been implicated in antioxidant status through its supportive role with Vitamin E to instill antioxidant protection through its supportive role with the important antioxidant enzyme glutathione peroxidase (Kidd 2004). Selenium has also been noted to impact disease resistance (Colnago et al. 1983, Dhur et al. 1990) and as such additional dietary selenium concentrations above NRC recommendations (NRC 1994) may improve immune function parameters (Kidd 2004)

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Carnitine (L-Carnitine) is a water-soluble quaternary amine, synthesized endogenously from the essential amino acids lysine and methionine (Mast et al. 2000). Physiologically, it is important in the oxidation of long chain fatty acids but has also been observed in an immune-stimulative role by increasing proliferative responses of lymphocytes, an important immune cell, in humans. Additionally, L-Carnitine may also support antibody production, which may potentially impart a lasting importance in enhancement of adaptive immune function (Mast et al. 2000).

Docosahexaenoic Acid, also known as DHA, is a long chain polyunsaturated fatty acid (PUFA) commonly referred to a n-3 fatty acids (Callicrate et al. 2010). Immune cells contain a high proportion of PUFA’s within their cell membrane and are reliant on the interaction, cooperation, and regulation of cell membrane associated events through protein and lipid mediators (Wu and Meydani 1998). These membrane interactions between protein and lipid mediators are essential in mounting a successful immune response and consequently appropriate immune responses may be vulnerable to variations in PUFA (i.e. DHA) content (Wu and Meydani 1998). Additionally, immune cells largely carry out functions through membrane associated activities such as secretion of antibodies, antigen reception, and cell lysis. In addition to its contribution to cell membrane associated activities critical for immune response, DHA has also been noted as an important component in mitigating the effects of an inflammatory response during infection and the resulting acute phase response (Korver and Klasing 1997). For example, Korver and Klasing (1997) observed positive immunostimulatory effects, reduction of the inflammatory response, and improved growth in juvenile chickens (Gallus gallus domesticus) fed elevated levels of DHA when exposed to a pathogenic challenge.

Vitamin D3 is a vitamin that may be derived from dietary sources or produced via conversion of 7-dehydrocholesterol by ultraviolet radiation to its active form known as calcitriol (Norman et al. 1982). Deficiencies of Vitamin D3 has been shown to induce growth depressing effects on the skeletal system and these is limited evidence tying Vitamin D3 to immune function. For example, diets deficient in Vitamin D3 resulted in

Texas Tech University, Drew G. Arnold, May 2019 reductions in growth rate, cell-mediated immune response, masses of immune supporting lymphoid organs (i.e. thymus, bursa of Fabricius, and spleen), and diminished counts of phagocytic macrophages in broiler chicks (Aslam et al 1998). However, within Aslam et al. (1998), it should be noted that similar reductions in humoral (adaptive) immune function as measured by antibody production was not observed, suggesting that Vitamin D3 is not relevant in adaptive immune function.

To reiterate, although numerous studies investigating the presence, distribution, and prevalence of diseases, parasites, and other harmful pathogens (e.g. viral infection) among wild bobwhites, very few researchers have examined the primary defense, the immune system, against the very pathogens that may affect bobwhites at a local and landscape scale. Likewise, those studies that have investigated immune function of bobwhites have done primarily by challenging bobwhites with antigens and other pathogens that bobwhites are unlikely to encounter in the wild. Matson et al. (2006) observed that life-history traits and previous exposure to infectious agents may be important determinants of immune responses. This suggests that exposure to novel challenges may be affected by inherent life history traits and may not yield a true indication immune responsiveness and contribute to difficulties in interpretation of responses. Moreover, the extent of knowledge about bobwhite immune response when exposed to improved nutritional supplementation is largely absent, although a few key studies have noted the importance of adequate nutritional quality on bobwhite immune function (Lochmiller et al 1993, Dabbert et al. 1996). What is largely missing from current research is information on bobwhite immune function in response to varying planes of nutritional quality and the effects of potentially immune enhancing nutrients. These effects are commonly studied and observed in the field of poultry science (e.g. vitamin, trace mineral, etc. as feed additives) in other gallinaceous species such as the Japanese quail (Coturnix japonica), domestic chicken (Gallus gallus) and domestic turkey (Meleagris gallopavo) (Friedman et al. 1998, Leshinsky and Klasing 2001, Sahin et al. 2006) Extensive research efforts have also been conducted on management activities to increase bobwhite population densities including but not limited to: habitat

Texas Tech University, Drew G. Arnold, May 2019 management, supplemental feeding, predator management, etc. Recently, supplemental feeding has increased in popularity following research from Texas and the Southeastern U.S. noting its positive effect on survival and subsequently increased productivity. It has been hypothesized that the positive effect on survival is due to improved physical condition of bobwhites receiving supplemental feed, especially during times when food might be limited (e.g. droughts, extended snow coverage, etc.) (Buckley et al. 2015, McLaughlin et al. 2018). It follows that bobwhites in improved physical condition might maintain a stronger immune system due to adequate resources to maintain and deploy an immune response if facing infection by a pathogen. A study supplementing Chinese ring- necked pheasants (Phasianus colchicus), a gallinaceous species, with varying amounts of protein and carotenoids noted that pheasants with a higher scoring body condition supported increased cell-mediated immunity (i.e. PHA injection) compared to those with lower body condition (Smith et al. 2006). Similarly, Bowers et al. (2014), observed that neonatal body condition and strength of a cutaneous immune response (i.e. PHA injection) was a strong determinant for house wren (Troglodytes aedon) survival and recruitment into the breeding population Additionally, during immune responses, upregulation of immune cells, requiring heightened need for metabolically important resources (i.e. glucose, carbohydrates, and lipids), can result in breakdown and consumption of the body’s protein reserves (i.e. muscle tissues). Moller et al. (1998) provides additional support for better body condition equating to improved immune function, detecting a correlational relationship between body condition and spleen size, an important immune supporting organ, across 1,095 individual birds from 20 separate species. Accordingly, Moller et al. (1998) hypothesized that birds in better body condition were likely to have more resources to dedicate to immune function. Therefore, it can be presumed that bobwhites in a better physiological state or condition may be better able to commit more resources to infection prevention or survive infection by maintaining adequate stores of resources to mobilize during infection. However, within bobwhite research, no such studies have addressed whether the immune function is affected by nutritional components beyond the addition of macronutrients such as protein and energy, and essential amino acids (Lochmiller et al. 1993, Dabbert et al. 1996, 56

Texas Tech University, Drew G. Arnold, May 2019

Dabbert et al. 1997). In fact, the nutrient requirements for bobwhites detailed by the National Research Council (1994) are largely incomplete in regard to micronutrients, vitamins, and trace minerals. Moreover, the nutritional requirements of bobwhites in the wild are not clearly discernable (Murphy 1994, Dabbert et al. 1996) although there is evidence to suggest that the nutritional needs are being met in the wild (Dabbert et al. 1996). However, whether these nutritional requirements are being met to the point of maintaining optimum immune function is unknown. Most studies of supplement feeding are reliant on provisioning of additional resources but do not address nutritional quality. The lack of research on the implications of nutritional quality on the immune system of bobwhites provides an opportunity to investigate the link between nutritional quality and immune function, especially in regard to the effects of vitamins, trace minerals, and antioxidant components. If nutritional quality elevated beyond known values improves immune function, especially when compared to commonly used supplemental food sources (e.g. Sorghum bicolor), then there may be merit in further research and development of a supplemental feed with nutritional quality beyond that of conventional supplemental feed to positively affect the immune system. Therefore, the objective of this study was to investigate the effects of elevated vitamins, trace minerals/metals, and other nutrients on the adaptive and innate immune function of captive bobwhites.

Research Justification It is well understood that nutrition plays an important role in moderating immune function, especially under environmentally adverse and energetically and physiological stressful conditions (temperature extremes, drought, etc.) (Lochmiller and Deerenberg 2000). While nutritional supplementation is commonly used as a management tool (i.e. supplemental feeding) by managers to buffer bobwhites against harsh conditions and stressful events, a scarcity of knowledge exists regarding the effect improved nutritional quality has on regulating bobwhite immune function, which subsequently may affect survival. Investigation of the bobwhite immune function in relation to elevated nutritional planes should yield insight into the implications that elevated nutrition beyond

Texas Tech University, Drew G. Arnold, May 2019 what might be consumed in the wild has on the bobwhite immune system which may consequently relate to bobwhite survival and persistence.

Hypothesis Nutrition has been observed to play an important role in the regulation and support of the avian immune system (Klasing 1988, Lochmiller et al. 1993, Klasing 1998, Kidd 2004, Korver 2012). Although numerous studies have studied the effects of nutrition on immune function in a myriad of avian species, very few have investigated nutritional effects of varying nutritional components on the bobwhite immune system (but see Lochmiller et al. 1993, Dabbert et al. 1996, Dabbert et al. 1997). Combined with the incomplete nature of recommended nutritional values (NRC 1994) and unknown nutritional values for wild bobwhite populations it follows that the addition of nutritional components noted for improving immune function in other avian species or at levels exceeding values for a similar gallinaceous species (Japanese quail), should have the potential to positively affect the immune system of bobwhites. I hypothesize that bobwhites fed diets supplemented with nutritional components a magnitude of five times the recommended nutritional requirements (maintenance diet - National Research Council – 1994) known as the high vitamin (HVTM) diet and a similar diet with the addition of key antioxidants (HVTM+A) will result in improved immunocompetence (i.e. increased efficacy in bacteria killing capacity of serum, increased levels circulating Immunoglobulin Y levels (via ELISA), and an increase in cutaneous immune responses (phytohaemagglutinin injection into patagium) of captive bobwhites when compared to bobwhites under a control diet (maintenance diet – NRC) and those fed a diet with similar composition to that consumed by bobwhites during the winter (NATURAL). I hypothesize that the immune function of bobwhites fed a diet equivalent to a diet consumed by wild bobwhites (i.e. 95% seed/vegetative matter/5% animal matter) (Jackson 1962) during the winter will yield lower immune function measures when compared to diets formulated from published nutritional requirements (NRC, HVTM, and HVTM+A) for Japanese quail.

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METHODS

Animal Care and Housing Individual male bobwhites were placed in maintenance cages measuring 25.4 cm wide x 60.96 cm long. Individuals were placed directly into maintenance cages from outdoor pens and were subsequently maintained as an all-male colony. The individuals were fed ad libitum a maintenance diet of commercially available game-bird feed (Purina) with a protein content of 19% and provided with clean water ad libitum. Bobwhites were allowed to acclimate to the environmental conditions of the animal care room (light and temperature regime) for a minimum of two weeks or until body mass stabilized for the majority of the birds as per recommendations by Bourgeon et al. (2010). Environmental conditions (temperature and lighting) were selected to imitate winter for this latitude within logistical parameters. Temperature was kept as cool as possible (~20°C), the lowest temperature setting possible for the facility’s HVAC system. Lighting duration was chosen to imitate light conditions for the middle of winter for the Rolling Plains of Texas. Winter is a suspected to be time of limited food availability, heightened energetic demands, and inadequate accessibility to nutritionally suitable foodstuffs (Nelson et al. 2002). Bourgeon et al. (2010) and Lochmiller et al. (1993) both noted that limitations of adequate nutrition can manifest in declines in function of the immune system in a short interval of time. Organisms within temperate environments are known to use photoperiod as a cue to modulate resource investment into physiological processes that might confer benefits to fitness and thus survival (Nelson 2002, Schultz et al. 2017). Photoperiod is an environmental cue utilized by a number of avian and mammalian species in the modulation of the immune system (Nelson 2004). Within avian species, the role of photoperiod and effect of seasonality in immune response is less obvious than mammalian species but appears to be driven by context dependent factors (species and immune parameters sampled) (Schultz et al. 2017). If nutrition and photoperiod can affect avian species within a short time, the prolonged nature of winter conditions may be an important time during an individual’s life under natural conditions in the maintenance and regulation of immune function, thus it was deemed appropriate to 59

Texas Tech University, Drew G. Arnold, May 2019 use lighting conditions pertinent to the locality. Lighting conditions for Lubbock, TX at the winter solstice approximate 10 hours light and 14 hours of darkness (10:14 l/d) (Astronomical Applications Dept.- U.S. Naval Observatory).

