ABSTRACT. Despite the degree of environmental protection and economic feasibility offered by

ABSTRACT ARBONA, DIANA VICTORIA. A Comparative Analysis of Free-Range vs. Caged Layer Hens: Egg Productivity & Quality, Humoral Immunocompetency, & Follicular Expression of Glucocorticoid Receptors. (Under the direction of Dr. Jacquelyn B. Hoffman.) Despite the degree of environmental protection and economic feasibility offered by traditional poultry houses, the practice of housing laying hens in cages has been widely criticized worldwide due to the public perception that caged housing systems provide poor welfare for laying hens. As a result of this increasing public interest in laying hen welfare, alternative management systems such as free-range environments have been resurrected and new legislation such as the California Proposition two is expediting a shift back towards extensive practices. The transition from intensive back to extensive practices coupled with the advent and increasing popularity of novel alternative layer housing management methods in recent years necessitates an examination of the influence of environmental stressors associated with different housing methods on layer egg production, immunological response, and reproductive well-being. The goal of this research is to begin to determine if alternative layer management methods such as the free-range environment improves the well-being of layers, in comparison to caged layers, while supporting the poultry industry s current standards for egg productivity.

Project I: Examines differences in rearing methods and production performance between hens reared in caged vs. free-range environments. By monitoring all rearing procedures, dietary regimens and vaccinations, and comparing egg productivity and quality of caged vs. free-range birds, it can be determined which layer management method (s) provide the most benefits to layer well-being while maintaining egg productivity and egg quality standards conducive to industry and consumer expectations. Project II: Assesses the influence of free-range and conventional layer housing management methods on humoral immune function and heterophil: lymphocyte ratios. Because stress may directly alter immune function it is necessary to determine whether different management methods increase hens susceptibility to disease by decreasing humoral immune function. By challenging hens reared in free-range vs. caged environments with a killed Newcastle s disease and measuring differences in humoral immune function between these birds, the impact of different environmental management methods on humoral immune function were determined. Project III: Aims to determine whether different housing management protocols can modulate layers reproductive well-being by increasing ovarian susceptibility to corticosterones as a result of altered mrna expression of follicular glucocorticoid receptors. By characterizing expression of the glucocorticoid receptor in ovarian follicles of free-range vs. caged hens, the impact of

environmental management methods on ovarian stress susceptibility may be ascertained. The overall objective of these three projects is to improve producers and consumers understanding of the impact of alternative management methods such as a free-range environment on hens productivity and welfare in comparison to traditional caged management methods. These studies will help to provide insight into the influence of intensive vs. extensive housing methods on layers welfare from both a production and physiological perspective, and concurrently add to the growing pool of research on effective methods for assessing welfare status in order to promote animal well-being and environmental adaptability.

A Comparative Analysis of Free-Range vs. Caged Layer Hens: Egg Productivity & Quality, Humoral Immunocompetency, & Follicular Expression of the Glucocorticoid Receptor in the Ovarian Follicles by Diana Victoria Arbona A thesis submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the degree of Master of Science Physiology Raleigh, North Carolina 2011 APPROVED BY: Glen Almond, D.V.M., Ph.D. Kenneth Anderson, Ph.D. Jacquelyn B. Hoffman, Ph.D. Committee Chair

ii BIOGRAPHY Victoria was born in Lowell, Massachusetts on September 8, 1983. Since then she has lived in eleven different states (and counting) and driven across the United States at least three times in the process. Growing up she wanted to train animals at Sea World and join the circus as an acrobat. She spent much time exploring her broad spectrum of interests by participating in sports, theatre and music, and she did much volunteer work through her church and local animal shelters. Between the years of receiving a Bachelor s degree from Virginia Tech in Theatre Arts and Scenic Design, and entering the graduate program at North Carolina State University, she discovered science and medicine as her passion, but never failed to be caught dancing in the lab or the elevators of Scott Hall at NC State. In June of 2009, Victoria became a Master of Science candidate in Physiology through the College of Veterinary Medicine and the Department of Poultry Science at NC State University in Raleigh, North Carolina. Under the direction of Dr. Jackie Hoffman, she attended and presented at conferences in Barcelona, Spain; Raleigh, NC; Atlanta, Georgia; and Denver, Colorado. She was involved in the University Grad Student Association as a representative to her department and the Chair of the Political Action Committee. During her two years at NCSU, the committee coordinated two holiday food drives and a blood drive among the students and faculty of the university. After graduation, she plans to pursue Veterinary School, or of course, join the circus.

iii ACKNOWLEDGMENTS First and foremost, I want to thank my Major Professor, Dr. Jackie Hoffman. Through her direction and her guidance I achieved much more than just my research goals; I realized my full potential. Thank you for making the difference in my graduate career and for being a major influence in my life, I could not have done it without you! I would also like to sincerely thank all the people that made these projects possible and supported me on my committee including Dr. Ken Anderson and Dr. Glen Almond. I want to thank my lab mate and undergraduate research student Lauren A. Bola, and my fellow graduate student friends who supported me and made the journey fun and unforgettable. My graduate career would not have been possible without the love of my family members, my parents, my sisters, and my brothers. Thank you so much for your continuing support and for all the long Gmail chats. One of the best parts of studying and writing was having my pups sleeping on my feet and taking them to the dog park and the lake during breaks! Jake, Dally, Hanners, Humphrey, Scout and Bailey you are the greatest friends a girl could ask for! To my wonderful friend, Quinn Henderson Thank you for dancing with me in the lab, never letting me forget who I am, and for being with me every step of the way. Finally, thank you to Christopher Kent, my best friend during my graduate career, whose never-ending love, has made all the difference.

