Duration: 10/01/2010 to 09/30/2013

Administrative Advisor(s):

NIFA Reps:

Non-Technical Summary

Statement of Issues and Justification

The current project (S1020 Enhancing Reproductive Efficiency of Poultry) began in September of 2004. This project was not as much new in nature as it was in scope. The core group of participants had long-standing membership in a multistate project pertaining to turkey reproduction. More to the point, these scientists had belonged to a series of multistate projects approved for the purpose of advancing turkey reproductive efficiency.

Historically, state experiment stations generally included poultry scientists, and the number of such scientists per experiment station were generally greatest in states with a significant poultry industry. However, those who worked with the domestic turkey were oftentimes a minority  either at a given experiment station or on a national level. This tendency, in part, reflected the seasonal nature of the U.S. turkey industry through the 1960s. Consequently, poultry scientists who specialized in turkeys tended towards management, nutrition, and disease. At this time, turkey management meant feeding, photostimulating, and treating broody hens with techniques that were more hopeful than based on empiricism. In general, photostimulation induces egg and semen production, and turkey hens tend to function like many wild birds. Specifically, photostimulation induces lay and once a clutch of eggs is laid, a hen tends to cease ovulating and incubate her clutch. Whereas 20 weeks of age is the conventional time at which chickens are photostimulated, turkeys are usually photostimulated at 30 weeks of age. Consequently, the production cost for a turkey breeding flock is high relative to chickens due to the combined effects of feed consumption prior to onset of lay, time to peak production, and duration of lay thereafter. In addition, it must be noted that effective photostimulation enables hen-day egg production to rapidly assume a maximal value. This is critical because subsequent hen-day egg production declines from this peak. Therefore, any hen that is managed properly to this point in time but becomes photorefractory thereafter constitutes a significant production loss. In review, turkey production tended to be seasonal until further processing began in the 1970s. This practice was stimulated, in part, by a public interest in low-fat, prepared foods.

The U.S. poultry industry is characterized by intense selection and multi-generational amplification of breeding stock. This approach enables hundreds to thousands of pedigree line birds to yield millions to billions of market birds. In the case of turkey breeding, rapid gains in body weight and body conformation produced parent stock that did not copulate effectively. Consequently, commercial turkey production became dependent upon artificial insemination. Therefore, the effectiveness of artificial insemination became critical to turkey reproductive efficiency along with maximizing egg production.

To summarize, turkey production began to increase in the 1970s as poultry science departments began to decline. However, the emergence of research tools such as the radio-immunoassay, electron microscope, and enzyme-linked immunoabsorbant assay afforded powerful techniques applicable to the study of turkey reproduction. During this time, turkey researchers employed by experiment stations and the Agricultural Research Service were concentrated in the Southeast (Maryland, Virginia, North Carolina, South Carolina), Midwest (Wisconsin, Missouri), and the West (California, Oregon, Utah). Due to geographical distribution, range of expertise, and value of the U.S. turkey industry, a multistate project addressing turkey reproduction was warranted. Over time, this group made many outstanding contributions to the discipline of poultry science. However, their number had diminished by the turn of the century due to experiment station funding priorities and retirement. This demographic change is evident in Appendix E of Multistate Project S285 (Reproductive Performance in Turkeys, 1998 to 2003) versus Appendix E of Multistate Project S1020 (Enhancing Reproductive Efficiency of Poultry, 2004 to 2009). S285 involved 18 scientists of whom 67% actively conducted research with turkeys. In contrast, S1020 lists 20 scientists of whom only 25% work with turkeys, and most of these participants are near retirement.

As S285 ended, it was deemed fitting and desirable to continue the broad goals and collegiality of those who formed the core of the historical turkey effort. This was done by expanding the scope of S1020 and was based upon the following argument.

Reproduction in birds is enabled (and adversely affected) by environ-mental stimuli that affect specific groups of central nervous system neurons. These neurons, in turn, control the pituitary gland and gonads. Hormones secreted from these organs enable the development and function of reproductive tracts and affect sexual behavior. Reproductive success is determined by the following environmental variables: photoperiod, diet, ambient temperature, pathogens, toxins, as well as social interaction among birds within a flock. Furthermore, the quality of a dams egg has a pronounced effect upon the performance of her offspring during incubation as well as post-hatch. Finally, poultry reproduction is profoundly affected by genetic selection. In summary, poultry reproduction constitutes a field of study with depth and breadth that has ready application within a major U.S. agribusiness.