Experimental Feed Development The feeds utilized in the nutritional modulation of the immune system portion of the study were previously developed through a collaboration with Dr. C.B. Dabbert and Dr. Charles Starkey during Dr. Starkey’s tenure at Texas Tech University. The feeds, with the exception of the feed designated “Natural”, were based on the National Research Council’s nutritional requirements for breeding Japanese quail (Coturnix japonica) (National Research Council 1994) due to incomplete known nutritional requirements for bobwhites. The feed diet designated as “NRC” was in fact the recommended nutritional requirements for Japanese quail. The diet designated “HVTM” was a high vitamin diet that amplified the nutritional requirements set forth in the NRC diet by a magnitude of five (i.e. 5 times the recommended nutrient concentrations). Recommended levels found within guiding literature (i.e. NRC (1994)) are often the result of quantitative studies determining the minimal level to maximize production characteristics (Klasing 1998). However, the levels for maximizing specific aspects of physiological systems (e.g. immune function) are relatively unknown. Although unknown, several studies have investigated the effects of elevated nutrient values beyond NRC (1994) values in domestic poultry production and have observed a dose dependence effect of various nutrients on the immune system (Sklan et al. 1994, Friedman et al. 1998, Korver and Klasing 1997). Rao et al. (2016) commented that for benefits of trace minerals, chromium, zinc, and selenium to be conferred to chickens that levels exceeding NRC (1994) recommendations 2 and 10 times greater were necessary. However, values of nutrients exceeding NRC (1994) recommendations have been observed to have negative consequences on the immune function of avian species (Friedman et al. 1998), or toxicity. Relevant to this project, a value of 5 times the NRC (1994) requirements was selected to ensure a measurable immune response might be observed and immunosuppressive effects avoided. The diet designated “HVTM+A” is 60

Texas Tech University, Drew G. Arnold, May 2019 likewise a high vitamin diet that amplified the nutritional requirements set forth in the NRC diet by a magnitude of five (i.e. 5 times the recommended nutrient concentrations) plus the addition of the antioxidants. I recognize that a magnitude of 5 times the NRC values is somewhat arbitrary. However, due to a dearth of information regarding optimal levels these concentrations were chosen with the expectation that a response may be elicited.

Feed Study Design Individual captive male bobwhites (n = 60) were assigned randomly into one of four experimental feed groups via a random number generator. Feed groups were designated as Natural (NATURAL), National Research Council (NRC), High Vitamin (HVTM), or High Vitamin + Antioxidants (HVTM+A). Birds were given approximately 100 -105 grams of appropriately assigned and labeled feed upon initiation of the study (Day 1). The initial mass of individual bobwhites was recorded on Day 1 of the study with a digital scale (Mettler Toledo, model no. MS4002S/03) and measured at the beginning of each subsequent week to monitor mass lost or gained. Birds that lost greater than 5% of their initial body mass were removed from the study due to animal welfare concerns for said individuals. Bobwhites were supplemented with the respective diets ad libitum over the course of 28 days. Upon reaching day 28 of the feeding trial bobwhites were randomly assigned via random number generator to one of twelve exertion groups or “time slots” for inclusion in the muscle damage modulation study (see Chapter III).

Biological Sample Collection Blood samples were procured on day one of the study via venipuncture of the brachial vein with a 25-gauge hypodermic needle and collected in 200 µl Saf-T-Fill capillary collections tubes (RAM scientific) and stored on ice packs or in a refrigerator until centrifuging could occur, typically within ~2 hours of collection. All blood samples were taken during the morning hours on the day of collection (800:1200). Puncture wounds were treated with styptic powder and pressure applied with a sterile cotton ball until bleeding ceased. Blood samples were centrifuged at 1500 RPM for 15 minutes in portable centrifuges (Fisher Scientific catalog no. 05-090-100) and the resulting separated

Texas Tech University, Drew G. Arnold, May 2019 serum component was removed via pipette and placed into Nalgene® cryo-tubes (Nalgene) (ThermoFisher Scientific catalog no. 5000-1012) and frozen at -20°C or -80°C for later analysis. Blood collection occurred on day one (Day 1) of the study and every other week until the completion of the study. Collection every other week was selected to allow puncture wounds to heal properly before the next blood collection event, reducing physical handling and possible associated stress. Blood collection occurred on day 1, day 14, and day 28 of the study.

Bactericidal Killing Capacity of Blood Serum The immune system is a complex physiological system. However, one of the most fundamental functions of the immune system is defense against bacteria, viruses, and non-self particles. The innate immune system is typically the first line of defense against pathogens outside of mucosal and integumentary membranes (Korver 2012). The innate immune system utilizes cytological, serological, soluble proteins, natural antibodies, and lysozyme among other components to accomplish pathogen recognition and destruction. The immune system has been hypothesized to be nutritionally, energetically, and physiologically costly to maintain (Sheldon and Verhulst 1996, Lochmiller and Deerenberg, 2000, Norris and Evans 2000) with immune response dependent upon a number of underlying individual factors (e.g. reproductive effort, nutritional state, infection, age, sex, species, etc.). This variability of immune response necessitates the measurement of multiple aspects of the immune system (i.e. innate vs. adaptive) to determine if immune function is affected similarly by different variables. The bacteria killing assay (BKA), as outlined in Millet et al. (2007) takes advantage of the natural antibodies and lysozymes present in the humoral fluid to destroy bacterial. The BKA is particularly well suited for evaluation of the innate immune system by evaluating multiple integrated aspects of innate immunity and the ability of the assay to be readily interpretable. That is to say that higher values of bacterial destruction should equate to greater ability of the innate immune system to deal with the particular bacterial species used (Matson et al. 2006a). We chose to use a modified protocol developed by Millet et al. (2007). For the assay Escherichia coli ATCC 8739 (3.1 x 107 CFUs per

Texas Tech University, Drew G. Arnold, May 2019 pellet, Epower Microorganisms no. 0483E7, MicroBioLogics, St. Cloud, MN) was reconstituted in 25 ml of tryptic soy broth and incubated for 24 hours at 37°C. After 24 hours the stock solution was centrifuged at 1200 relative centrifugal force (rcf) for 10 minutes and tryptic soy broth removed and replaced with 25 ml sterile PBS. Twenty-five

µl of previously collected and frozen serum (diluted 1:4 with CO2 independent media plus 4 mM L-glutamine (catalog no. 18045; Gibco-Invitrogen, Carlsbad, CA) yielding a final volume of 100 µl (25µl serum:75 CO2 independent media). To the diluted serum samples, we added a 20µl aliquot of E. coli ATCC 8739 serially diluted to contain approximately 200 colony forming units (CFU). The day prior to assaying samples, serial dilutions were performed and plated on agar plates at varying concentration to assess stock solution concentrations. Diluted serum samples were incubated for 40 minutes at 37°C and then 50 µl aliquots promptly spread on plates in duplicate. Control samples were included for every 10 samples run. Control samples consisted of 100µl of

CO2 independent media with the addition of 20µl of E. coli ATCC 8739 working solution. Plated samples were incubated at 37°C overnight. Viable colonies (i.e. colony forming units) were enumerated per plate and the average of CFUs per duplicate calculated ((Plate #1 + Plate #2)/2). Bactericidal capacity of bobwhite serum was measured as the proportion of bacteria (CFUs) “killed” between the control sample average CFU count and the serum sample plate average CFU count, ((Control CFU average – Serum Sample CFU average)/Control CFU average). It should be noted that the dilution 1:4 and time stop point of 40 minutes was used based on a prior experiment to optimize the best time stop point and dilution factor.

Phytohemagglutinin Assay Phytohemagglutinin (PHA) is a plant lectin, derived from red kidney beans, that elicits a general inflammatory response characterized by a recruitment and proliferation of lymphocytes and phagocytes, important cellular components of the cell mediated immune system, at the injection site producing a measurable swelling of the skin (Smits et al. 1999). The pathways responsible for this general immune response are derived from both the innate and adaptive immune systems but recent research has indicated that

Texas Tech University, Drew G. Arnold, May 2019 the innate immune system is primarily responsible (Vinkler et al. 2014). Following the protocol of Dabbert et al. (1997) birds were injected with 0.5 mg of PHA dissolved in 0.1 ml of phosphate buffered saline (PBS) in the right “wing web” or patagium. The right and left “wing web” thickness was measured just prior to injection and 24 hours after injection which has been noted in many bird species to be the optimal response time (Smits et al. 1999). I did deviate from the Dabbert et al. (1997) protocol by omitting the injection of the left-wing web with sterile PBS as Smits et al. (1999) demonstrated that the control PBS injection was unnecessary. A “wing web” index was calculated, as an indicator of cell-mediated immune response, as the difference between “wing web” thicknesses at PHA injection site versus “wing web” thicknesses at control sites (i.e.) the un-injected “wing web”. PHA injections were carried out on day 21 of the study. Timing of injection at Day 21 was based on data from a feed study performed by Lochmiller et al. (1993) and due to concerns for the possibility of swelling stemming from blood collection within the “wing web” area carried out on days 14 and 28. Day 21 falls on a non-blood collection week and the nearby puncture site area should be healed.

Adaptive Immune Assay Adaptive immune function was assessed by measurement of total IgY levels within the serum of bobwhites. IgY is the most important circulating plasma protein within the humoral immune system of avian species (Roitt et al. 2001) and is the avian equivalent to IgG mammalian species in its function (Hegemann et al. 2017). Measurement of total IgY can used as an indicator of overall adaptive humoral immune function, although we acknowledge that caution should be used when interpreting results, as high IgY may indicate high parasite loads or current infections (Morales et al. 2004, Demas et al. 2011). The protocol of Killpack et al. (2013) (Originally Fassbinder-Orth (2013)) was used to determine total IgY levels. Flat bottomed 96 well microplates were coated with 100 µL of bobwhite serum diluted to 1:100 in coating buffer (0.015 M Na2CO3, 0.035 M NaHCO3, pH 9.6) in duplicate. Negative controls consisting of coating buffer and positive controls of a composite serum sample (i.e. a mixture of 10 randomly selected serum samples) were also included on every plate. Plates were then

Texas Tech University, Drew G. Arnold, May 2019 incubated overnight at 37°C after which the coating buffer was removed and 200 µL of blocking buffer (PBS with 5% non-fat dry milk, 0.05% Tween) was added to each well of the plate. The plate was incubated at room temperature for 30 minutes and then washed with wash buffer (PBS with 0.05%Tween) four times. Fifty µL of detecting conjugate goat anti-bird IgY – horse radish peroxidase (Bethyl Laboratories, Inc., Montgomery, TX) was then added at a dilution of 1:1000 in blocking buffer, incubated for 1 hour at 37°C and then washed away with wash buffer. One hundred µL Tetramethylbenzidine (TMB)-peroxidase substrate (catalog no. 34028; ThermoFischer Scientific, Waltham, MA) was then added to each well and incubated for 5 minutes, and the reaction was stopped with 100 µl 1 M H2SO4. Optical density (OD) was then read with an automated microplate reader SpectraMax 340PC (Molecular Devices, Sunnyvale, CA) at A450. Total IgY was reported as (OD of sample) – (OD of negative control sample).

Statistical Analysis Repeated measures ANOVA’s (Prism, GraphPad Software, San Diego, CA) were used to examine differences in innate immune function (BKA) and adaptive immune function (IgY concentration - ELISA) over three time points (Day 1, 14, and 28) among diet treatments (NATURAL, NRC, HVTM, and HVTM+A) for two replicates of the feed study. The feed study replicates were pooled for analysis. One-way Analysis of Variance (ANOVA) (Prism, GraphPad Software, San Diego, CA) were used to compare cell- mediated immune response (i.e. wing web swelling post PHA injection) among diet treatments (NATURAL, NRC, HVTM, and HVTM+A). Statistical significance was set at P < 0.05 for both repeated measures ANOVA’s and one-way ANOVA.

Note: During the feed study, three trials were performed, each starting with 60 bobwhites, with 15 individuals per feed treatment. Trial #1 was discarded from all analyses due to large amounts of missing data resulting from low biological sample (i.e. serum) volumes to perform assays and failure of environmental controls (i.e. lighting and ambient temperature control) throughout the study.

All aspects of this research were in accordance with Texas Tech University Animal Care and Use Committee Protocol # 114.47-06 65

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RESULTS

Innate Immune Function – Bacteria Killing Assay Trial #2 had 10 bobwhites censored from analysis due to mortality during the trial or from inadequate serum volumes for assay performance. Two, 2, 4, and 2 bobwhites from the HVTM, HVTM+A, NATURAL, and NRC feed groups were removed respectively from analysis. Trial #3 had 13 bobwhites censored from analysis due to mortality or inadequate serum volume. Three, 4, 3, and 3 bobwhites in the respective feed groups HVTM, HVTM+A, NATURAL, and NRC were removed from analysis. There was no observed difference in bacteria killing capacity between diet treatments (n = 4, DF = 3, p = 0.5542). However, there was a significant difference between time points (Days 1,14, and 28) over all groups (n = 3, DF = 2, p = ˂0.0001). The interaction between time and diet was not supported (n = 7, DF = 6, p = 0.5464), signifying all diets responded in a similar manner over respective time points (Day 1, Day 14, Day 28).

Adaptive Immune Function – ELISA Trial #2 had 2 bobwhites, both from the NATURAL feed group censored from analysis due to mortality or inadequate volume of serum for analysis. Trial #3 had 3 bobwhites censored from analysis with 1 bobwhite excluded from each of the respective groups, HVTM, NRC, and NATURAL. Concentrations of circulating immunoglobulin Y (IgY) were not significantly different between diet treatments (n = 4, DF = 3, p = 0.0832) but were significantly different across time points (Day 1, Day 14, Day 28) (n = 3, DF = 2, p = ˂0.0001). The interaction between time and groups was not significant (n = 7, DF = 6, p = 0.4295), signifying that there was no significant difference in feed group effect on circulating IgY over time.