iv TABLE OF CONTENTS LIST OF TABLES... x LIST OF FIGURES... xi CHAPTER I STRUCTURE, DEVELOPMENT, & REGULATION OF THE DOMESTIC HEN S REPRODUCTIVE SYSTEM...1 1. THE DOMESTIC HEN S OVARY...2 1.1 STRUCTURE OF THE OVARY...2 1.2 HISTOLOGICAL ORGANIZATION OF THE OVARIAN FOLLICLES...4 1.3 FOLLICULAR DEVELOPMENT...5 1.4 REPRODUCTION: OVULATION OF THE OVARIAN FOLLICLES...6 1.5 THE HYPOTHALAMIC-PITUITARY-GONADAL (HPG) AXIS...7 1.6 SUMMARY...8 CHAPTER II INTERACTION & DYSFUNCTION OF THE HYPOTHALAMIC- GONADAL (REPRODUCTIVE) & HYPOTHALAMIC-PITUITARY-ADRENAL (STRESS) AXES...10 2. STRESS...11 2.1 THE HYPOTHALAMIC-PITUITARY-ADRENAL (HPA) AXIS...11 2.2 INTERACTION OF THE HYPOTHALAMIC-PITUITARY- ADRENAL & HYPOTHALAMIC-PITUITARY-GONADAL AXES...11 2.3 DYSFUNCTION OF THE HYPOTHALAMIC-PITUITARY- ADRENAL AXIS & IMPACTS ON THE HYPOTHALAMIC- PITUITARY-GONADAL AXIS... 12 2.4 DYSFUNCTION OF THE HYPOTHALAMIC-PITUITARY-GONADAL AXIS... 13

v 2.5 INFLUENCE OF GLUCOCORTCOIDS ON AVIAN OVARIAN FUNCTION...14 2.6 REGULATION OF GLUCOCORTICOID SECRETION VIA CIRCADIAN RHYTHMS...16 2.7 ENZYMATIC REGULATION OF CORTICOSTERONE PRODUCTION & ACTIVITY...17 2.8 THE GLUCOCORTICOID RECEPTOR...18 2.9 SUMMARY...20 CHAPTER III INTENSIVE VS. EXTENSIVE COMMERCIAL EGG PRODUCTION MANAGEMENT SYSTEMS...21 3. COMMERCIAL EGG PRODUCTION MANAGEMENT METHODS...22 3.1 STRESSORS IMPACTING EGG PRODUCTIVITY & EGG QUALITY... 22 3.2 DEVELOPMENT OF INTENSIVE COMMERCIAL EGG PRODUCTION PRACTICES...23 3.3 INTENSIVE COMMERCIAL EGG PRODUCTION MANAGEMENT SYSTEMS IN THE UNITED STATES...24 3.4 CHALLENGES ASSOCIATED WITH INTENSIVE COMMERCIAL EGG PRODUCTION MANAGEMENT SYSTEMS...25 3.5 DEVELOPMENT OF EXTENSIVE ALTERNATIVE MANAGEMENT SYSTEMS FOR U.S. COMMERCIAL EGG PRODUCTION...27 3.6 CHALLENGES ASSOCIATED WITH ALTERNATIVE MANAGEMENT SYSTEMS FOR COMMERCIAL EGG PRODUCTION...28 3.7 IMPACT OF GROUP SIZE...28 3.8 FREEDOM OF MOVEMENT...29

vi 3.9 COMPLEXITY OF THE ENVIRONMENT...30 3.10 HYGIENE...30 3.11 AIR QUALITY...31 3.12 ENVIRONMENTAL EXPOSURE TO BACTERIA, VIRUSES, & PARASITES...32 3.13 SUMMARY...35 CHAPTER IV ASSESSMENT OF LAYER HEN STRESS AND WELFARE...36 4. ASSESSMENT OF STRESS IN LAYER HENS...37 4.1 CURRENT METHODS USED TO ASSESS STRESS IN LAYER HENS...37 4.2 ENVIRONMENT BASED VS. ANIMAL BASED PARAMETERS...37 4.3 INVASIVE SAMPLING METHODS...38 4.4 IMMUNE FUNCTION ASSESSMENT...38 4.5 BLOOD PLASMA CORTICOSTERONE MEASUREMENTS...40 4.6 SCORING OF INTEGUMENT...40 4.7 POST-MORTEM STUDIES...42 4.8 RECENT DEVELOPMENT OF NON-INVASIVE METHODS TO ASSESS LAYER HEN WELFARE...44 4.9 EGG CORTICOSTERONE CONCENTRATIONS...44 4.10 BEHAVIOR, SPACE & RESOURCE UTLIZATION, & ELECTORNIC MONITORING...45 4.11 SUMMARY...47

vii CHAPTER V FREE-RANGE VS. CAGED REARING PARRAMETERS & SINGLE CYCLE EGG PRODUCTION PERFORMANCE...49 5. A COMPARATIVE EXAMINATION OF REARING PARAMETERS & LAYER PRODUCTION PERFORMANCE FOR BROWN EGG-TYPE PULLETS GROWN FOR EITHER FREE-RANGE OR CAGE PRODUCTION..50 5.1 SUMMARY...51 5.2 DESCRIPTION OF PROBLEM...51 5.3 MATERIALS AND METHODS...53 5.3.1 Pullet Rearing...53 5.3.2 Single Cycle Egg Production Performance...57 5.3.3 Statistical Analysis...57 5.4 RESULTS AND DISCUSSION...57 5.4.1 Pullet Age...57 5.4.2 Rearing Environment...58 5.4.3 Single Cycle Egg Production Performance...59 CHAPTER VI FREE-RANGE VS. CAGED HUMORAL IMMUNE FUNCTION...65 6. A COMPARISON OF HUMORAL IMMUNE FUNCTION IN RESPONSE TO A KILLED NEWCASTLE S VACCINE CHALLENGE IN CAGED VS. FREE-RANGE HY-LINE BROWN LAYERS...66 6.1 ABSTRACT...67 6.2 INTRODUCTION...68 6.3 MATERIALS AND METHODS...72 6.3.1 Pullet Rearing...72