In this regard, the importance of the proposed work must be understood within a broad conceptual and technical context, i.e. genetics. This discipline has undergone a series of scientific revolutions since the mid-20th century. Advances include understanding the structure of DNA, gene transcription, the ability to copy and manipulate genes, genome sequencing, transcriptomics, and bioinformatics. In essence, the discipline of genetics now pertains to molecular information in addition to patterns of inheritance. It is noteworthy that three sub-disciplines would not have emerged apart from concomitant advances in computing.

To reiterate a previous point, the U.S. poultry industry depends upon intense genetic selection. Even though this assertion was true throughout the latter half of the 20th century, the technology that enables genetic selection at present either did not exist then. Furthermore, the emergence of systems biology affords a means of explaining biological processes in terms of gene networks. In this regard, the proposed work constitutes a point of application. Whereas the environmental factors affecting poultry reproduction were by and large outlined within the 20th century, the gene networks affecting reproduction will be outlined in the 21st century. This outcome is inevitable. However, where this advance occurs is another matter altogether. Historically, federal funding has empowered Land Grant and ARS scientists to study poultry reproduction. This project, if approved, would enable such scientists to build upon their accomplishments and collaborate towards a new goal: defining gene networks that enable poultry reproduction.

In review, the proposed research addresses poultry reproduction, which is a critical challenge to a major U.S. agribusiness that depends upon intense genetic selection and multi-generational amplification of breeding stock. Reproduction is a physiological process, and any physiological process can be explained in terms of cellular networks. Research performed during the 20th century served to outline these networks as well as the molecular mechanisms upon which they depend. By the turn of the century, advances in the discipline of genetics changed the practice of poultry breeding. For example, the first and second assemblies of the chicken genome were released in 2004 and 2006, respectively. The third assembly is anticipated in 2010. In addition, the release of the first assembly of the turkey genome is imminent. Such advancements change the nature of animal breeding, e.g. SNPlotype-based selection. In summary, the ultimate goal of the proposed multi-state project is a paradigm that links the reproductive process with the information inherent to DNA. This advancement will strengthen the U.S. poultry industry in a fundamental and long-term manner.

Related, Current and Previous Work

In general, the expertise of S1020 participants was commensurate with the scope of study outlined above. However, the number of active participants was not, and the issue of critical mass is compounded by the imminent retirement of several members. Nevertheless, a new project is warranted, and a rationale follows.

Based upon information provided by Mark Mirando, who is currently transitioning from the USDA-CSREES Competitive Grants Program into the new National Institute of Food and Agriculture, 45% of the S1020 participants have been awarded a grant from the NRICGP within the past 8 years. Therefore, nearly half of the current participants conduct innovative, state-of-the-art research. Even though such grantsmanship reflects independent effort, it also shows potential for substantive collective effort.

In this regard, it is proposed that a new project address fundamental questions within three broad areas: photoperiod, gametes/embryos, and the neural basis for reproductive behavior. Supporting reasons follow. Furthermore, it is proposed that collaboration  as defined by this proposal -- serve as a requirement for individual participation. Though this was one goal of S1020, it was not broadly achieved. However, it must be noted that S1020 was transitional in nature. Finally, it is proposed that collaboration be based upon the following criteria: (1) development and sharing of research techniques and resources, (2) generation or use of bioinformatic data bases, (3) assistance with statistical analyses, and (4) field application.

The research topics stated above were chosen for the following inter-related reasons. First, the role of photoperiod is central to poultry reproduction. Second, the brain controls not only the hypothalamo-pituitary-gonadal axis but the hypothalamo-pituitary-adrenal axis as well, and the latter can affect the former. The next point cannot be appreciated apart from understanding the study of poultry reproduction through time. This perspective reveals a chronology in which organs, processes, cells, signal molecules, and signal transduction mechanisms have been identified and explained. At present, we are on the verge of understanding poultry reproduction in terms of gene networks. Even though the S1020 membership includes but one geneticist, this is where the future leads. In fact, SNPlotyping and bioinformatics are already key elements of commercial poultry breeding programs.

It is also proposed that annual meetings serve, in part, to define common challenges within the U.S. poultry industry. Though this may be viewed as tangential to the purpose of multistate research projects, it is the chairpersons contention that industry input in this case could be used to build consensus and rank priorities. Such direction would enable groups of participants to draft compelling proposals for submission to the new National Institute of Food and Agricultures applied research area. In this manner, the project could be used to channel scientific expertise towards substantive problems in a timely manner. This would create a feed-forward atmosphere within the group and funding for collaborative work.