Cell Mediated Immune Function – PHA injections/Wing Web Swelling Trial #2 had 3 bobwhites censored from analysis due to mortality during the trial, with 2 bobwhites and 1 bobwhite from the NATURAL and HVTM feed groups removed

Texas Tech University, Drew G. Arnold, May 2019 respectively. Trial #3 had 2 bobwhites, both from the HVTM+A feed group removed from analysis due to mortality prior to assay implementation. A total of 115 bobwhites were assessed for cell-mediated immune response via PHA injection with 28, 30, 28, and 29 bobwhites in HTVM, HVTM+A, NATURAL, and NRC feed groups respectively. There was no difference observed between diet treatments in respect to cell-mediated immune response (n = 4, DF = 3, p = 0.7687). Average HVTM and HVTM+A cell- mediated immune responses were arithmetically greater (HVTM; µ = .8535, n = 28; HVTM+A; µ = .8577, n = 30) than NRC and NATURAL (NRC; µ = .8355, n = 29; NATURAL; µ = .7596, n = 28) yielding an 11% increase of HVTM+A (the highest average) over NATURAL (the lowest average). However, this difference but this was of no statistical significance.

DISCUSSION

Immune function is directly associated with the support of the immune system in avian species and within gallinaceous birds, particularly in poultry production systems, has been widely implicated in modulating immune function (Klasing 2004, Kogut and Klasing 2009, Korver 2012). While I observed a change in immune function over the course of the feed study (i.e. time effect) for both concentrations of circulating IgY (ELISA) and bactericidal killing capacity of the serum, I did not observe a significant effect between diet treatments over the course of the 4-week feed trial. For example, I observed increases in circulating IgY of 6.1%, 27.4%, 19.3%, and 22.8% in NATURAL, NRC, HVTM, and HVTM+A diets respectively from day 1 to day 28 of the study. This indicates that there was a significant difference in circulating IgY between time points. However, it appears that circulating IgY responded in a similar manner between diets at respective sampling time points (i.e. Day 1, Day 14, and Day 28).

The lack of significance observed between diets and immune parameters may stem from the removal of one trial from analysis due to inadequate volumes of serum for

Texas Tech University, Drew G. Arnold, May 2019 immune assay and failure of environmental controls (i.e. lighting and temperature). Removal of Trial #1 from analysis resulted in reduced sample sizes, with most diets composed of less than 30 individuals (n = <30) for most diets when trials were combined for analysis. I suspect that the limited sample sizes may have reduced my power to detect a difference between diets in corresponding immune assays. Generally, high power studies (i.e. more observations) provide more informative results than low power studies (i.e. limited observations) (Button et al. 2013) and researchers should strive to collect as many observations as logistically possible. However, this generality does not always hold true for all studies and appears to be constrained on an individual study basis. Wagenmakers et al. (2015) provides evidence that low power studies have the capability to provide strong informative evidence and alternatively for high-power studies to provide weak informative evidence. Therefore, it is conceivable that this study does provide informative evidence regarding nutrition and immune parameters. However, as a generality I recommend future studies attempt to bolster observations and increase statistical power of such studies.

Innate Immune Function – Bacteria Killing Assay The lack of differences in diet treatment on bactericidal killing capacity did not support my hypothesis that diets containing elevated nutrient levels beyond NRC (1994) values (NRC diet) and a natural diet analog (NATURAL diet) would increase bactericidal killing capacity of serum when compared to NRC and NATURAL diets. Although there were no differences between diets, the observed time effect provides some evidence that the diets provided to bobwhites in this study were able to increase bactericidal killing capacity of serum beyond that of a commercially available maintenance diet. From Day #1 of the trials to Day #14 increases in bactericidal killing capacity occurred with bobwhites on the NATURAL, NRC, HVTM, and HVTM+A diets increasing killing capacity 53.36%, 52.95%, 41.80%, and 40.68% respectively. I consider this data to suggest that the commercially available maintenance diet was adequate for maintenance but likely did not optimize innate immune function. Interestingly, after initially spiking in bactericidal killing capacity at Day #14, killing capacity of serum declined for all diets

Texas Tech University, Drew G. Arnold, May 2019 except for HVTM which saw a minimal increase of 2.52% from Day #14 (See Figure 2.1). The NATURAL, NRC, and HVTM+A diets experienced a -1.82%, -17.096, and - 41.61 change in killing capacity from Day #14 to Day #28.

Although there was no significance of bactericidal killing capacity between groups, it is possible that the lack of observed significant effects can be attributed to the relatively unknown nutrient values for bobwhites (NRC 1994). The diets were based on NRC (1994) values for Japanese quail (Coturnix japonica), with the exception of the NATURAL diet, and while this species has a similar life history and is morphologically similar (e.g. mass), these values might not truly reflect the nutritional requirements of bobwhites. The ability of the innate immune system has been hypothesized to be tied to investment in physiological life history aspects of a particular species (reproduction, growth, etc.) and where that species falls on the slow-fast pace continuum (i.e. short- lived, high productivity vs. long-lived, low productivity) (Ricklefs and Wilkelski 2002). Species exhibiting alternate life history investment patterns have been observed to likewise invest in immune system function differentially, particularly in relation to ability to kill bacteria (Matson et al. 2006a). Additionally, prior exposure to a particular infectious agent, can affect the innate immune system’s ability to control bacterial infections (Roitt 1997). Similarly, immune function is considered to be affected by life- history traits and organisms are known to invest in traits that might have the most immediate effect on survival (Norris and Evans 2000). Therefore, the lack of significance between dietary effect on bactericidal killing capacity might arise from life- history traits that have developed in the evolution of bobwhites to prioritize bacteria killing capacity despite nutritional state.

The specific nutrient requirements of bobwhites are largely incomplete (NRC 1994). Moreover, the specific nutritional requirements necessary to optimize resistance to infectious agents in bobwhites are largely unknown and even in the rather mature field of poultry nutrition and production is difficult to ascertain (Klasing 1998). Although, no significant differences were observed in the bactericidal killing capacity of the innate immune system, I suspect that the diets with elevated nutrient concentrations (HVTM and

Texas Tech University, Drew G. Arnold, May 2019

HVTM+A) were not sufficient in magnitude to elicit effects on the innate immune system above that of diets of lower nutritive value. Conversely, the elevated nutrient diets (HVTM and HVTM+A) may have induced negative effects on the innate immune system. For example, Leshchinsky and Klasing (2001) found that Vitamin E, a known antioxidant and immunostimulatory compound, exhibited a dose dependent interaction with immunity in chickens (Gallus gallus). This dose dependence effect can be perceived as excessive or inadequate concentrations of Vitamin E may not yield optimum immunostimulatory effects when compared to diets of less nutrient content. Likewise, certain nutrients may have negative effects on immune function if supplied in excessive amounts. For example, both Vitamin A and Vitamin E have been observed to increase immune function in gallinaceous species (i.e. chicken and turkeys) but above a particular concentration reduced immune function (Sklan et al. 1995, Friedman et al. 1998) Therefore, it is plausible that diets with elevated nutrient contents were beyond what is optimum for bobwhite innate immune function and dampened the ability of said diets to contribute to the bacteria killing ability of the innate immune function. However, observed differences over time suggest that all diets were able to boost innate immune function over baseline which can be considered as Day #1. All diet groups were elevated beyond baseline (Day #1) bacterial killing capacity at the cessation of the trial (Day #28). Diets NATURAL, NRC, HVTM, and HVTM+A experienced a 53%, 45%, 43%, and 16% increase in bactericidal killing capacity of serum respectively from Day #1 to Day #28. Interestingly, the effects of diet on bacterial killing capacity appear to remain relatively static or decline slightly from Day #14 to Day #28 but do not return to baseline conditions. While not statistically significant, there is no such literature to my knowledge, acknowledging the magnitude of elevated immune function in relation to biological relevance, though several have theorized that higher immune function may impart benefits in regard to resistance and resilience to infectious agents and fitness (Klasing 1998, Tielman et al. 2005, Matson et al. 2006b). The observed “levelling” of bactericidal killing capacity at Day #28 is interesting as I expected the bactericidal killing capacity to continue to increase. However, it is possible that there was some sort of threshold at which nutritional contributions were no longer able to affect bacterial killing 70

Texas Tech University, Drew G. Arnold, May 2019 capacity. Differential investment into one branch of the immune system over another (i.e. innate vs. adaptive) has been observed in avian species when subjected to pathogenic challenges. For example, (Forsman et al. 2008) observed measures of adaptive immune function (antibody titers) were negatively associated with cell-mediate immunity (PHA injection) in house wrens (Troglodytes aedon) suggestive of a differential investment of resources into a particular immune function component. Bobwhites were injected at Day #21 of the feed studies. The implications of mounting an immune response in terms of mobilizing resources towards one branch of the immune system may have resulted in depression of one aspect of the innate immune system. The cell-mediated response following PHA injections has been largely attributed to activity of the innate immune system although aspects of the adaptive immune system have also been implicated (Vinkler et al. 2014). It follows that PHA induced cell-mediated immune response may have depressed bactericidal capacity, resulting in the observed pattern of declines or relatively little change from Day #14 to Day #28.

Despite the lack of significant differences in bacteria killing capacity between diet treatments, the results of the BKA suggests that the innate immune system of bobwhites can be nutritionally modulated. This statement can be justified due to observed elevations between Day #1, Day #14 and Day #28 which while no significant effect was observed between diets, still resulted in elevated bacterial killing capacity beyond baseline (Day #1) conditions for all diets. Regarding the lack of significance, I acknowledge that lack of significance between diets may be the result of low sample sizes and consequently lacked adequate power to detect significant differences. Regardless of shortcomings, the implications of nutritionally modulating bobwhite immune function in the wild to improve resistance to or resilience during infection is currently unknown. Likewise, through which nutritional pathways the immune system is being improved was not revealed in this study. As such, I can only comment on the potential for future studies. Because of the uncertainty of nutritional pathways in addition to the observed increase in bacterial killing capacity in the NATURAL diet, I suspect that a diet consumed by bobwhites in the wild may be adequate in sustaining

Texas Tech University, Drew G. Arnold, May 2019 innate immune activity under natural conditions. This study may not be directly comparable to wild bobwhite populations that experience food resources reductions in the winter and spring (Robel et al. 1979) concomitant with potentially higher exposure to infectious agents from social behaviors (covey formation) and extreme temperatures may exert stress on bobwhites not experienced by bobwhites in this study. Additionally, in a review of immune function in avian species, Hasselquist and Nilsson (2012) comment that the effects of food restriction or supplementation on immune function is likely the result of the effects of food on stress and thus condition of an organism rather than the direct effect of food on the immune system. As such, I recommend that future studies focus efforts on determining baseline immunological parameters during relevant times of the year when infectious agents may be most prevalent or during stressful periods (i.e. thermal stress, food shortages, etc.). Additionally, determining the relative nutritional state or condition of bobwhites using potential proxies such as body mass, availability of food resources, and/or nutritive value (macro and micro nutrients) of important food sources when compared to immune function parameters should provide insight into the relationship between immune function, nutrition, and bobwhites in the wild.

Adaptive Immune Function – IgY via ELISA The adaptive immune system, specifically humoral immunity, represented in this study by circulating immunoglobulin Y (IgY), has been observed to be influenced in avian species through nutritional pathways (Friedman et al. 1998, Pihlaja et al. 2006). However, the data derived from this study did not detect any effect of diet on the concentration of circulating IgY; although, there appears to be a significant effect of time on circulating IgY. IgY concentrations appear to decline from baseline levels at Day 1 to Day 14 of the study and subsequently become elevated from Day 14 to Day 28. Stress and the subsequent upregulation of the stress hormone corticosterone has been implicated in the depression of the immune system (Råberg et al. 1998) as a potential adaptive trait to minimize risk of autoimmunity. Similarly, the depression of the immune system during stressful situations has been hypothesized as beneficial in mitigating the effects of reactive oxygen species and oxidative stress generated during elevated metabolism during

Texas Tech University, Drew G. Arnold, May 2019 stressful situations (Svensson et al. 1998). The bobwhites in our study were allowed to acclimate to their cages for a minimum of two weeks prior to the start of the trial. Upon Day #1 of the trials, bobwhites were handled to record morphometric measurements (e.g. mass) and collect blood samples. Handling procedures have the capability to affect the immune system. For example, Chin et al. (2013) observed reduced innate immunity in ring-billed gull (Larus delawarensis) chicks after a short-term restraint stressor but failed to detect a similar response in the adaptive immune system. Similar immunosuppressive effects of acute stressors (i.e. handling) have also been noted passerines, affecting cell- mediated immunity (Berzins et al. 2008). While reductions of adaptive immunity have not been observed from acute stressors, chronic stressors are known to affect the adaptive immune system in a number of avian species (Bourgeon and Raclot 2006, Merino et al. 2006). It is possible that the sudden initiation of handling (i.e. a stressor) may have induced immunosuppression of the adaptive immune system resulting in the observed slight decline of circulating IgY from Day #1 to Day #14. Likewise, the stress of repeated handlings and potential increase in the stress hormone corticosterone throughout the trials may have been sufficient enough to suppress the adaptive immune system and mask the effect of diets.