viii 6.3.2 Killed Newcastle s Vaccine Challenge & Heterophil:Lymphocyte Ratios...75 6.3.3 Statistical Analysis...76 6.4 RESULTS...76 6.4.1 Humoral Immune Response...76 6.4.2 Heterophil:Lymphoctye Ratios...76 6.5 DISCUSSION...77 CHAPTER VII GLUCOCORTICOID RECEPTOR EXPRESSION IN THE OVARIAN FOLLICLES OF FREE-RANGE VS. CAGED LAYING HENS...81 7. A COMPARATIVE EXAMINATION OF GLUCOCORTICOID RECEPTOR EXPRESSION IN THE OVARIAN FOLLICLES OF HY-LINE BROWN LAYER HEN FLOCK MATES REARED IN A FREE-RANGE OR CAGE PRODUCTION ENVIRONMENT...82 7.1 ABSTRACT...83 7.2 INTRODUCTION...84 7.3 MATERIALS AND METHODS...87 7.3.1 Pullet Rearing...87 7.3.2. Animals...90 7.3.3 Tissue Collection...91 7.3.4 Primer/Probe Design...91 7.3.5 RNA Extraction & Two-Step Real-Time Polymerase Chain Reaction...92 7.3.6 Quantification of the Real-time PCR products...92 7.3.7 Statistical Analysis...93 7.4 RESULTS...93

ix 7.4.1 GR Expression in F1-F4 Theca & Granulosa Free-Range vs. Caged Tissue Samples...93 7.4.2 Combined Theca & Granulosa Free-Range vs. Caged Tissue Samples...93 7.5 DISCUSSION...94 REFERENCES...99

x LIST OF TABLES Table 1. Effect of pullet age and rearing environment on pullet weight, gain, feed conversion and total feed consumption...62 Table 2. Effect of rearing environment on livability and flock uniformity...63 Table 3. Single cycle egg production performance parameters...63 Table 4. Single cycle egg production distribution of egg grades, checks, and Losses...63 Table 5. Single cycle distribution of egg weights and sizes...64 Table 6. Single cycle production egg quality parameters...64 Table 7. Free-Range vs. Caged Newcastle s Disease (ND) Antibody Titres...80 Table 8. Table 9. Free-Range vs. Caged H:L Ratios in Response to a Killed Newcastle's Vaccination Challenge...80 Sequences for the oligonucleotide primer pairs and probes for real time PCR...96

xi LIST OF FIGURES Figure 1. The relative expression of GR mrna as determined by real time PCR using total RNA isolated from free-range and caged hens theca tissues of the F1-F4 hierarchical follicles..97 Figure 2. The relative expression of GR mrna as determined by real time PCR using total RNA isolated from free-range and caged hens granulosa tissues of the F1-F4 hierarchical follicles.98 Figure 3. The relative expression of GR mrna as determined by real time PCR using total RNA isolated from free-range and caged hens combined granulosa/theca tissues of the small yellow (SY) non-hierarchical follicles...99 Figure 4. The relative expression of GR mrna as determined by real time PCR using total RNA isolated from free-range and caged hens combined granulosa/theca tissues of the large white (LWF) non-hierarchical follicles..100

1 CHAPTER I STRUCTURE, DEVELOPMENT, & REGULATION OF THE DOMESTIC HEN S REPRODUCTIVE SYSTEM

2 1. THE DOMESTIC HEN S OVARY 1.1 STRUCTURE OF THE OVARY The laying hen s ovary is a particularly useful biological model for the study of follicular maturation and differentiation, selection, and ovulation due to its unique properties. One of the most prominent differences in the development of the hen s ovary in comparison to mammalian species, is the development of only the left ovary as a result of the inhibitory effects of Mullerian Inhibiting Substance (Mishra, et al., 2005), a glycoprotein growth factor produced by male and female gonads of avian and mammalian species during early embryological development (Hutson, et al., 1981). In mammalian male embryos, MIS prevents the formation of the Mullerian ducts, which later develop into the fallopian tubes. In the laying hen, however, MIS leads to the regression of the right ovary while the left ovary reaches full maturation (Hutson, et al., 1981). The development of the left ovary is protected from the effects of MIS, due to estrogen s suppressive action on MIS in conjunction with increased expression of estrogen receptors on the left ovary (Hutson, et al., 1982; Johnson, et al., 2008; Teng, 2001). In addition to the unique asymmetrical development of the avian ovaries, the hen s ovary also serves as an excellent biological model due to the large size of the ovarian follicles, which allows for easy visualization of a follicle s stage of maturation based upon differences in size. The avian ovary consists of several pools of follicles connected and innervated by a follicular stalk to the ovarian tissue (Johnson, 1990a). The ovary contains 5 to 8 large yellow-yolk filled follicles that are arranged in a hierarchy on the basis of size, which inherently represents the sequence in which the follicles will be ovulated. Each of

3 these large pre-ovulatory hierarchical follicles consists of yellow yolk and has a diameter ranging from approximately 12-40 mm, based upon their stage of maturation. The largest of the pre-ovulatory follicles is designated as the F1 follicle and will be ovulated within the next 24 hours. Subsequent to the F1 follicle, the F2 follicle is the next largest in size and the remaining pre-ovulatory hierarchical follicles are named in a subsequent fashion. After the F1 follicle is ovulated, the F2 follicle continues to mature and is now designated as the F1 follicle. Similarly, the succeeding follicles in the hierarchy each advance one place and an additional follicle is recruited into the hierarchy from a pool of small, yellow, nonhierarchical follicles each time an F1 follicle is ovulated. The small, non-hierarchical follicles are arranged in a pool of small, yellow-yolk filled follicles (SYF) that are approximately 5-12 mm in diameter and a pool of white follicles that are less than 5 mm in diameter. The white follicles consist of white yolk and are further classified by size into two groups: the large white follicles (LWF, 2-5 mm) and the small white follicles (SWF, < 2 mm). Recruitment of follicles into the hierarchy occurs following each consecutive ovulation of the F1 follicle. One follicle is recruited from the pool of SYF and thus enters the group of pre-ovulatory hierarchical follicles that are destined for ovulation while several LWF advance to become SYF. Follicular recruitment into the hierarchy is, however, a highly selective process. In fact, it has been estimated that for every 20 ovarian follicles in the hen s ovary that grow to a size of 6-8 mm in diameter, only one will be selected into the pre-ovulatory hierarchy (Gilbert, et al., 1983). The remaining follicles will undergo follicular atresia (Gilbert, et al., 1983).