The proposed project is neither redundant in scope nor detail as evidenced by a comprehensive CRIS search. In this regard, a CRIS search was done by objective. Twenty-eight related projects were identified. Of these, 96% were projects involving participants in either the capacity of a federal grant or the previous multi-state project (S1020). It is noteworthy that the single exception was an NCSU Hatch Project entitled Identification of genes underlying traits of economic importance in poultry. This effort, however, is directed at phenomena such as immune function and nutrient utilization as opposed to reproduction.

In conclusion, this document requests permission for a new multistate project in poultry reproduction. This document explains the history underlying the emergence of S1020. This document also describes how the brain and genome have become contemporary focal points for understanding poultry reproduction in the broadest sense. Finally, we have proposed how highly effective people could be used to greater effect relative to the U.S. poultry industry. In this regard, the S1020 chair proposed that photoperiod, gametes, and the neural basis for behavior serve as a conceptual nexus for a new project. Therefore, this section will conclude with a brief overview of each of these research areas.

Photoperiod

The domestic turkey is a seasonal breeder. As such, the turkeys reproductive cycle is regulated by photoperiod. The goal of the turkey industry is meat production. Therefore, turkey breeders have selected for growth rate as opposed to reproductive ability. Consequently, the domestic female turkey continues to manifest attributes of its wild ancestors, which includes becoming photorefractory, i.e. an inability to respond to photostimulaton. This physio-logical state leads to termination of reproductive neuroendocrine function, which results in regression of reproductive organs and molting. The basis for cessation of reproduction remains unresolved.

Photorefractoriness appears to have a neuroanatomical rather than a purely endocrine basis. In this regard, a breakthrough made by S1020 members involves the identification of a dopaminergic and melatonergic neurons in the premammillary nucleus of the turkey hypothalamus. Note that the central nervous system, of which the hypothalamus is a part, contains neurons organized as nuclei (aggregates of similar neurons at designated locations that share specific structural or functional characteristics). The neurons of interest synthesize melatonin as well as dopamine and appear to be photoreceptive (Kang et al., 2007; Thayananuphat et al., 2007a; Thayananuphat et al., 2007b). Co-localized dopamine and melatonin cycle rhythmically and with opposite phases in these neurons. As evidenced by such preliminary data, it is hypothesized that photorefractoriness may stem from a disruption of the circadian rhythms within these neurons of interest. This disruption likely terminates a photoinducible circadian rhythm and thereby inactivates neuroendocrine mechanisms that promote reproduction. In this regard, a relationship has been established between photostimulation, glial cell function, thyroid hormone, and gonadotropin secretion in the tuberal hypothalamus. As such the effect of the premammillary nucleus upon this information pathway warrants clarification (Steinman et al., 2008).

Gametes

Work in this area is not as narrowly defined as that described above. There are three reasons for this. First, much of this work is performed by those who work with chickens rather than turkeys. Second, these scientists are divided into groups based upon expertise in female reproduction, male reproduction, and embryonic development. In regard to female reproduction, there is yet another division; for those interested in egg production tend to work with either egg-type or meat-type chickens. Another factor that has limited convergent research directed at a short list of fundamental problems is time. Though participants have complementary expertise, many have yet to collaborate over a comparable interval of time as have those on the brink of defining the basis for turkey photorefractoriness. In this regard, a new project would afford participants with a context in which comparable focus and collaboration can emerge.

Proposed work is based upon breakthroughs along three broad fronts: (1) the biological roles of adiponectin and anti-mullerian hormone, (2) sperm mobility, a quantitative trait discovered in the mid-1990s, and (3) elaboration of the reproductive tract transcriptome and DNA isolation for high throughput PCR. It is noteworthy that each of these advances is based upon work performed within the laboratories of S1020 participants (Bailes et al. 2007; Froman et al., 2006; Froman, 2007; Froman, 2009; Hendricks et al., 2009; Johnson et al., 2005; Johnson et al., 2008; Maddineni et al., 2005; OcÌn-Grove et al, 2008; Panzica et al., 2007; Ramachandran et al., 2007).