The most striking difference observed within circulating IgY occurred within the NATURAL diet, with an approximately 70% reduction in IgY from Day #1 to Day #14 of the study. Although all diets were observed to decline at Day #14, the diets increased modestly above Day #1 levels at Day #28. Of note, the NATURAL diet only climbed 6% more at Day #28 versus Day #1 levels while all other diets exceeded <15% above baseline levels at Day #1. While not significantly different from the other diets in regard to circulating IgY at Day #28, the lower observed IgY at Day #28 for the NATURAL diet may be biologically relevant, especially when encountering infectious agents. That is to say that the lower levels of observed IgY in the NATURAL diet may not be adequate for defense against pathogens. IgY is an important component of the adaptive immune system particularly in immature birds, and is maternally derived and transferred to chicks via egg yolks (Grindstaff et al. 2003). The relevance of IgY in the wild may be related to

Texas Tech University, Drew G. Arnold, May 2019 the nutritional quality and condition of bobwhite hens such that bobwhite hens in better nutritional condition may be better able to allocate more immunoglobulins to chicks to improve overall immunity. Additionally, chicks in better condition due to maternally derived IgY in addition to adequate nutrition to sustain other physiologically important processes (i.e. growth) may result in a more robust adaptive immune system and thus better survival. For example, circulating IgY concentrations vary with respect to nutritional status and/or chick condition (Pihlaja et al. 2006, Chin et al. 2006). Nevertheless, the levels at which circulating IgY might confer biologically relevant protection from infectious agents is to my knowledge unknown in wild bobwhites and warrants addition research.

Cell-Mediated Immunity Cell-mediated immunity (CMI) as conveyed by response to PHA injections is known to be influenced by nutritional pathways and nutritional status of avian species (Alonso-Alvarez et al. 2001). The data derived from the CMI response from PHA injection does not support the hypothesis that diets of nutritional quality elevated beyond recommended levels and a wild diet analog would yield higher CMI responses when compared to less nutritious diets. Although no significant differences were observed, I would be negligent to note that the nutritionally elevated diets (HVTM and HVTM+A) were arithmetically greater than what I consider diets of lesser nutritional quality. Adequate protein intake has been associated with stronger CMI in avian species (Lochmiller et al. 1993, Saino et al. 1997). While not significant, the differences in CMI response may arise from differences in protein content in diets. For example, the NATURAL diet contained approximately 11.3% digestible protein compared to the HVTM+A diet which contained approximately 14.3% digestible protein. Respective of digestible protein, CMI responded in a fashion suggestive of the effect of increasing protein supplementation, increased CMI in bobwhites (see figure 2.3). Similarly, Tella et al. 2008 hypothesized that CMI response was likely due to dietary intake of proteins to support immune cells and commented that lack of CMI responses observed in captive studies (e.g. Alonso-Alvarez et al. 2001) were not apparent due to the ad libitum nature of

Texas Tech University, Drew G. Arnold, May 2019 feed and adequate intake of feed. Indeed, for bobwhites it appears that protein intake is often a limiting factor for physiological important processes (i.e. growth, reproduction, etc.) (Lochmiller et al. 1993, Giuliano et al. 1996). Giuliano et al. (1996) observed that bobwhites could detect dietary deficiencies in energy content in feed and compensate by increasing intake but could not detect or compensate for protein deficiencies. Given the importance of protein and nutritional state in the CMI response (Alonso-Alvarez et al. 2001) it is possible that no significant differences were observed due to the relatively consistent protein content among diets (See Table 2.4). Body mass or body mass corrected for size has been likewise associated with increases in CMI response to PHA injection (see Alonso-Alverez et al. 2001 for review). While we did not specifically investigate the role of dietary supplementation on body mass there could potentially be some significance to body mass in CMI.

The lack of significance observed in the response of CMI to elevated nutrient levels may be the result of upregulation of the immune system of all bobwhites in the study. Photoperiod exerts a strong regulatory effect on the immune system of many organisms, owing to reduction of input into other energetically costly physiological processes (e.g. reproduction, growth, etc.) (Nelson 2004). Avian species have been observed to upregulate the immune function, specifically cell-mediated immunity in response to shortened photoperiod and subsequently elevated levels of the hormone melatonin (Bentley 2001). The photoperiod utilized for this study was selected to simulate the photoperiod for Lubbock, TX at the winter solstice with approximately 10 hours light and 14 hours of darkness (10:14 l/d) (Astronomical Applications Dept.- U.S. Naval Observatory). Thus, the observed lack of differences between cell-mediated immune response between diets can conceivably be due to the immunostimulatory effects of shortened photoperiod and increased melatonin expression. Consequently, future studies should determine the effects of photoperiod on bobwhite cell-mediated immunity along with other immune parameters.

Diet composition and characteristics have been known to modulate the immunological status of avian species (Klasing 1998). The lack of effect noted on all

Texas Tech University, Drew G. Arnold, May 2019 three immune parameters, bactericidal killing capacity, circulating IgY, and cell-mediated immune response (wing web swelling) via PHA injection suggests that the feeds utilized in this study were likely insufficiently different from each other to bolster immune function in respective aspects when compared to each other. However, the observed significant time effect does yield evidence that the immune system in bobwhites responds to nutritional pathways, which has been demonstrated in past studies (Lochmiller et al. 1993, Dabbert et al. 1996). Due to the incomplete nutrient requirements for bobwhites and the paucity of research indicating the exact nutrients that might benefit the bobwhite immune system, nutrient concentrations or inclusion of nutrients into diets can only be hypothesized largely based on studies using other gallinaceous species (i.e. domestic chicken and Japanese quail). Unfortunately, the responses of these substitute species might not align with optimal function of the immune system, especially for wild bobwhites who likely encounter dynamic environmental, seasonal, and infectious agent stressors and subsequently higher physiologically demands than captive birds in artificially stable environments. Likewise, the nutritional pathways of bobwhites in the wild are influenced by a number of environmental factors such as precipitation, successional state, and land use among other factors with variations in nutritional quality. These factors combined with the numerous effects of environmental conditions and physiologically demanding activities (i.e. growth, reproduction, thermoregulation, etc.) may confound the ability of future research to adequately identify the exact inclusion or rates of nutrients to improve immune function in wild bobwhites. Therefore, I propose that future research focus on investigating factors that might negatively affect bobwhite immunological parameters in the wild such as environmental conditions (i.e. precipitation, thermal extremes, etc.) in conjunction with physiologically demanding processes (i.e. reproduction) and/or availability and nutritional quality of natural food sources important to bobwhites.

Texas Tech University, Drew G. Arnold, May 2019

Table 2.1 Composition of complete diet base mix for feed trials. TTU Quail Tech Complete Complete diets diets diets

Ingredient Kg/ton Lbs./ton

Milo 651.13 1435.466

Soy bean meal (47%) 212.40 468.26

Fat 6.20 13.66

Limestone 11.52 25.4

Oystershell 0.00

Salt 2.36 5.2

Biophos(monocal) 16.81 37.06

Methionine DL 99 1.91 4.21

VTM Premix 4.54 10

Na Bicarb 1.88 4.14

Choline chloride 0.60 1.32

Threonine 0.13 0.284

Total 907.2 2000

Texas Tech University, Drew G. Arnold, May 2019

Table 2.2 Composition of premix added to base mix to achieve experimental feed nutrient concentrations. Ingredient NRC 5X 5X + Antioxidants

Vitamin A, IU/kg feed 1650 8250 8250

Vitamin D, IU/kg feed 750 3750 3750

Vitamin E, IU/kg feed 12 60 250

Vitamin K, mg/kg feed 1 5 5

B1 Thiamin, mg/kg feed 2 10 10

B2 Riboflavin, mg/kg feed 4 20 20

B6 Pyridoxine, mg/kg feed 3 15 15

B12 Cyanocobalamin, mg/kg feed 0.003 0.015 0.015

Niacin, mg/kg feed 40 200 200

Panthothenic acid, mg/kg feed 12 60 60

Folic acid, mg/kg feed 1 5 5

Biotin, mg/kg feed 0.03 0.15 0.3

Manganese, mg/kg feed 60 300 300

Zinc, mg/kg feed 25 125 125

Iron, mg/kg feed 120 120 120

Copper, mg/kg feed 5 25 25

Iodine, mg/kg feed 0.3 1.5 1.5

Cobalt 0 0 0

Selenium, mg/kg feed 0.2 0.3 0.6

Canthaxanthin, mg/kg feed 0 0 12

DHA, mg/kg feed 0 0 100

Texas Tech University, Drew G. Arnold, May 2019

Table 2.2 Continued

Ingredient NRC 5X 5X + Antioxidants

Hy-D, mg/kg feed 0 0 0.092

Vitamin C, mg/kg feed 0 0 200

Chromium 0 0 500

Carnitine 0 0 100

Texas Tech University, Drew G. Arnold, May 2019

Table 2.3 Concentrations of nutrient additives in grams and milligrams added to base mix (see table 2.2) Bobwhite Diets

Control High VTM Antioxidants + High VTM

NRC 5x 5x + antioxidants

Ingredient G mg g Mg G mg

Vitamin A, IU/kg feed 0.4 374.6 1.9 1872.8 1.9 1872.8

Vitamin D, IU/kg feed 0.3 340.5 1.7 1702.5 1.7 1702.5

Vitamin E, IU/kg feed 61.9 61909.1 309.5 309545.5 1289.8 1289772.7

Vitamin K, mg/kg feed 0.7 687.9 3.4 3439.4 3.4 3439.4

B1 Thiamin, mg/kg feed 22.5 22455.2 112.3 112276.2 112.3 112276.2

B2 Riboflavin, mg/kg feed 1.1 1135.0 5.7 5675.0 5.7 5675.0

B6 Pyridoxine, mg/kg feed 82.5 82505.5 412.5 412527.3 412.5 412527.3

B12 Cyanocobalamin, mg/kg feed 1.0 1031.8 5.2 5159.1 5.2 5159.1

Niacin, mg/kg feed 9.2 9171.7 45.9 45858.6 45.9 45858.6

Pantothenic acid, mg/kg feed 2.8 2751.5 13.8 13757.6 13.8 13757.6

Texas Tech University, Drew G. Arnold, May 2019

Table 2.3 Continued

Bobwhite Diets

Control High VTM Antioxidants + High VTM

NRC 5x 5x + antioxidants

Ingredient G mg g Mg G mg

Folic acid, mg/kg feed 11.4 11350.0 56.8 56750.0 56.8 56750.0

Biotin, mg/kg feed 31.0 30954.5 154.8 154772.7 309.5 309545.5

Manganese, mg/kg feed 42.6 42562.5 212.8 212812.5 212.8 212812.5

Zinc, mg/kg feed 16.0 15985.9 79.9 79929.6 79.9 79929.6

Iron, mg/kg feed 90.8 90800.0 90.8 90800.0 90.8 90800.0

Copper, mg/kg feed 4.5 4504.0 22.5 22519.8 22.5 22519.8

Iodine, mg/kg feed 1.5 1474.0 7.4 7370.1 7.4 7370.1

Selenium, mg/kg feed 75.7 75666.7 113.5 113500.0 227.0 227000.0

Texas Tech University, Drew G. Arnold, May 2019

Table 2.3 Continued

Bobwhite Diets

Control High VTM Antioxidants + High VTM

NRC 5x 5x + antioxidants

Ingredient G mg g Mg g mg

Beta Carotene, mg/kg feed 0.0 0.0 0.0 0.0 22.7 22700.0

Canthaxanthin, mg/kg feed 0.0 0.0 0.0 0.0 27.2 27240.0

Hy-D, mg/kg feed 0.0 0.0 0.0 0.0 151.9 151883.6

Vitamin C, mg/kg feed 0.0 0.0 0.0 0.0 129.7 129714.3

DHA, mg/kg feed 0.0 0.0 0.0 0.0 133.5 133529.4

Chromium mg/kg 0.0 0.0 0.0 0.0 56.8 56750.0

Carnitine mg/kg 0.0 0.0 0.0 0.0 454.0 454000.0

Texas Tech University, Drew G. Arnold, May 2019

Table 2.4. Analysis of nutritional components performed by A & L Plains Analytic Laboratory (302 34th St, Lubbock, TX 79404).