4 Follicular atresia is defined as the break down or reabsorption of the ovarian follicle and is normally characterized by rupture of the follicular wall (Johnson, 1990b). 1.2 HISTOLOGICAL ORGANIZATION OF THE OVARIAN FOLLICLES Each ovarian follicle consists of several concentric layers of tissue. The theca and granulosa tissues comprise the outermost layers of the follicle with a basal lamina separating them. The theca layer, or most external layer, can be divided into the theca externa and theca interna, both of which contain much of the follicle s vasculature. Deep in the theca interna lays a basement membrane that separates the theca interna from the granulosa cell layer. Beneath the granulosa cells is the perivitelline substance followed by the plasma membrane of the ovum (Sturkie, 2000). In the white-yolk containing non-hierarchical follicles the theca layer is less developed while the granulosa cells comprise several layers. In contrast, yellow-yolk containing hierarchical follicles have a monolayer of granulosa cells that can be easily dissected away and separated from the theca cell layer. The granulosa cells of nonhierarchical SYF and LWF are arranged in several densely packed layers and are cuboidal in shape (Etches, 1990). As the follicles mature and grow, granulosa cells spread out significantly, leaving hierarchical follicles with a granulosa cell layer that is only one cell thick. The histological structure of the hen s pre-ovulatory hierarchical follicles allows for the manual separation of the theca and granulosa tissues with ease and minimal contamination (Hernandez, 2001) making it an ideal model for the conduction of expression studies that are tissue specific in avian species.

5 Such studies are not as feasible in mammals where the theca and granulose cells of the follicle are more intermixed and not easy to separate manually. The germinal disc region (GDR) of the pre-ovulatory hierarchical follicles is also easily identified as it is white in color and stands out on the surface of the yellow-yolk containing follicles. The GDR consists of the germinal disc, also known as the blastodisc, where sperm will penetrate during fertilization and the embryo will begin to form, in addition to the granulosa cells that lie over it. The GDR of pre-ovulatory hierarchical follicles contains all of the hen s genetic material needed for procreation. Destruction of the GDR induces apoptosis and atresia of the follicle, suggesting that the GDR produces a factor that is essential for ovulation and follicle viability (Hernandez, 2001). Apoptosis is defined as programmed cell death and is the mechanism by which the granulosa cells of an ovarian follicle die off and detach from the basal lamina. The oocyte of each immature follicle dies and at this point it is considered to have undergone atresia at which point it is termed an atretic follicle. Eventually the entire structure undergoes phagocytosis and resorption.

6 1.3 FOLLICULAR DEVELOPMENT Maturing follicles undergo vitellogenesis, or yolk accumulation, which occurs in three phases: 1) a slow growth phase occurs in follicles 60-100 µm in diameter over the course of several months and possibly years whereby the yolk deposited into the follicle consists mainly of white neutral lipids to comprise the large white follicles, 2) an intermediate growth phase where the diameter of the follicle increases from 2 to 6 mm as yolk protein begins to be deposited into the follicle over a period of 60 days and the follicle matures into a small yellow follicle, and 3) a rapid growth phase, which occurs during the final days before ovulation when the bulk of yellow yolk consisting of nutrients, proteins, and lipids is deposited into the follicle and the diameter and volume of the follicle increase significantly per day (Johnson, 1990b). The number of follicles in the rapid growth phase remains consistent as these follicles make up the hierarchical follicles. A follicle in the intermediate growth phase only enters the hierarchy and the rapid growth phase after the largest follicle (F1) is ovulated (Zakaria, et al., 1984). Regulation of growth, differentiation, and ovulation of avian pre-ovulatory hierarchical and non-hierarchical follicles is dependent upon the timely release of the gonadotropin hormones: follicle stimulating hormone (FSH) and luteinizing hormone (Von Engelhardt and Groothius) from the anterior pituitary gland. LH primarily acts upon the F1 and F2 follicles in order to prepare these follicles for ovulation, while the F3-F5 follicles undergo significant follicular growth as a result of FSH stimulation (Hernandez, 2001).

7 1.4 REPRODUCTION: OVULATION OF THE OVARIAN FOLLICLES Avian species serve as excellent biological models for reproductive studies in comparison to mammalian species due to their consecutive ovulations, which occur, depending upon the species, approximately every 24 or 48 hours. Ovulation is easily predicted in domesticated poultry because it occurs approximately 20-40 minutes after ovipositioning, or egg laying. Similar to maturation and proliferation, ovulation is highly dependent upon the regulatory activities of gonadotropin releasing hormone (GnRH) and its stimulatory effect on FSH and LH, all of which are secreted as part of the hypothalamicpituitary-gonadal (HPG) axis. The HPG axis is comprised of the synergistic relationship between the hypothalamus, the anterior pituitary gland, and the ovaries (female gonads). The HPG axis modulates growth and ovulation through the interaction of steroids and hormones secreted by these three components. Ovulation is induced by a rise in FSH secretion from the anterior pituitary gland 12-16 hrs prior to ovulation (Scanes, et al., 1977) and a pre-ovulatory surge of LH also from the anterior pituitary gland 4-6 hrs prior to ovulation(johnson and van Tienhoven, 1980). The LH surge is accompanied by simultaneous increases in progesterone (P4) and estrogen secretion from the F1 follicle (Etches, 1990).