With regard to egg production, different types of chickens have been bred with particular goals in mind. For example, egg-type chickens have been selected for prolific ovulation rates and relative small body size. In contrast, meat-type chickens have been selected for rapid growth and heavy bodies. Such selection has enabled desired traits to be realized but to the detriment of other important traits. Consequently, meat-type hens tend to have poor ovarian follicular development with disorganized hierarchies. These terms warrant brief explanation. Hens  be they chicken or turkey hens  have an ability to lay one hard-shelled egg each day for many days when photostimulated. However, eggs are laid in clutches rather than a single series. As such, consecutive clutches are separated by one or more days when no egg is laid. In effect, genetic selection for egg production has increased clutch size and decreased the interval between clutches. The ability to lay a series of eggs is evident at the level of the ovary. This may be explained as follows. The ovary contains a finite set of oocytes, each within a follicle. Follicular diameter increases as an oocyte accrues yolk. However, yolk accrual is staggered in time. Consequently, the ovary contains follicles characterized by an incremental increase in diameter. This graded series of follicles is called a follicular hierarchy. Hens with disorganized or multiple hierarchies are not effective layers. The nature of this problem is beginning to be understood, and contemporary ovarian research is converging along three lines. These include: (1) the effect of a new adipose-related hormone (adiponectin) on ovarian steroidogenesis, (2) oocyte signaling, and (3) interaction between oocytes and granulosa cells, which form the interface between an oocytes surface and the inner face of the follicular wall. The relationship between feed intake, body weight, and ovulation is of particular importance to broiler breeder hens.

Insofar as male reproduction is concerned, the on-going analysis of sperm mobility has enabled a gene-based definition of semen quality. To date, semen quality  in chickens and turkeys alike  has been assessed in terms of ejaculate volume, sperm cell concentration, sperm viability, and motion. However, the number of mobile sperm produced per day is a major determinant of male fertility in the chicken. Moreover, such output is subject to genetic selection. The difference between sperm motility and mobility is critical. Whereas all mobile sperm are motile, not all motile sperm are mobile. Specifically, a motile sperm cell must have a velocity > 30 µm per second in order to be mobile in vitro. This distinction is biologically significant because sperm mobility phenotype predicts male fertility when hens are inseminated with a fixed number of viable sperm.
At the cellular level, phenotype is determined by the proportion of immobile sperm within an ejaculate that contain dysfunctional mitochondria. The time course for mitochondrial failure begins prior to ejaculation, and percentages of affected sperm range from 10 to 100%. This variation is attributed to a genetic predisposition that puts sperm cells at risk as they pass through the deferent ducts of the testis. Sperm mobility is heritable (h2 = 0.30), and phenotype is influenced by a maternal additive genetic effect. SNP-based, genome-wide association studies have revealed three major loci on two chromosomes (Gga6 and Z). The trait has been studied most thoroughly in experimental lines but is found in pedigree lines of commercial chickens. In summary, semen quality is a production trait that can be quantified in terms of sperm output from the testes, sperm transport through the deferent ducts, and the percentage of mobile sperm ejaculated thereafter.

Neural Basis for Reproductive Behavior

Broiler breeder flocks have shown a reduction in fertility and hatchability over the past few decades. Thus, selection for increased growth rate, improved feed conversion and yield seem to have exerted a negative effect upon reproductive fitness. Mating behavior has emerged as a contributing variable. In economic terms, male fertility is critical in naturally mated flocks because one male copulates with many hens. Thus, any given sire affects the quality of hatching eggs laid by many hens.

To date, this male effect has been countered by intensive management where inexperienced younger males are introduced to a breeding flock. This practice is called spiking. This practice has become more common in recent years in an attempt to increase fertility as a breeding flock ages. Though effective, this practice imposes significant additional production costs. This practice also illustrates why reproductive competence can no longer be assumed simply because healthy males and females are housed together at a reasonable sex ratio and properly fed. Historically, reproductive fitness was defined in terms of semen production and hen-day egg production, in part, because such variables are readily measured. Though these variables are critical, reproductive success very much depends upon less tractable factors. These include male dominance and competition, female acceptance and cryptic choice, and all of this within a complex social hierarchy in which members are confronted with feed restriction.

Whereas chicken reproductive behavior has been well-documented for many decades, the neurological basis for this behavior is only beginning to be understood. For example, recent breakthroughs (Jurkevich et al. 2005; Mikhailova et al., 2007; Jurkevich et al., 2008; Madison et al., 2008;) linking neuroanatomy, neuroendocrinology, and stress will enable the study of reproductive behavior to help define fitness within commercial flocks.

Summary

Life science is undergoing a transformation in which phenomena-based paradigms are being explained in terms of bioinformatics  as evidenced by the prominence and utility of the terms genomics and proteomics. Therefore, it is important to understand topics such as photostimulation, the hypothalamo-pituitary-gonadal axis, oogenesis, and sperm cell function within a contemporary context. The work proposed herein affords a community of scientists with the opportunity to begin this process and address limitations to reproduction within commercial breeding flocks.