Sample ID X A Z Y B

Sample Diet ID NRC NATURAL HVTM+A HVTM HVTM

Moisture % 0 - 13.65 0 - 10.70 0 - 13.20 0 - 13.05 0 - 12.55

Crude Protein % 17.19 - 19.90 14.56 - 16.31 18.25 - 21.03 17.50 - 20.13 18.31 - 20.94

Digestible Protein % 12.55 - 14.53 10.63 - 11.90 13.32 - 15.35 12.78 - 14.69 13.37 - 15.29

Crude Fat % 2.75 – 318 8.25 - 9.24 2.8 - 3.23 2.85 - 3.28 3.25 - 3.72

Acid Detergent Fiber 3.89 - 4.50 4.55 - 5.10 2.89 - 3.43 3.35 - 3.85 3.62 - 4.14

Ash % 5.90 - 6.83 2.60 - 2.91 6.50 - 7.49 6 - 6.90 6.0 - 6.86

Total Digestible Nutrients % 76.76 - 88.89 78.84 - 88.28 78.11 - 89.99 77.87 - 89.56 78.06 - 89.27

Nitrogen % 2.75 - 3.18 2.33 - 2.61 2.92 - 3.36 2.8 - 3.22 2.93 - 3.35

Manganese ppm 115 – 133 25 - 28 286 - 329 349 – 401 339 – 388

Copper ppm 13 – 15 7.0 - 8.0 32 – 37 54 – 52 29 – 33

Zinc ppm 43 – 50 49 - 55 95 – 109 90 – 104 217 – 248

Selenium ppm < 2.00 < 2.00 < 2.00 < 2.00 < 2.00

Chromium ppm 1.71 - 1.98 < .50 2.93 - 3.38 1.66 - 1.91 1.94 - 2.22

Texas Tech University, Drew G. Arnold, May 2019

Effect of Diets on Bobwhite Plasma Bactericidal Killing Capacity

80 HVTM HVTM+A Natural

NRC y

t 60

)

i 40

(

a B

1 4 8 y 1 2 a y y D a a D D

Days on Diet

Figure 2.1 Bactericidal killing capacity of diets for Trial #2 and Trial #3 combined in percentage bacterium, Escherichia coli ATCC 8739, killed compared to controls over the duration of the feed trials (28 days)

Texas Tech University, Drew G. Arnold, May 2019

Effect of Diets on Bobwhite Plasma IgY

1.5

HVTM HVTM+A Natural

NRC )

I i

1.0

( P

0.5

1 4 8 y 1 2 a y y D a a D D Days on Diet

Figure 2.2 Circulating immunoglobulin Y (IgY) of diets for Trial #2 and Trial #3 combined observed as the optical density (OD) at wavelength λ450 over the duration of the feed trials (1, 14, and 28 days).

Texas Tech University, Drew G. Arnold, May 2019

PHA Induced Web Wing Swelling

2.0

e n

k 1.5

g n

i 1.0

)

(

n 0.5

e r

e 0.0

i D

-0.5 l M A a C T + r R V M tu N H T a V N H Diets

Figure 2.3. Wing web swelling in response to phytohemagglutinin (PHA) injections of Trial #2 and Trial #3 combined at Day 21. Values presented as a wing web swelling index calculated as the difference between wing web thicknesses at PHA injection site versus thicknesses at un-injected sites.

Texas Tech University, Drew G. Arnold, May 2019

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Dunham, N. R., A. Bruno, S. Almas, D. Rollins, A. M. Fedynich, S. M. Presley, and R. J. Kendall. 2016. Eyeworms (Oxyspirura petrowi) in northern bobwhites (Colinus virginianus) from the rolling plains ecoregion of Texas and Oklahoma, 2011−13. Journal of Wildlife Diseases 52:562–567. Dunham, N. R., L. A. Soliz, A. M. Fedynich, D. Rollins, and R. J. Kendall. 2014. Evidence of an Oxyspirura petrowi epizootic in northern bobwhites (Colinus virginanus), Texas, USA. Journal of Wildlife Diseases 50:552–558. Errington, P. L. 1939. The comparative ability of the bobwhite and the ring- necked pheasant to withstand cold and hunger. The Wilson Bulletin 51:22–37. Fassbinder-Orth, C. A., V. A. Barak, and C. R. Brown. 2013. Immune responses of a native and an invasive bird to Buggy creek virus (togaviridae: alphavirus) and its arthropod vector, the swallow bug (Oeciacus vicarius). L. L. Coffey, editor. PLoS ONE 8: e58045. Fenoglio, S., M. Cucco, and G. Malacarne. 2002. The effect of a carotenoid-rich diet on immunocompetence and behavioural performances in Moorhen chicks. Ethology Ecology & Evolution 14:149–156. Finkel, T., and N. J. Holbrook. 2000. Oxidants, oxidative stress and the biology of ageing. Nature 408:239–247. Forsman, A. M., S. K. Sakaluk, C. F. Thompson, and L. A. Vogel. 2010. Cutaneous immune activity, but not innate immune responsiveness, covaries with mass and environment in nestling house wrens (Troglodytes aedon). Physiological and Biochemical Zoology 83:512–518. Fraps, G. S. 1947. The composition and utilization of Texas feeding stuffs. Texas Agricultural Experimental Station Bulletin No. 461. Friedman, A., I. Bartov, and D. Sklan. 1998. Humoral immune response impairment following excess vitamin E nutrition in the chick and turkey. Poultry Science 77:956–962 Giuliano, W M., R. S. Lutz, and R. Patiño. 1996. Reproductive responses of adult female northern bobwhite and scaled quail to nutritional stress. Journal of Wildlife Management 60:302-309. Grindstaff J.L., E.D. Brodie, and E.D. Ketterson. 2003. Immune function across generations: integrating mechanism and evolutionary process in maternal antibody transmission. Proceedings of the Royal Society of London. Series B: Biological Sciences 270:2309–2319. Guthery, F. S. 1997. A philosophy of habitat management for northern bobwhites. The Journal of Wildlife Management 61:291–301. Guthery, F. S., A. R. Rybak, S. D. Fuhlendorf, T. L. Hiller, S. G. Smith, W. H. Puckett, and R. A. Baker. 2005. Aspects of the thermal ecology of bobwhites in North Texas. Wildlife Monographs 159:1–36. 90

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Merino, S., J. Moreno, G. Tomás, J. Martínez, J. Morales, J. M.-D. L. Puente, and J. L. Osorno. 2006. Effects of parental effort on blood stress protein HSP60 and immunoglobulins in female blue tits: a brood size manipulation experiment. Journal of Animal Ecology 75:1147–1153. Miller, R.S. 2011. Foraging behavior of northern bobwhite in relation to resource availability. Thesis, Mississippi State University, Starkville, MS, USA. Millet, S., J. Bennett, K. A. Lee, M. Hau, and K. C. Klasing. 2007. Quantifying and comparing constitutive immunity across avian species. Developmental and Comparative Immunology 31:188–201. Møller, A. P., and N. Saino. 2004. Immune response and survival. Oikos 104:299–304. Møller, A. P., P. Christe, J. Erritzøe, and J. Mavarez. 1998. Condition, disease, and immune defence. Oikos 83:301–306. Moller, A.P., C. Biard, J.D. Blount, D.C. Houston, P. Ninni, N. Saino, and P. Surai. 2000. Carotenoid-dependent signals: indicators of foraging efficiency, immunocompetence or detoxification ability? Avian and Poultry Biology Reviews 11: 137-159. Monaghan, P., N. B. Metcalfe, and R. Torres. 2009. Oxidative stress as a mediator of life history trade-offs: mechanisms, measurements and interpretation. Ecology Letters 12:75–92. Morales, J., J. Moreno, S. Merino, G. Tomás, J. Martínez, and L. Z. Garamszegi. 2004. Associations between immune parameters, parasitism, and stress in breeding pied flycatcher (Ficedula hypoleuca) females. Canadian Journal of Zoology 82:1484–1492. Murphy, M. E. 1994. Amino acid compositions of avian eggs and tissues: Nutritional implications. Journal of Avian Biology 25:27–38. National Research Council. 1994. Nutrient Requirements of Poultry. Ninth edition. National Academy Press, Washington, DC, USA. Nelson, R. J. 2004. Seasonal immune function and sickness responses. Trends in Immunology 25:187–192. Nelson, R. J., G. E. Demas, S. L. Klein, and L. J. Kriegsfeld. 2002. Seasonal patterns of stress, immune function, and disease. Cambridge University Press, Cambridge, UK. Norman, A. W., J. Roth, and L. Orci. 1982. The vitamin D endocrine system: steroid metabolism, hormone receptors, and biological response (calcium binding proteins). Endocrine Reviews 3:331–366. Norris, K. and M.R. Evans. 2000. Ecological immunology: life history trade-offs and immune defense in birds. Behavioral Ecology 11:19–26.

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Wilcoxen, T. E., R. K. Boughton, and S. J. Schoech. 2010. Selection on innate immunity and body condition in Florida scrub-jays throughout an epidemic. Biology Letters 6:552–554 Wiley, D. 2017. Northern bobwhite reproduction in the rolling plains of Texas. Thesis, Texas Tech University, Lubbock, USA. Williams, C. K., F. S. Guthery, R. D. Applegate, and M. J. Peterson. 2004. The northern bobwhite decline: scaling our management for the twenty-first century. Wildlife Society Bulletin 32:861–869. Wood, K. N., F. S. Guthery, and N. E. Koerth. 1986. Spring-summer nutrition and condition of northern bobwhites in South Texas. The Journal of Wildlife Management 50:84–88. Wu, D., and S. N. Meydani. 1998. n-3 Polyunsaturated fatty acids and immune function. Proceedings of the Nutrition Society 57:503–509.

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CHAPTER III

NUTRITIONAL MODULATION OF MUSCLE DAMAGE

Introduction The capture, handling, and tagging (e.g. radio-tagging) of wildlife is a necessary component for evaluating critical aspects of the biology and ecology of wildlife. Capture, handling, and radio-tagging (i.e. radio transmitters) of avian species is regularly utilized to gather data on the physiology, morphology, and behavior of avian species in addition to the collection of biological samples. Bobwhites are regularly subjected to capture, handling, and deployment of tagging or marking devices (i.e. radio transmitters, bands, etc.) to estimate survival rates (Pollock et al 1989, Burger et al. 1995, Faircloth et al. 2005). Since inception and implementation of radio-telemetry studies, there has been much debate and concern over the practice of radio-marking and subsequent inferences collected on avian species using radio-tagging (White and Garrott 1990, Guthery and Lusk 2004, Barron et al. 2010, Casas et al. 2014). A number of detrimental risks to individual fitness stemming from capture, handling, and/or radio-tagging have been documented on wild avian species including muscle damage/capture myopathy, reduced survival compared to non-tagged cohorts, variable behavior, amplified stress indicators, deterioration of physical condition and altered immune function (Nicholson et al. 2000, Höfle et al. 2004, Cox et al 2004, Casas et al 2014, B.R. Buckley and C.B. Dabbert, Texas Tech University, unpublished manuscript) Bobwhites have likewise been the subject of many radio-tagging effects studies. A number of these studies have supported the contention that application of radio transmitters reduces bobwhite survival or affects bobwhite physical or behavioral characteristics in contrast to unmarked counterparts (Osborne et al. 1997, Cox et al. 2004, Guthery and Lusk 2004, Abbott et al. 2005). However, these criticisms have been equally met with evidence of null effects of radio- marking on the behavior, physical condition, or reductions in survival (Palmer and Wellendorf 2007, Sisson et al. 2009, Terhune et al. 2007). Despite conflicting evidence over utilization of radio-telemetry studies and resultant data on bobwhite ecology, to our

Texas Tech University, Drew G. Arnold, May 2019 knowledge there has been limited research on the effects of the capture and handling process of bobwhites.

Parallel to the associated effects and concerns mentioned prior, one additional concern associated with capture and handling especially among avian species is a condition known as capture myopathy. Capture myopathy is an acute degeneration of muscle tissue resulting from intense muscular exertion and trauma caused by chase, struggle, or transport (Hulland 1993). Capture myopathy from intense muscular exertion and trauma triggers a physiological response in animals, shunting blood flow away from muscle tissue following exhaustion of initial aerobic energy, resulting in decreased oxygen and nutrient delivery to tissues (Beringer et al. 1996). The consequences of these physiological mechanisms result in cellular death and metabolic acidosis due to elevated lactic acid levels and secondary muscle necrosis, renal failure, and depressed immune function (Harthoorn et al. 1983, Spraker 1993, Williams and Thorne 1996, Nicholson et al. 2000, Deguchi et al. 2014). In conjunction with skeletal muscle damage, other physiological systems may be similarly affected such as the cardiac muscle damage and necrosis which may result in mortality for an individual even if it appears in good condition following capture (Williams and Thorne 1996, Nicholson et al. 2000) Another consequence of these metabolic processes is the release intracellular enzymes such as aspartate aminotransferase (AST) and creatine kinase (CK) from damaged muscle into the bloodstream, both of which have been used to indicate muscle damage in several avian species (Bollinger et al. 1989, Dabbert and Powell 1993, Guglielmo et al. 2001, Franson et al. 1985).