8 1.5 THE HYPOTHALAMIC-PITUITARY-GONADAL (HPG) AXIS The hypothalamic-pituitary-gonadal axis (HPG) plays an essential role in regulating female reproduction as it mediates follicular maturation, development, and ovulation (Chrousos, et al., 1998). Gonadotropin releasing hormone (GnRH) also formerly known as luteinizing hormone releasing hormone (LHRH) is synthesized and stored in the hypothalamus. Specifically, GnRH is secreted by neurons in the pre-optic and arcuate nuclei of the hypothalamus into the hypophyseal portal system, or blood vessels, which link the hypothalamus and the pituitary gland (Chrousos, et al., 1998). When secreted, GnRH binds to its receptors at the anterior pituitary gland stimulating the synthesis and release of FSH and LH. At the level of the ovary, FSH regulates granulosa cell differentiation and has been detected in the follicle 15 hours prior to ovulation in the domestic hen (Scanes, et al., 1977). LH administration has been found to increase plasma concentrations of progesterone, androgens, and estrogens and can result in follicular atresia or premature ovulation of follicles, depending upon the follicle s position within the ovulatory sequence (Gilbert, et al., 1983). In addition to acting on several target tissues in the reproductive and central nervous system, the gonadal steroids secreted from the follicles: progesterone (granulosa cells), androgens and estrogens (theca cells) exert feedback at the level of the anterior pituitary gland further mediating release of FSH and LH (Chrousos, et al., 1998).

9 Due to the detection of increased LH plasma concentrations in hens 4-7 hours prior to ovulation in conjunction with increased levels of progesterone (Lague, et al., 1975), researchers were led to investigate the relationship between increased levels of gonadal steroids and the pre-ovulatory release of LH in the laying hen (Wilson and Sharp, 1976). Intramuscular injections of progesterone and estrogen in ovariectomized hens were observed to exert a positive feedback action on LH secretion, leading to the conclusion that LH release is facilitated by the combined actions of estrogen and progesterone(wilson and Sharp, 1976). The HPG axis and its effects on ovulation and follicular maturation are not only modulated by feedback from the gonadal steroids but also by interactions with the hypothalamic-pituitary-adrenal (HPA) axis, also known as the stress axis (Gore, et al., 2006).

10 1.6 SUMMARY Overall, the histological structure and hierarchy of the hen s ovary provide an excellent paradigm for studying the modulation of follicular selection, growth, development, survival and atresia, due to the highly regulated and visually apparent avian follicular hierarchy and the ease with which the histological structure can be manipulated and analyzed. Understanding the growth phases of follicular development, and the influence of the hormones, which regulate maturation and ovulation of the avian ovarian follicles leads to analysis of the relationship between the hormones of the HPG axis and their collaborative roles in influencing reproduction. In order to fully understand how gonadal function is modulated, it is important to examine the role of stress mechanisms in homeostasis and in regulating the HPG axis. These factors are essential in order to comprehend the impact of stress on ovarian function.

11 CHAPTER II INTERACTION & DYSFUNCTION OF THE HYPOTHALAMIC-PITUITARY GONADAL (REPRODUCTIVE) & HYPOTHALAMIC-PITUITARY-ADRENAL (STRESS) AXES

12 2. STRESS 2.1 THE HYPOTHALAMIC-PITUITARY-ADRENAL (HPA) AXIS The hypothalamic-pituitary-adrenal axis (HPA axis) involves interactions among the hypothalamus, the anterior pituitary gland, and the adrenal glands (Harvey, et al., 1984). Interaction of these endocrine glands regulates the body s neuroendocrine response to stress and influences essential bodily functions such as digestion, immunity, reproduction, and metabolism of nutrients (Harvey and Hall, 1990). Corticotropin Releasing Hormone (CRH) is released from the hypothalamic neurons in response to stressors (Harvey and Hall, 1990). CRH stimulates the release of adrenocorticotropic hormone (ACTH) from the anterior pituitary gland. As a result, CRH and ACTH directly regulate daily variation in the production of glucocorticoids; cortisol (in mammalian species) or corticosterone (in avian species) at the level of the adrenal glands (Carsia and Harvey, 2000), which are located on top of the kidneys. In turn, these glucocorticoids may exert negative feedback control on the hypothalamic release of CRH and the anterior pituitary gland s release of ACTH. 2.2 INTERACTION OF THE HYPOTHALAMIC-PITUITARY-ADRENAL & HYPOTHALAMIC-PITUITARY-GONADAL AXES In addition to regulation of the HPA axis, increased plasma concentrations of glucocorticoids have been found to be inhibitory to reproductive function in many species (Gore, et al., 2006) indicating that the stress axis modulates the normal functions of the HPG axis.

13 Other studies have found that administration of ACTH and dexamethasone, a synthetic glucocorticoid, can cause an increase and decrease, respectively, in plasma corticosterone concentrations in the laying hen (Etches and Cunningham, 1976). 2.3 DYSFUNCTION OF THE HYPOTHALAMIC-PITUITARY-ADRENAL AXIS & IMPACTS ON THE HYPOTHALAMIC-PITUITARY-GONADAL AXIS Research suggests that the HPA axis plays an important role in regulating reproduction through a variety of endocrine regulatory points (Dobson and Smith, 2000). For example, stress can hinder reproductive function by inhibiting or interfering with the timing of reproductive hormone release in the HPG axis (Dobson and Smith, 2000). Reproductive hormones or gonadotropins such as LH and FSH, which have the ability to affect growth of the gonads are essential in the process of steroidogenesis at the level of the ovary (Johnson, 1990b). LH stimulates androstenedione production in theca cells and using theca cells as a substrate, androstenedione is aromatized into estrogens and androgens. Studies in Wistar rats have shown that hypo- and hyper-corticosteronisms can impair ovarian endocrine and exocrine function (Valli, et al., 2000). Corticosterone administered to Wistar rats resulted in a decrease in FSH, LH, and estradiol levels (Valli, et al., 2000). Such interference occurs at the level of the hypothalamus when secretion of corticotrophin releasing hormone (CRH) from the hypothalamus exerts an inhibitory effect on GnRH secretion, blocking secretion of LH and FSH from the anterior pituitary gland and limiting production of progesterone and estrogen at the level of the ovary (Chrousos, et al., 1998; Matsuwaki, et al., 2006; Saketos, et al., 1993).

14 As a result, limited production of progesterone and estrogen leads to disruptions in functional follicular maturation (Kawate, et al., 1992; Tetsuka, 2007). In addition to exerting an effect at the level of the hypothalamus, the HPA axis can also inhibit the HPG axis at the level of the target tissues (Chrousos, et al., 1998). The avian ovary is not able to produce corticosterone de novo, and as a result, corticosterone that impacts the ovary is synthesized in the adrenal cortex and secretion of it is mediated by the hypothalamic-pituitary-adrenal axis (Omura and Morohashi, 1995). Anatomically, glucocorticoid receptors and ovarian CRH receptors, in addition to components of the HPA axis are located near reproductive tissues and modulate their activity (Chrousos, et al., 1998). Due to the close anatomic location of the adrenal gland to the left ovary of the laying hen, the left ovary is highly innervated by the adrenal gland, which further suggests interactions between the two (Etches, et al., 1984a). When high levels of corticosterone are induced from lack of food and water (dietary stress) or infused daily, ovarian regression is observed and a decrease in the gonadotropin, LH is also observed (Etches, et al., 1984b). The total number of large yellow-yolk filled hierarchical follicles decreases and the number of atretic, pre-hierarchical follicles increases (Etches, et al., 1984b).

15 2.4 DYSFUNCTION OF THE HYPOTHALAMIC-PITUITARY-GONADAL AXIS As the laying hen ages, and levels of gonadotropin hormones fluctuate, dysfunction of the hypothalamic-pituitary-gonadal axis may occur (Williams and Sharp, 1978). LH positive feedback mechanisms normally modulate the release of progesterone, but as ovaries age and lose function, regulating steroids such as estrogen and progesterone are less concentrated in the blood to feedback into the HPG axis, resulting in lowered levels of LH (Williams and Sharp, 1978). Lowered function of the LH positive feedback mechanism is thought to be responsible for the fall in the rate of lay towards the end of the hens laying cycle (Williams and Sharp, 1978). Ovariectomized hens showed lowered function of the LH positive feedback mechanism until injected with proper doses of progesterone and estrogen (Williams and Sharp, 1978). This led researchers to conclude that even with the onset of age, the ovary may potentially be fully functional and that changes in the rate of lay and fluctuations of gonadotropin levels may also be due to functional changes in the central nervous system (HPG axis) (Williams and Sharp, 1978). 2.5 INFLUENCE OF GLUCOCORTCOIDS ON AVIAN OVARIAN FUNCTION Due to the significant interactions between the HPA and HPG axes, it is important to understand the role of corticosterone, the avian glucocorticoid, in avian ovulation and egg production. Glucocorticoids affect ovarian function through three primary modes: (1) glucocorticoids act on the HPG axis and exert an inhibitory effect at the level of the anterior pituitary gland (Dobson and Smith, 2000).

16 (2) Glucocorticoids may influence levels of ovarian growth factors and metabolic hormones, which are essential to ovarian development and function (Thakore and Dinan, 1994);(Kritsch, et al., 2002) and (3) glucocorticoids can exert direct effects at the level of the ovary(tetsuka, 2007). The effects of glucocorticoids at the level of the anterior pituitary gland can result in disruption of follicular maturation due to increased amounts of progesterone and estrogen in the theca and granulosa tissues of the follicle. Additionally, receptors in the ovary for the enzyme P450 aromatase, involved in estrogen biosynthesis, and LH are necessary prerequisites for proper follicular maturation (Tetsuka, 2007). Glucocorticoids have been shown to suppress the expression of the enzyme P450 aromatase (Hsueh, 1978) and LH receptors (Schoonmaker and Erickson, 1983) in cultured rat granulosa cells, impacting follicular growth and development. Similar results have been found in cultured bovine (Kawate, et al., 1992) and swine (Danisova, et al., 1987)granulosa cells. Steroidogenesis occurs primarily in the granulosa and theca cells of the ovarian follicle (Johnson, 1990b). The granulosa cells are considered to be the primary source of secreted progesterone. In contrast, the theca cells produce estrogens and androgens (Johnson, 1990b). Glucocorticoids are believed to interfere in this process by suppressing the aromatization of androstenedione into estrogen in cultured rat granulosa cells (Hsueh, 1978). Glucocorticoids were also found, on the other hand, to stimulate the production of progesterone in cultured rat granulosa cells and in bovine granulosa and theca cells (Hsueh, 1978; Kawate, et al., 1992; Spicer and Chamberlain, 1998).

17 These results suggest that glucocorticoids cause both inhibitory and stimulatory effects at the level of the ovary (Hsueh, 1978). Because increased levels of progesterone play a role in the induction of ovulation (Richards, et al., 2002) these results also suggest that glucocorticoids play a more important role in ovulation than in follicular maturation (Tetsuka, 2007). Glucocorticoids can also affect the ovulatory process by inhibiting the synthesis and expression of enzymes produced by follicles, which are essential for ovulation, such as prostaglandin E2 (PGE2) (Davis, et al., 1999), and enzymes such as phospholipase A2 and cyclooxygenase 2 that are responsible for PGE2 production (Ben-Schlomo, et al., 1997). According to these findings, excessive concentrations of glucocorticoids may thus block ovulation (Tetsuka, 2007). Additionally, glucocorticoids exert a direct affect on the ovary by playing a role in mediating the processes of follicular rupture and apoptosis. Follicular rupture can occur when proteases act on the follicular wall. Tissue-type plasminogen (tpa) is an example of a protease that glucocorticoids have been shown to increase activity and expression of in cultured rat granulosa cells (Wang and Leung, 1989). Alternatively, confounding studies have found that glucocorticoids prevented apoptosis in human and rat granulosa cells (Amsterdam and Sasson, 2002); (Sasson, 2003), while also inducing apoptosis in invading leukocytes, which protect follicular cells (Amsterdam and Sasson, 2002).