The capture event and radio-tagging for a wildlife species is often treated as though the event has no negative effects upon the captured individual and the subsequent survival estimation of a marked individual (White and Garrot 1990). In other words, captured and marked individuals should behave, react physiologically, and present the same survival probability of those without tags or transmitters. However, there is evidence that capture events may produce effects on the survival of individuals beyond the commonly used censor periods for survival analysis of avian species of 2 to 14 days

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(White and Garrot 1990). For example, Mueller et al. (1999) found that for every 1000 u/L increase in creatine kinase, bobwhite survival decreased by 14% over a 16-week study period. Likewise, Deguchi et al. (2014) noted that albatross chicks restrained for transmitter attachment suffered from moderate muscle damage which subsequently resulted later fledging and shorter sustained fledging flights, which they hypothesized could impact survival although they could not determine whether this muscle damage impacted long-term survival due to a limited monitoring period. Alternatively, Abbot et al. (2005) failed to find any statistically significant relationship between survival and capture effects, based on enzyme indicators of muscle damage, CK and AST. A shortcoming described in some muscle damage studies utilizing CK and AST as muscle damage indicators is the disparity between species and individuals in the quantifiable amount of handling/exercise necessary for significant increases in enzyme activities indicative of muscle damage (Nicholson et al. 2000, Shutz et al. 2010). It should also be noted that studies have taken into account the effect that capture method (e.g. rocket nets, walk-in or swim in traps, etc.) have upon muscle damage (see Bollinger et al. (1989), Dabbert and Powell (1993), Höfle et al. (2004)). Bobwhites are typically captured via a Stoddard walk-in trap (Stoddard 1931). Walk-in type traps appear to have a reduced effect on muscle damage indicated via CK and AST levels versus other trap types (mist nets, rocket nets, etc.). For instance, Bollinger et al. (1989) concluded that CK and AST levels in mallards (Anas platyrhynchos) were lower in individuals trapped with walk-in type traps versus an alternate rocket netting method. Similarly, Toepfer et al. (1987) noted that greater prairie chickens (Tympanuchus cupido pinnatus) captured via walk-in traps presented lower mortality than those trapped using rocket nets. Whether mortality was due to muscle damage incurred from the trapping techniques of Toepfer et al. (1987) was not studied but the study’s results suggest it is prudent to consider trapping techniques when studying capture myopathy, muscle damage and the implications it may have on overall survival of the study species. Höfle et al. (2004) found that histopathologic lesions indicative of muscle damage from capture myopathy were more common in the leg muscles of red-legged partridge, a Galliform species with a similar life-history as bobwhites. However, Höfle et al. (2004) did note that all birds affected by 101

Texas Tech University, Drew G. Arnold, May 2019 capture myopathy also had self-inflicted wounds due to struggling with attached transmitters and may have exacerbated the original muscle damage from capture and handling. This suggests that not only should the effects of the initial capture be considered but also subsequent individual interaction with attached transmitters/tags when investigating potential causative actions of muscle damage. However, this is not within the scope of our study and this aspect of capture induced muscle damage will not be investigated.

Beyond the investigations of the effects of capture, handling, and marking of wildlife species (i.e. capture myopathy), researchers have attempted to minimize incidence of capture myopathy. Methodologies utilized to minimize the risk of capture myopathy in wildlife have emphasized the use of establishing species specific guidelines for trapping and handling methodologies. For example, Nicholson et al. (2000), recommended that wild turkeys (Meleagris gallapavo) in Oklahoma should not be captured when winter temperatures exceed 10° C, relative humidity is below 40%, and handling and release times should be kept to a minimum. Similarly, Bollinger et al. (1989) observed elevated levels of the serum enzymes creatine kinase (CK) and aspartate aminotransferase (AST) in mallards (Anas platyrhynchos) were correlated to trap type used and suggested that minimizing the time mallards spent struggling and handled might be useful in reducing capture myopathy. Likewise, Spraker et al. (1987) suggested that increased incidence of capture myopathy was associated with capture method used and recommended researchers use caution when using certain methodologies (e.g. drop nets). Outside of establishing capture and handling guidelines to minimize incidence of capture myopathy, relatively few studies exist addressing treatment of capture myopathy once present. Those studies that do exist appear to have had limited success in rehabilitation of capture myopathy (Businga et al. 2007). Although, the efforts of Rogers et al. (2004) appeared to have been relatively successful in rehabilitation of capture myopathy effected Great Knots (Calidris tenuirostris) and Red Knots (Calidris canutus), the researchers acknowledge that their techniques should be used as a last resort due to associated time intensive constraints. The techniques used by Rogers et al. (2004) included the

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Texas Tech University, Drew G. Arnold, May 2019 maintenance and rehabilitation of knot species over a series of weeks under conducive artificial conditions (i.e. a quiet air-conditioned room with rehabilitators). Such conditions are not typically at the disposal or logistically possible for many wildlife studies and as such may have limited feasibility as an option for treatment of capture myopathy in many wildlife species. Susceptibility to capture myopathy has been suggested to be the result of nutritional deficiencies (Herbert and Cowan 1971, Hulland 1985). Likewise, nutritional deficiencies have been speculated to affect the oxidative status and increase the potential for oxidative damage following intense or stressful exertional activities and ensuing depletion of energy stores and catabolism of skeletal muscle (i.e. migration flights) (Constantini et al. 2007). Antioxidants play a major role in cell protection from reactive oxygen species and prevention of lipid peroxidation and thus inhibition of oxidative damage. Specifically, Vitamin E and selenium deficiencies have been suspected to contribute to capture myopathy possibly due to their antioxidant properties. Several other antioxidants, for example, Vitamin C, folic acid, carotenoids and the mineral zinc have also been implicated in reducing oxidative stress and damage in gallinaceous species during stress (Sahin et al. 2002, Harsini et al. 2012). These specific nutrients may play an important role in minimizing the effects of capture myopathy by reducing the effects of free radical production experienced after exhaustive muscular exertion (Viña et al. 2000). The supplementation of Vitamin E and selenium has been recommended to minimize effects of capture myopathy in veterinary literature for avian species (Carpenter et al. 2001, Olsen and Orosz 2000). Within wildlife studies, Dabbert and Powell (1993) provided diets containing varying concentrations of Vitamin E and selenium to mallards (Anas platyrhynchos) but were unable to detect a response in the enzymes indicating capture myopathy (CK and AST) due to feeds containing apparently sufficient concentrations of both nutrients for mallards. Abbott et al. (2005) intradermally injected wild captured bobwhites with a Vitamin E and selenium following capture, handling, tagging and transport to a translocation site in order to examine the effects of injections on survival. Abbott et al. (2005) failed to detect a statistically significant difference in survival between injected bobwhites vs. control bobwhites for the duration of the study period. Although the lack of significance was largely attributed 103

Texas Tech University, Drew G. Arnold, May 2019 to small sample sizes, there was evidence to support greater survival occurred in bobwhites injected with Vitamin E and selenium compared to control bobwhites 45- and 66-days post capture, when adequate sample sizes were available. Guglielmo et al. (2001) further associates the role of nutrition might play in capture myopathy by suggesting that migratory shore birds (western sandpipers and bar-tailed godwits) performing longer more intense flights may exhibit longer stopover times in order to restore fuel loads and allow muscle damage to heal. Related to nutritional modulation of muscle damage, body condition has been documented as a potential contributor to limiting the effects of exhaustive exercise or stressful events. For instance, Constantini et al. (2007) noted higher levels of fat and protein stores in barn swallows and garden warblers were associated with higher circulating anti-oxidants following exhaustive migration flights indicating individuals in better condition were potentially able to defend against oxidative damage and potentially muscle damage by maintaining adequate reserves of dietary anti-oxidants. Because capture myopathy is suspected to be the result of metabolic processes and the subsequent generation of free radicals and oxidative damage, it follows that antioxidants beyond those typically investigated (i.e. Vitamin E and selenium) in capture myopathy may likewise be effective at mitigating the effects of capture myopathy. Additionally, there exists a lack of information directly evaluating the effects of nutrition plane on capture myopathy. Therefore, an opportunity exists to evaluate the role such factors may play in capture myopathy. The objective of this study was to assess how varying nutritional planes, specifically those exceeding established nutritional recommendations and the addition of antioxidants effect muscular damage from exhaustive exercise. The implications of the successful moderation of muscle damage from exhaustive exercise via nutritional supplementation could drive future research of nutritional modulation of capture myopathy and subsequently limit mortality resulting from capture myopathy.

Bobwhites are a species that have important economic and ecological value (Conner et al. 2007, Burger et al.1999). However, population declines across much of their range raises concern for conservation efforts (Brennan et al. 1991, Hernandez et al.

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2013). As with most birds, bobwhites rely on flight to survive and perform daily functions. Flight in birds have many functions to their survival such as escape and avoidance of predators, search for food, searching for mates, and movement among habitats. Bobwhites differ from many birds due to their sedentary, non-migratory nature and preference to fly primarily when encountering a threat such as a predatory escape event or as predator avoidance mechanism. Some forms of flight can be strenuous and have energetic costs such as those short bursts of flight typically found in bobwhites when escaping or avoiding perceived threats. For example, Perkins et al. (2014) determined that bobwhites may be able to differentiate between threats, with individuals choosing differing degrees of flight patterns, flight speeds, and vegetative escape cover dependent upon the perceived threat, mammalian, aerial raptor attack, hunter, and researcher scenarios. Perkins et al. (2014) also noted the perception that bobwhites in the study were aware of approaching researchers and posited that bobwhites chose to hide or hold tight to cover until it was no longer a viable avoidance option. Guthery and Lusk (2004) also noted that radio-tagged bobwhites were less likely to flush than non-tagged counterparts when encountered by researchers, although this was based on anecdotal evidence. Consequently, hiding/avoidance behavior may be of some benefit in regard to energetics, avoiding a possible strenuous and energetically costly function until absolutely necessary. Nudds and Bryant (2000) revealed that short flights by zebra finches (Taeniopygia guttata) was more energetically expensive than stationary control birds or those in sustained flight. Nudds and Bryant (2000) attributed this energetically costly flight to the low speeds at which the zebra finches were flying, leading to additional required power from muscles to generate lift as well as take-off acceleration (see Pennycuik (1989) for flight theories). Time spent flying and take off acceleration accounted for variations in energy expenditure in individuals in this study. While bobwhites have a different flight pattern from that of zebra finches, Perkins et al (2014) provide evidence that bobwhites alter their flight speed and path dependent on the perceived threat. Bobwhites in Perkins et al. (2014) flew at higher speeds for longer periods with no detectable deceleration phase and a more irregular but less arced flight path when faced with an aerial threat from a raptor versus a reduced flight speed, 105

Texas Tech University, Drew G. Arnold, May 2019 straighter albeit arced flight path, and noted deceleration phase with characteristic wing setting and gliding when faced with mammalian, hunter, or researcher threats. It follows that behavioral responses to stimuli resulting in intense bouts of flights may be energetically costly to bobwhites and the threat response behaviors may serve as mitigation to such an energetically costly function. Not only could flight be perceived as energetically costly for wild birds but there is evidence that escape/avoidance events may have other physiological consequences. For example, Guglielmo et al. (2001) noted that intense or enduring flights of migratory shore birds resulted in elevated plasma enzymes symptomatic of muscle damage (e.g. creatine kinase). Although creatine kinase concentrations were elevated, the magnitude of concentrations suggested that muscle damage was moderate. Guglielmo et al. (2001) posited that the moderate amounts of muscle damage observed might not truly be representative of the population but rather a by-product of physiological or behavioral consequences of individuals suffering from intense muscle damage dropping out of the population due to immunosuppression, susceptibility to predation, or increased stopover time for muscle recovery. Interestingly, Guglielmo et al. (2001), noted that highest concentrations of creatine kinase were associated with capture of shore birds in mist nets, despite rapid removal from mist nets, rather than the consequences of flight itself. Dabbert and Powell (1993) likewise demonstrate that intense bouts of physical exercise resulting from capture and struggle of mallards (Anas platyrhynchos) in differing types of capture nets resulted in increased levels of the indicator enzymes of creatine kinase (CK) and aspartate aminotransferase (AST). The short intense periods of flight experienced by bobwhites during escape processes in the wild may have the potential to induce muscle damage as well as affect the immune function of individuals. The immune system is important for survival in free living organisms (Lochmiller and Deerenberg 2000) and as such has been noted as energetically expensive to maintain often requiring tradeoffs in life history functions of many avian species (Norris and Evans, 2000). Nebel et al. (2012) reported that European starlings (Sturnus vulgaris) displayed reduced constitutive immune function after intense bouts of flight. Matson et al. (2012) also noted non-migratory intense flight exercises on an avian species (Columba livia) yielded reduced immune responses. 106

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However, Matson et al. (2012) were unable to link flight speed (a metric of exertion) as an indicator of the magnitude of immune response but were able to provide evidence that the consequences of flight can influence the immune function of an avian species. Moreover, Matson et al. (2012) points out that “Understanding how all forms of physical exertion, including flight, influence immunological indices will undoubtedly prove useful in the broader goal in accounting for immunological variation within and among species” the ramifications of which could provide managers and researchers with a better understanding of the physiological consequences of exertion on fitness and ultimately survival in bobwhites.

The ultimate consequences (mortality) of exertion and muscle damage related to capture are not well documented in gallinaceous birds outside of mortalities directly observed during or immediately after capture events (Spraker et al. 1987, Nicholson et al. 2000). The physiological consequences of exertion resulting from capture and handling or adaptationally relevant escape behaviors are poorly understood, especially for bobwhites. However, many have suggested that the effects of these activities may have fitness consequences and subsequently lead to mortality (Spraker et al. 1987, Guglielmo et al. 2001, Matson et al. 2012). With a scarcity of relevant data on the consequences of exertion on bobwhites in conjunction with a poor understanding of how consequences might be alleviated, the objective of this study was to determine if muscle damage from exertion can be modulated through nutritional pathways.