18 These findings have led researchers to suggest that glucocorticoids may be involved in minimizing damage caused by the inflammatory ovulatory process by promoting repair and luteinization of ovulated follicles (Hillier and Tetsuka, 1998); (Tetsuka, 2007). 2.6 REGULATION OF GLUCOCORTICOID SECRETION VIA CIRCADIAN RHYTHMS Previous studies have shown that hens maintained on a constant lighting schedule (e.g. 14 hours of light and 10 hours of darkness) exhibit pre-ovulatory surges of LH during the period of darkness, suggesting circadian regulation of ovulation (Wilson and Sharp, 1973);(Williams and Sharp, 1978). Corticosterones may be part of the mechanism responsible for modulating the responsiveness of the hypothalamus to circadian rhythms due to findings that include a daily pattern of circulating corticosterone levels in chickens(seigel, et al., 1976);(Etches, 1979);(Johnson and van Tienhoven, 1980);(Wilson and Cunningham, 1981);(Wilson, et al., 1984). In hens exposed to 14 hours of light and 10 hours of darkness, a surge of corticosterone was detected 2 hours preceding ovulation during the dark period and was concluded to be due to coincident ovipositioning (Johnson and van Tienhoven, 1980). Another minimally invasive study on circadian rhythms involved the insertion of a cannula into individual hens to take blood samples of corticosterone, which showed a clear, daily rhythm of corticosterone (Beuving and Vonder, 1977).

19 In studies using metyrapone, a drug that blocks corticosterone synthesis, to manipulate the effectiveness of exogenous adrenocorticotropic hormone (ACTH), results suggested that environmental stimuli such as light can act via the adrenal gland to modulate the timing of the pre-ovulatory release of LH in the hen, therefore demonstrating the possible inverse relationship between plasma concentrations of LH and corticosterone (Wilson and Cunningham, 1980). 2.7 ENZYMATIC REGULATION OF CORTICOSTERONE PRODUCTION & ACTIVITY The ovary modulates normal levels of active glucocorticoids locally during follicular maturation and ovulation with two isoforms of the enzyme 11β-hydroxysteroid dehydrogenase (11β-HSD1 and 11β-HSD2), which catalyze the conversion between active and inactive glucocorticoids. 11β-HSD1 is an oxidoreductase which is expressed in the liver, adipose tissue, brain, lung, vasculature, ovary, central nervous system and other glucocorticoid target tissues (Monder, 1993) and (Stewart and Krozowski, 1999). In order for 11β-HSD1 to modulate glucocorticoid activity, it must be activated when there is a substantial pool of substrate 11-ketosteroids (cortisone, 11-dehydrocorticosterone) available which are inactive precursors to glucocorticoids. The oxo-reductase activity of 11β-HSD1 increases levels of active glucocorticoids during ovulation while in contrast the dehydrogenase activity of 11β-HSD2 suppresses active glucocorticoids during follicular maturation (Tetsuka, 2007).

20 11β-HSD2 is expressed in aldosterone-selective target tissues such as the distal nephron, colon, sweat glands, and the placenta (Seckl, 2001) and has been shown to be highly down regulated in human and rat granulosa cells during the ovulatory process triggered by the HPG axis while 11β-HSD1 is up-regulated (Tetsuka, 1997), (Tetsuka, 1999). The presence of both isoforms has been characterized in various ovarian tissues of humans(tetsuka, 1997);(Rae, 2004a);(Albiston, et al., 1994), rats(benediktsson, et al., 1992); (Roland and Funder, 1996);(Waddell, et al., 1996);(Tetsuka, 1999), mice (Condon, et al., 1997) and cattle (Tetsuka, et al., 2003). 2.8 THE GLUCOCORTICOID RECEPTOR Glucocorticoids modulate ovarian function by binding to glucocorticoid receptors (GRs). The fact that glucocorticoids can be inhibitory and stimulatory at the same time in ovarian tissue may be explained by identifying the GR as the mediator of glucocorticoid action in the ovary (Schreiber, et al., 1982). Therefore analysis and characterization of the expression of functional GRs in target tissues can be a direct measurement of the potential action of glucocorticoids on that tissue (Bamberger, et al., 1995). The GR is a type I Nuclear Receptor, member of subfamily 3, group C, member 1 that binds the mammalian glucocorticoid, cortisol, and the avian glucocorticoid, corticosterone. GR expression has been characterized in human (Rae, 2004a), and rat (Schreiber, et al., 1982); (Waddell, et al., 1996); (Tetsuka, 1999); (Towns, et al., 1999) ovarian follicles and corpora lutea.

21 The complete cdna for mammalian GR in the human (Hollenberg, et al., 1985), mouse (Francke, 1980), rat (Miesfeld, et al., 1986), and rabbit (James, 2003) has been characterized and cloned in addition to teleost fish such as trout (Gao, et al., 1994). The entire cdna for avian species GR had not been cloned until the cdna for chicken GR was cloned and characterized in its entirety from the chicken kidney by (Kwok, et al., 2007). GR was detected and found to be widely expressed in 12 avian tissues: brain, pituitary, lung, heart, liver, kidney, intestine, pancreas, breast muscle, spleen, testis and the ovary (Kwok, et al., 2007). Relatively high expression of GR was detected in pituitary, muscle, ovary, and kidney tissues (Kwok, et al., 2007). While tissue specific characterization of GR in the chicken ovary has yet to be attempted, the GR has been characterized in the follicles and corpus luteum of the human (Rae, 2004b) and rat (Schreiber, et al., 1982); (Waddell, et al., 1996); (Tetsuka, 1999); (Towns, et al., 1999). In humans, the GR was characterized in the ovarian surface epithelial cells (Rae, 2004b) while in the rat, GR mrna was detected mainly in pre-antral follicle granulosa cells (Schreiber, et al., 1982). Additionally, bovine GR mrna has been detected in granulosa and theca tissues (Tetsuka, 2010). This study also examined differences in expression levels of GR in dominant and small developing follicles, and even though GR expression in granulosa and thecal tissues did not differ between different types of bovine follicles (Tetsuka, 2010), it is important to continue to examine for this possibility. 2.9 SUMMARY

22 The HPA (stress) axis regulates normal reproduction by modulating the release of hormones within the HPG axis. Dysfunction of either of these two axes can occur for a variety of reasons and can impact the level of hormones released that may act directly on target tissues of the ovary. Glucocorticoids, the primary class of stress hormones released by way of the HPA axis, affect ovulation and egg production by modulating the HPG axis and influencing the levels of growth factors and hormones acting on the ovary. Secretion of glucocorticoids via the HPA axis is regulated via circadian rhythms and enzymatic activity. In order to better understand the influence of glucocorticoids in modulation of gonadal function, characterization of the glucocorticoid receptor has occurred in a variety of species in several tissues including the reproduction tract. Further tissue specific characterization is necessary in the laying hen s hierarchical and non-hierarchical follicles in order to examine the role of glucocorticoids in regulation of follicular recruitment and development. Additionally, examination of differences in GR expression in hens reared in different environmental management methods may give insight into how such management methods impact reproductive fitness due to potentially adverse effects of glucocorticoids on follicular development, maturation, and ovulation.