For this study we are attempting foremost to modulate bobwhite immune function via nutritional supplementation of elevated levels vitamins and minerals above published nutritional recommendations for Japanese Quail (National Research Council 1994). Our secondary goal for this study was to minimize the effects of induced muscular damage of bobwhites via nutritional pathways. Studies were carried out in a laboratory setting in order to reduce variables and biases that might arise under natural conditions. Exertion trials were also performed under laboratory conditions to allow for the collection of muscle tissue samples for visual examinations to identify muscular lesions as performed by Spraker et al. (1987) Höfle et al. (2004) and Ruder et al. (2012) which could not occur

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Note on Capture and Handling of Bobwhites Peer reviewed literature is currently lacking in regard to bobwhite behavioral and exertional responses to capture and handling and/or time spent in traps prior to investigator arrival. This paucity in data is likely due to the prevailing assumption among a number of bobwhite researchers that capture, and handling of bobwhites does not affect survival (Pollock et al. 1989, Palmer and Wellendorf 2007, Sisson et al. 2009, Terhune et al. 2007). Personal communication with peers also revealed bobwhites tended to be confined to Stoddard walk –in traps baited with milo (Stoddard, 1931) for a minimum of several minutes to possibly several hours. Once in the traps and upon approach by a researcher, bobwhites may take on what can be described as panicked behavior with intense bouts of running and escape flight behavior within the trap often resulting in physical contact with cage boundaries and abrasions to the head, wing tips, breast and feet. Upon reaching the trap bobwhites are removed to either one communal cotton bag or to individual cotton bags as rapidly as possible and placed within the confines of a research vehicle. The average number of bobwhites caught per capture event varies based on individual trapping event. Average bobwhite covey size during trapping season (October – March) averages 12 individuals (Hernandez and Guthery 2012) so it is conceivable a whole covey could be successfully trapped yielding 12 or more bobwhites. Bobwhites are typically processed within the confines of the research vehicle, and morphometric data and blood samples collected, radio transmitter attachment and leg banding performed. Elapsed time of processing largely depends on individual experience in processing of bobwhites. Bobwhites in communal bags were often described as agitated or easily stirred up by external stimuli. Bobwhites placed in individual cotton bags were described as easier to work with and less agitated or stressed due to lack of external stimuli or by conspecifics. The time bobwhites spent within the confines of the truck in cotton bags was dependent upon the number of birds captured at any one capture event. Likewise, it should be noted that handling time is largely dependent on expertise

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Texas Tech University, Drew G. Arnold, May 2019 and experience of investigator and bobwhites processed by unexperienced investigators may experience prolonged handling times. Something that would be prudent to consider during this processing time would be the behavior or bobwhites in hand during processing, which was described as calm or non-resistant to handling (i.e. no to little struggling). What is important to take from these personal accounts are the times that bobwhites are constrained to intense exertion, struggle, or stress. For this study emphasis is placed on muscle recovery following intense bouts of exercise, such as those resulting from capture events as described above or of those associated with predatory escape/avoidance behaviors as described in the previous section. From the above account we can see that bobwhites may be exposed to intense bouts of exercise for short a short duration assuming that bobwhites are executing intense escape behavior the entirety of the time within a trapping event, which is quite doubtful. One must also consider the effect of increased stress and frantic escape behaviors that may occur due to predatory approaches and attacks that occasionally happen before the researcher may encounter the trapped birds (e.g. hawks, bobcats, raccoons, coyotes, etc.).

METHODS

Feed Study Design Individual male bobwhites (n = 60) were randomly assigned into one of four experimental feed groups via a random number generator. Feed groups were designated as Natural (NATURAL), National Research Council (NRC), High Vitamin (HVTM), or High Vitamin + Antioxidants (HVTM+A). See Chapter II for complete details of diets. +Groups were composed of five bobwhites (N = 5). Upon assignment bobwhites were then subjected to the following protocol on day 29 of the study to examine a novel approach to mitigate muscle damage incurred from intense exertion via nutritional modulation.

Protocol Muscular exertion was stimulated via movement of the researcher’s inserted arm as would be done by a researcher in a walk-in trap to remove bobwhites, referred to as 109

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“hand grappling”. Dabbert and Weeks (Texas Tech University, unpublished data) performed a similar study where bobwhites were exposed to muscular exertion by similar means of “hand grappling” for varying amounts of time. Hand grappling can be defined as gently pursuing and grabbing a bobwhite within the confines of a trap in order to secure said bobwhite and subsequently releasing the bobwhite. During capture, bobwhites often escape the grasp of the investigator due to the bobwhite’s escape behaviors. Hand grappling was meant to simulate the process of capture and handling often experienced by wild bobwhite. The results of Dabbert and Weeks (Texas Tech University, unpublished data) found that creatine kinase concentrations were disparate as measured at 4 hours but were similar among trials (time of exposure) at 24 hours. Bobwhites were exposed to “hand grappling” for approximately 15 minutes total of simulated capture and handling, alternating 1 minute of simulated capture and 1 minute of rest for a total of 30 minutes per group per exertion trial. Bobwhites were then immediately removed from the trap and placed back into their assigned maintenance cages. Blood was then drawn 4 hours and 24 hours post exertion for respective groups via venipuncture of the brachial vein with a 25-gauge hypodermic needle and collected in 200 µl Saf-T-Fill capillary collections tubes (RAM scientific). Blood samples were stored on ice packs or in a refrigerator until centrifuging could occur, typically within ~2 hours of collection. Blood samples were centrifuged at 1500 RPM for 15 minutes in portable centrifuges (Fisher Scientific catalog no. 05-090-100) and the serum component was removed via sterile pipette and placed into Nalgene® cryo-tubes (ThermoFisher Scientific catalog no. 5000-1012) and frozen at -20°C or -80°C for later analysis. After collection of blood 24 hours post exertion, the respective group was euthanized via CO2 asphyxiation and cervical dislocation. Muscle biopsies were taken from the right thigh muscle and left pectoral muscle and preserved in individually marked tissue cassettes and placed in formalin specimen cups for later examination. Blood and serum were frozen for later examination. Indication of muscle damage was evaluated by determining the concentration of creatine kinase (CK) and aspartate aminotransferase (AST) within the serum. Muscle damage was also indicated through microscopic muscle cell examination for lesions and hemolysis. 110

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Creatine Kinase and Aspartate Aminotransferase Concentration Creatine kinase (CK) and Aspartate aminotransferase (AST) are enzymatic indicators of muscular damage, often the result of extreme exertion (Franson et al. 1985, Spraker et al. 1987, Bollinger et al. 1989). Circulating CK and AST values were determined using a VetTest 8008 chemistry analyzer ((Idexx Laboratories, Rydalmere, NSW, Australia) located within the Dabbert laboratory (Goddard ANNEX, 15th and Detroit Lubbock, TX 79407). Briefly, individual serum samples were diluted 1:20 (20 µl serum:400 µl PBS) in sterile phosphate buffered saline (PBS) and 40 µl of diluted sample aliquoted into a pipette vial within the analyzer for distribution onto dry slides for analysis. Twenty µl of serum total is necessary to run analysis for both CK and AST. Appropriately matching CK and AST analysis dry slides (Idexx Laboratories, Rydalmere, NSW, Australia) were placed into the VetTest 8008 and chemistry analysis performed as per the manufacturer’s recommendations. Manufactured dry slides for AST and CK are species specific and typically are manufactured for common avian species seen in veterinary offices (e.g. parakeets, cockatoos, parrots, etc.) for this specific analyzer (Vettest 8008). Dry slides manufactured for cockatiels were utilized due to similarity in mass between cockatiels and bobwhites. Serum samples that were overly hemolytic or lipemic were excluded from analysis as these factors could skew chemistry analysis of enzyme concentrations. Samples exceeding the capable linear range of values for the VetTest 8008 (> 2,036 IU) were diluted to 1:40 and re-analyzed to determine values. Values of 0 were diluted 1:10 and reassessed to determine values. All values were multiplied by their dilution factor (e.g. 1:20 = multiplied by 20) to determine true concentration value of CK and AST.

Microscopic Muscle Cell Examination (Tissue Biopsies) Microscopic examination of muscle cell damaged was performed by Zoo/Exotic Pathology Service (C/O: Dr. Drury Reavill, 6020 Rutland Drive #14, Carmichael, CA 95608). Breast and thigh muscle tissues were examined using standard histological examination methods for lesions indicative of muscle damage. Morphologic lesions were characterized as being focal, multifocal, multifocal to confluent, or diffuse. The severity

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Texas Tech University, Drew G. Arnold, May 2019 of individual lesions was graded from minimal, mild, moderate, and severe. Although severity and presence of muscle damage lesions varied among bobwhites, for this study we simply recorded whether lesions indicative of muscle damage were present and did not indicate severity in the analysis.

Statistical Analysis One-way Analysis of Variance (ANOVA’s) (Prism, GraphPad Software, San Diego, CA) were used to compare the effects of diets (NATURAL, NRC, HVTM, and HVTM+A) on muscle damage indicators (CK and AST). The effects of diets on the prevention of positive muscle damage lesions designated as a binary variable (0 = no lesions, 1= lesions present) were analyzed using a chi square test. The criterion for significance was set at P < .05.

All aspects of this research were in accordance with Texas Tech University Animal Care and Use Committee Protocol # 114.47-06

RESULTS

Creatine Kinase/Aspartate Aminotransferase Trial #2 had 9 bobwhites censored from analysis due to mortality during the trial or from inadequate serum volumes for assay performance. Two, 5, and 2 bobwhites from the HVTM+A, NATURAL, and NRC feed groups were removed respectively from analysis. Trial #3 had 7 bobwhites censored from analysis due to mortality or inadequate serum volume. Two, 3, 1 and 3 bobwhites in the respective feed groups HVTM, HVTM+A, NATURAL, and NRC were removed from analysis.

Serum samples collected following muscular exertion with sufficient volumes for analysis were available for 105 bobwhites. Fifteen serum samples were excluded from analysis due to lipemia, hemolysis, error readings, or due to incomplete data (i.e. sampled at 4 hours but not 24 hours) due to mortality. Creatine kinase (CK) concentrations

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Texas Tech University, Drew G. Arnold, May 2019 exceeded the upper limits (< 2,036 U/L) for the Vettest 8008 chemistry analyzer in 22 samples at 4 hours post exertion and 46 samples 24 hours post exertion. Aspartate aminotransferase (AST) concentrations did not exceed the upper limits (<2,036 U/L) for the Vettest 8008 chemistry analyzer in any samples.

When CK activity was evaluated as an interaction between diets and time over the course of 4 and 24 hours a significant difference was detected between the diet treatments of NATURAL and NRC at 4 hours post exertion (P = 0.0391). The NRC diet was on average 8,690 +/3,572 U/L. No differences in CK activity were observed between other diet treatments over the 4 and 24-hour time points post exertion (P = 0.2584). Differences in CK concentrations were detected between time points 4 hours and 24 hours post exertion but not between diet groups (P = <0.0001).

There were no differences detected between diet groups at 4 hours post exertion (P = 0.1616). Likewise, at 24 hours post exertion no significant differences in CK serum concentrations were detected (P = 0.1007). Serum concentrations of CK were greatest in the NRC diet at 4 hours post exertion (µ = 24,071 ± 2,838 U/L) but was not significantly greater than other diets. Serum concentrations of CK were greatest in the HVTM diet 24 hours post exertion (µ = 33,436 ± 2,225 U/L) but was not significantly higher.

Serum concentrations of AST were not different between diets when evaluated as an interaction between diets over the course of 4 and 24 hours (P = 0.1938). A significant time effect was observed for AST serum concentrations (<0.0001) between 4 and 24 hours post exertion. serum concentrations of AST were not different between diet groups (0.1693). There were no differences detected in AST serum concentrations between diet groups at 4 hours post exertion (P = 0.5703). No significant differences in serum AST concentrations were observed between diet groups at 24 hours post exertion (P = 0.1742). AST concentrations were highest 4 hours post exertion in the HVTM diet group (µ = 893 ± 138 U/L) but was not significantly higher than other diets. AST concentrations were greatest 24 hours post exertion in the HVTM diet group (µ = 2,713 ± 475 U/L) but was not significantly greater compared to other diets.

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Differences in the incidence of muscle lesions were detected between diets. Incidence of leg lesions was significant between diet treatments (X2 = 9.653, df = 4, P = 0.0467). Incidence of breast lesions was significant between diet treatments (X2 = 9.949, df = 4, P = 0.0413). Incidence of muscle damage lesions was greatest in the HVTM and HVTM+A diets within breast tissues with 11 incidences observed for each diet. Incidence of muscle damage lesions was greatest for the NRC and HVTM+A within leg tissues with 13 and 10 incidences observed respectively.

DISCUSSION

No differences in diet treatment on the concentration of muscle damage enzymes, with the exception of differences observed between the NATURAL and NRC diets, is suggestive that the nutrient elevated diets (HVTM and HVTM+A) were not effective at reducing incidence of muscle damage following exertion. The differences observed between the NATURAL and NRC diet is perplexing, considering the NATURAL diet, was not different from the other diets with elevated nutritional value (HVTM and HVTM+A). This is even more interesting when taking the incidence of muscle damage lesions between diet groups into account. The NATURAL diet had the lowest incidence of muscle damage lesions during both Trials #2 and #3. However, I am unsure of the underlying reasons why the NATURAL diet might buffer bobwhites against muscle damage especially with regard to the HVTM+A diet. Sorghum bicolor, which was used as the base for the diet (95% of content) has been shown to have fairly high levels of antioxidants (Dykes and Rooney 2006), sometimes exceeding values observed in fruits and vegetables. It is possible that the lower rates of muscle damage enzymes CK and AST observed in the NATURAL diet is due to high antioxidant concentrations within in the diet. It might also be why the NATURAL diet was no different than the HVTM and HVTM+A which both have elevated nutrients with the potential to act as antioxidants thus maintaining a surplus of antioxidants that might be used to combat oxidative damage following exertion early on (~ 4 hours post exertion). The HVTM+A diet with its 114

Texas Tech University, Drew G. Arnold, May 2019 antioxidants theoretically should be able to stave off the effect of oxidative damage known to occur during muscular exertion. However, it appears that elevated nutritional diets were less beneficial to bobwhites post exertion, than the NATURAL diet. In fact, bobwhites fed diets supplemented with additional nutrients and antioxidants (HVTM and HVTM+A) in general exhibited higher levels of the muscle damage indicator enzyme, Creatine kinase at 24 hours post exertion. Counter to this, the diet simulating a natural diet of bobwhites on the Rolling Plains of Texas, NATURAL, appears to have generally resulted in lower levels of both muscle damage enzymes, CK and AST over both 4 and 24 hours post exertion. While the observed differences between diet treatments were not significant, it is important to note that this trend held for both feed trials. Despite a lack of significance, levels of AST were consistently higher for the HVTM diet treatment for both trials. The NRC diet appeared to start off higher than other treatments 4 hours post exertion but did not exhibit as great a change as other diets at 24 hours post exertion.

The lack of significance of diet treatments on CK and AST concentrations might be the result of the excessive demands of the exhaustive exercise procedure. Bobwhites were exposed to capture and handling procedures for 15 minutes to ensure muscle damage was incurred. I am not aware of any study where bobwhites have been subjected to a standardized exercise protocol and associated times besides an unpublished preliminary study by Dabbert and Weeks (Texas Tech University, unpublished data). However, Tripp and Schmitz (1982) appeared to utilize a standardized protocol to induce enzyme levels indicative of muscle damage in 10-week old turkey (Meleagris gallapavo) poults without apparent negative effects. For my study, negative effects were quite conspicuous during and following the exertional protocol. For example, there were many occasions where bobwhites were observed to involuntarily constrain movement by approximately 5 minutes into the 15-minute process. Behavioral and physical attributes of these bobwhites were consistent with capture myopathy and included weakness, paralysis, locomotion abnormalities in addition to drooping wings and heads, accelerated respiration and/or gular flutter, and in extreme cases, mortality before the end of the capture and handling procedure. Likewise, several bobwhites died prior to blood

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Texas Tech University, Drew G. Arnold, May 2019 sampling at 4 and 24 hours post exertion. Despite the lack of knowledge regarding the magnitude or duration of handling and exertion necessary to induce elevated CK and AST concentrations and muscle damage, I observed similar levels to those of Abbott et al. (2005). Although not directly comparable due to methodological differences, the values observed by Abbott et al. (2005) reflect that even when attempting to minimize capture, handling, and transport time and attenuate muscle damage via supplementation of nutrients that enzyme values representative of muscle damage can occur. It is conceivable, that the capture and handling procedure was excessive for bobwhites and the procedure produced artificially high levels of muscle damage than what might be experienced under typical capture and handling procedures in the field although this is unlikely due to similar values observed for Abbott et al. (2005)

Studies to distinguish nutritional requirements to maximize physiological processes are common in the poultry industry to produce desirable traits, with many investigating the nutritional requirements to maximize muscle growth or maximum reproduction. However, the nutritional requirements for bobwhites are largely incomplete (NRC 1994). Additionally, the nutritional requirements for wild bobwhites to maximize physiological processes (i.e. muscle growth, reproduction, etc.) are unclear and often context dependent (Lochmiller et al. 1993, Dabbert et al. 1996). In an ideal setting, provision of diets at varying concentrations (i.e. 1, 2, 5, 10 order of magnitude) or the elevation of single nutrients would provide more insight into the role of specific nutritional pathways managing physiological processes underlying muscle damage. However, it must be recognized that studies are often constrained by time and resource limitations. As such, it is conceivable that the lack of observed significant effect on mitigating muscle damage may be the result of insufficient levels of certain nutrients important for processes minimizing muscle damage. Similarly, levels of nutrients necessary for processes managing muscle damage from exhaustive exercise might be excessive and interactions between nutrients may underlie the observed elevations of CK and AST in the HVTM and HVTM+A diet treatments.

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Serum concentrations of CK and AST were substantially lower in birds not exposed to the muscular exertion procedure. Bobwhite serum concentrations from Trial #2 for combined feed treatments were approximately 4.2 and 31 times larger for AST and CK respectively, than for published baseline levels for bobwhites (Hill and Murray 1987). Bobwhite serum concentrations from Trial #3 for combined feed treatments were approximately 4.5 and 32 times larger for AST and CK respectively, than for published baseline levels for bobwhites. When Trial #2 and Trial #3 were combined, AST and CK concentrations were 2 and 22 times larger at 4 hours post exertion than for published baseline levels for bobwhites. AST and CK values were 5 and 31.5 times higher at 24 hours post exertion when compared to published baseline concentrations for bobwhites. When compared to control birds not exposed to muscular exertion protocol, AST and CK concentrations were 4.5 and 41 times higher for Trial #2. For Trial #3, AST and CK concentrations for combined diet treatment means were 3.4 and 24.5 times higher respectively when compared to bobwhites not exposed to the muscular exertion trial. When data for Trial #2 and Trial #3 were combined, AST and CK concentrations were 1.7 and ~22 times higher at 4 hours post exertion than mean values of control bobwhites. AST and CK values were 4.5 and 31 times larger 24 hours post exertion than values observed for control bobwhites. The observed consistency of values in relation to magnitudes higher than control or published baseline levels suggests that the control birds were adequate in representing baseline conditions. Likewise, these observed differences are in accordance with finding by Abbot et al. (2005), who noted mean AST and CK concentrations 3 and 100 times above respective normal levels following a capture, handling, and transport protocol. Although, my concentrations for CK values were less than half those of Abbott et al. (2005), this difference is likely due differing methodologies such as the use of wild bobwhites versus captive bobwhites. Domestication of wild animals can result in less active and aggressive animals that are easy to catch and handle (Archard and Braithwaite 2010). Alternatively, captivity can alter physiological responses to stressors (i.e. capture, transport, captivity, thermal temperatures, etc.) with upregulation or downregulation of physiological responses that may affect fitness (Dickens et al. 2009). It is plausible the lower levels of serum enzymes 117

Texas Tech University, Drew G. Arnold, May 2019 were due to physiological responses resultant from use of captive game farm bred bobwhites.

Differences in incidence of muscle damage as indicated via pathologically identified lesions was significant for bobwhites within the feed study. The lesions appeared most concentrated in the HVTM+A group for both leg and breast tissues. It is interesting that lesions were concentrated in a diet treatment that was predicted to lessen the degree of muscle damage during and following intense exertion. The addition of antioxidants in the HVTM+A were justified by the presumed antioxidant effect these nutrients would exert on pro-oxidants produced from muscular exertion (Viña et al. 2000). The oxidative status of an organism is reliant on the ability of antioxidant defenses to neutralize pro-oxidants or reactive oxygen species (ROS). When pro- oxidants/ROS levels begin to exceed the capacity of antioxidant defenses, an imbalance occurs which may lead to oxidative stress and damage (Monaghan et al. 2009). In addition to imbalances resultant from excessive metabolic production of ROS, certain nutrients have been known to become pro-oxidants. For example, Vitamin E in reacting with ROS is itself turned into a pro-oxidant (Monaghan et al. 2009). However, Vitamin E produced pro-oxidants can be “revived” for antioxidant capacity through interaction with other antioxidants, notably carotenoids and Vitamin C (Surai 2002). These potential factors in conjunction with lack of knowledge regarding optimal concentrations for most nutrients in the bobwhite’s diet and high incidence of muscle damage lesions in elevated nutrient diets (HVTM and HVTM+A), there are several conceivable reasons for observed incidences of muscle damage lesions. However, I am unable to discern through what pathways or interactions these observed incidences occurred and as such refrain from speculating without further studies.

Management Implications From this study, it is unclear if elevated nutrients and the addition of antioxidants is beneficial in reduction of muscle damage. However, it appears that elevated nutrients and addition of antioxidants were either not sufficient to reduce muscle damage or 118

Texas Tech University, Drew G. Arnold, May 2019 possibly excessive, resulting in elevated indicators of muscle damage. Interestingly, the diet treatment, NATURAL, although not significantly different than other diets, did result in lower levels of CK and AST at both 4 and 24 hours post exertion. This suggests that naturally occurring food sources might be sufficient to manage the muscle damage resultant from capture and handling. While not directly comparable to muscular damage, increased oxidative damage can have fitness consequences when considered from a life- history standpoint. For example, escape flights are considered to be physiologically different than long distance flights, imposing its own demands and may be a proxy for fitness for many species (Irschick and Garland 2001). The supplementation of antioxidants has been shown to reduce the incidence of oxidative damage in avian species following strenuous exercise (Larcombe et al. 2008). Concurrent with antioxidant supplementation, regular exercise training can provide protection from oxidative damage, with repeated periods of physical activity enhancing the ability of an animal to handle extra pro-oxidants produced during exercise. Indeed, such observations related to exercise training has been observed in avian species. Larcombe et al. (2010) noted that budgerigars exposed to several bouts of short duration escape flights were able to ameliorate oxidative damage indicators due to antioxidant protection from exercise training. Larcombe et al. (2010) go on to suggest that because the physical activity of wild animals is typically greater than those of laboratory animals, the physiological protection from exercise may be elevated at all times, and thus may require additional antioxidants from dietary sources to prevent pro-oxidants and oxidative damage. Relevant to this study, it is possible that bobwhites in the wild are able to defend themselves more effectively from oxidative damage and muscle damage due to their increased physical activity. Because the bobwhites within the feed trials were confined to relatively small areas, and repeated exercise was not administered, the effects of the capture and handling procedure is not directly comparable to the activity of wild bobwhites, that may experience an abundance physical activity and may subsequently be better able to cope with pro-oxidant production. Bobwhites also can experience drastic changes in dietary quality from one season to the next which may result in deficiencies of important nutrients required for growth, reproduction, and survival (Peoples et al. 1994). 119

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However, I observed lower CK and AST concentrations in the NATURAL diet, an analog for a wild bobwhite diet during winter, than for elevated nutrient and antioxidant supplemented diets. While not significant, the lower levels may indicate that bobwhites utilizing natural food stuffs or supplemental feed (i.e. Sorghum bicolor) may be more less likely to incur muscle damage from than manufactured feeds. Considering the specialization of equipment and limited access to nutritional supplements (i.e. vitamins, minerals, etc.) required for specialty feed formulation in addition to limited preference for manufactured feeds by bobwhites (Larson et al. 2012), I do not foresee supplementation of a manufactured feed as a feasible option for bobwhite management in order to reduce muscle damage from capture and handling. In lieu of nutritional supplementation, I recommend that muscle damage from capture and handling be minimized by establishing species specific capture and handling guidelines and adopting methodologies to minimize muscular exertion as suggested by numerous researchers (Spraker et al. 1987, Bollinger et al. 1989, Nicholson et al. 2000). Additionally, the implementation of management activities to improve habitat conditions and provision of ample food resources should be given priority in order to ensure bobwhites are in good condition, thus reducing the likelihood of muscle damage during a capture situation.

Future investigations on the effects of elevated or additional specific nutrients to the diet should focus on determination of efficacy of individual nutrients at varying concentration to reduce muscle damage. Likewise, optimizing a proxy for capture and handling procedures that adequately represent capture and handling methodologies applied in the field are needed to best determine how bobwhites might respond under similar situations.

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Effects of Diet on Plasma CK After Muscular Exertion

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Figure 3.1 Response of liver enzyme, creatine kinase (CK), among diet treatments for both Trial #2 and Trial #3 combined at 4 hours and 24 hours post muscular exertion.

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Figure 3.2 Response of creatine kinase (CK), among diet treatments for Trial #2 and Trial #3 combined at 4 hours post muscular exertion.

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Effects of Diet on Plasma CK 24-hours After Muscular Exertion

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Figure 3.3 Response of liver enzyme, creatine kinase (CK), among diet treatments for Trial #2 and Trial #3 combined at 24 hours post muscular exertion.

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Figure 3.4 Response of liver enzyme, aspartate aminotransferase (AST), among diet treatments for Trial #2 and Trial #3 combined at 4 hours and 24 hours post muscular exertion.

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) 1000

/ U

( 500

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Figure 3.5 Response of liver enzyme, aspartate aminotransferase (AST), among diet treatments for Trial #2 and Trial #3 combined at 4 hours post muscular exertion.

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Figure 3.6 Response of liver enzyme, aspartate aminotransferase (AST), among diet treatments for Trial #2 and Trial #3 combined at 24 hours post muscular exertion.

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