23 CHAPTER III INTENSIVE VS. EXTENSIVE COMMERCIAL EGG PRODUCTION MANAGEMENT SYSTEMS

24 3. COMMERCIAL EGG PRODUCTION MANAGEMENT METHODS 3.1 STRESSORS IMPACTING EGG PRODUCTIVITY & EGG QUALITY In commercial egg production, a variety of stressors have been found to significantly impact the laying hen s adrenocortical responses, thereby influencing reproduction and egg productivity. Common stressors include: physiological stressors due to rapid growth resulting from genetic selection (Freeman, 1987), feeding and nutritional stressors due to shortage of nutrients or feed intake issues (Ben-Nathan, et al., 1981; Etches, et al., 1984b), social and psychological stressors resulting from pecking order and associated fear behaviors (Beuving and Vonder, 1981; Gross and Siegel, 1981), physical stressors from handling,, transportation, and injections (Gregory, et al., 1992; Jones, et al., 1988), and environmental stressors such as changes in photoperiod (Bédécarrats, et al., 2009) and environmental management methods (Saino, et al., 2005; Webster, 2004). Of particular recent concern is the impact of environmental management methods and living conditions on population viability, productivity, and overall welfare of layer hens (Saino, et al., 2005). Stress-induced ovarian dysfunction is of major concern to the poultry industry due to the economic costs that can ensue due to poor productivity and quality in addition to a significant increase in public scrutiny concerning the welfare status of poultry. As a result of such scrutiny, numerous studies have been conducted to assess layer hen welfare in different housing environments. It is necessary, however, to first understand the development and history of the most commonly used environmental management methods in commercial egg production, and how they came into practice, and their impact on the poultry industry.

25 3.2 DEVELOPMENT OF INTENSIVE COMMERCIAL EGG PRODUCTION PRACTICES The transition from backyard coops to intensive production practices occurred as the human population and the demand for food increased and small family farms utilizing small scale, extensive production techniques were unable to meet these demands in a costeffective manner (UEP, 2010). Backyard flocks remained in use throughout the 1940s, where it was customary to use laying chickens for meat after they had a decline in egg production levels. Eggs were not sold fresh in the winter since backyard hens went into molt during the cold months and ceased laying. Production numbers were relatively low, due partially to higher mortality rates among backyard flocks because of exposure to disease and the elements, as well as high rates of contamination of produce as a result of lacking microbial control methods (UEP, 2010). As urbanization took over, flock managers began to find quicker and more economical ways to supply eggs to growing cities. Commercial poultry houses were developed to protect birds from environmental extremes that adversely affected growth rates, immune function, and fertility and egg production. With the advent of the modern cage system and large scale, intensive production, came optimized bird welfare and labor through mechanization, which greatly reduced labor costs, improved stocking density of birds, and allowed for larger production units (Abrahamsson and Tauson, 1998; UEP, 2010).

26 New modern caged housing was also increasingly cost-effective due to a lower incidence of cannibalism (Hilbrich, 1985), disease, dust and ammonia levels (Engstrom and Schaller, 1993); and (Hauser, 1988), as well as better management of flock nutrition and more effective waste management which provided cleaner eggs for safer consumption (UEP, 2010). 3.3 INTENSIVE COMMERCIAL EGG PRODUCTION MANAGEMENT SYSTEMS IN THE UNITED STATES Intensive practices were developed, in part, to save land resources and to manage birds in the most efficient manner possible. In 2010, 95% of commercial egg production in the U.S. and 90% of the world s egg production was obtained from caged layers, while the remaining 5% of eggs produced in the U.S. were derived from alternative systems of management, which include cage free and free-range systems. Conventional battery style and stair step cages are made of wire or plastic mesh and are stacked in three to five tiers. Under UEP certified guidelines for cage production, cages are required to allow the bird to fully stand upright and on a floor, which is not sloped more than 8 degrees. Each cage should allow for 67 to 86 square inches of usable space per bird in order to allow for natural behaviors to occur. Birds should also have continuous access to water and be able to feed at the same time in order to minimize aggression. Cages are classified based on two different methods of manure collection. In battery style cages, manure is collected via a belt system and must be configured such that manure from birds in upper tiers does not come in contact with lower tier cages. After collection, the manure is transported out of the barn (Chore-Time, 2007).

27 In conventional stair-step cages, there is a high-rise system where manure falls into a collection and storage area beneath the cages where it is removed one or more times per year (Chore-Time, 2007). With respect to egg collection, two primary methods exist for collecting eggs, in-line systems and off-line systems. In both systems, when the egg is laid it rolls to the front of the cage due to the slight incline in the cage and lands on a nylon belt. The belt then transports the egg out of the hen house directly to an egg processing facility (in-line) or to a storage cooler facility (off-line). At the egg processing facility, eggs are cleaned in a high temperature solution, visually inspected, and graded for packaging. Off-line eggs are stored in a cool room for 2-3 days and then transported to a similar egg processing facility via a refrigerated truck. Feeding and watering is usually automated to most efficiently deal with the large number of birds. Feed is delivered to birds along the front of cages via a chain system at specific times of the day, and water is always available via nipple drinkers in each cage, which are supplied by a pipe spanning the length of the house. 3.4 CHALLENGES ASSOCIATED WITH INTENSIVE COMMERCIAL EGG PRODUCTION MANAGEMENT SYSTEMS Despite the stringent welfare guidelines that must be met in order to maintain caged production, numerous critiques of intensive practices have led to the development of alternative management systems for commercial egg production.