Objectives

1. Characterize mechanisms enabling rhythmic regulation of dopaminergic-melatoninergic neurons within the hypothalamus of photosensitive turkey hens.
   
2. Characterize molecular mechanisms affecting egg production in chickens.
   
3. Identify chromosomal regions affecting phenotypic variation in sperm mobility, a primary determinant of male fertility
   
4. Characterize the mating behavior of contemporary broiler breeders and define central pathways affecting reproductive behavior.
   

Methods

OBJECTIVE 1 Participants: California and Minnesota (Millam and El Halawani). Minnesota and California will exchange reagents such as in situ hybridization probes and antibodies used in immunohistochemistry. Likewise, brain tissue collected under different physiological conditions will be exchanged. Minnesota will take the lead in all work related to the premammillary nucleus and genomic analysis of turkeys in different reproductive states. Californias complementary effort will focus on the role of the tuberal hypothalamus and glial cell function in mediating photosensitivity photorefractoriness. Specific Aim 1: Establish clock gene rhythms in neurons within turkey hens exposed to a photoperiod that induces gonad function. Quiescent adult female turkeys housed under a short photoperiod (LD 6:18; lights on at 0800 h) will be used in this experiment. Half of these birds will be exposed to a long photoperiod (15:9; light on at 0800 h) for 10 days during their photoinducible phase in order to activate the reproductive portion of their neuroendocrine systems. Light onset time (0800 h) will serve as circadian time zero, which will be a temporal reference point for comparison photoperiods. The daily pattern of expression of the clock genes Per2, Per3, Cry1, Cry2, Clock, and Bmal1 mRNAs within the premammillary nucleus will be determined by in situ hybridization using hens exposed to short and long photoperiods at circadian times 3, 14, and 20 hours. Specific Aim 2: Establish the expression of clock genes in neurons within photosensitive hens during night interruption during dark phase. The expression of turkey circadian clock genes (Per2, Per3, Cry1, and Cry2, Clock, Bmal1) within the premammillary nucleus will be determined in response to night interruption. This term denotes brief exposure to light of adequate intensity to photostimulate but during the dark phase of the photoperiod. This experiment is needed to further define the relationship between neuronal function, clock genes and the effect of photoperiod on reproductive function. Specific Aim 3: Establish circadian profile of clock genes in neurons within photorefractory and photosensitive turkey hens. This study will entail two experimental approaches. First, the clock gene expression will be determined based upon mRNA from Per2, Per3, Cry1, Cry2, Clock, and Bmal1 using RT-PCR with samples from photorefractory and photosensitive hens. Thereafter, measurements will be limited to those clock genes whose expression differ between physiological states. These genes will be further investigated utilizing immunocytochemistry and in situ hybridization to test whether clock gene expression occurs within neurons of interest, i.e. the dopaminergic-melatoninergic neurons within the premammillary nucleus of the hypothalamus. Specific Aim 4: Establish glial cell status relative to the operation of the hypothalamo-pituitary-gonad axis within photorefractory and photosensitive turkeys hens. Thyroid hormones help regulate seasonal reproduction reproductive in birds. Whereas thyroid hormones circulate within the bloodstream and affect metabolic rate, these hormones are lipid soluble and therefore can enter the brain by diffusing through the blood-brain barrier. Recent work has demonstrated that photostimulation serves to up-regulate type 2 iodothyronine deiodinase (Dio2) within the tanycytes of the ependymal layer in the basal hypothalamus (Yoshimura et al. 2003). The significance of this can be explained as follows. The hypothalamus is a small, but critical region of the brain. Hypothalamic neurons regulate homeostasis as well as enable motivated behavior, which includes reproduction. The hypothalamus forms the left, right, and bottom walls of the third ventricle, one of four fluid-filled chambers within the vertebrate brain. The ventricular surface is composed of a sheet of ependymal cells that separates underlying neural tissue from the cerebrospinal fluid within the ventricle. Tanycytes are one type of cell found within this epithelial boundary. Neuronal function is oftentimes affected by bordering glial cells. In this regard, it is noteworthy that glial cells are the predominant type of cell found within the vertebrate CNS. The tanycyte is one type of glial cell. In this case, Dio2 converts the inactive form of thyroid hormone to its active form, which facilitates neurosecretion of GnRH-I/LH (Nakao et al, 2008; Yamamura et al., 2004). It is highly noteworthy that photostimulation fails to induce expression of Dio2 within the tanycytes of photorefractory hens (Steinman et al., 2008). However, the basis for this is unknown. This effort will assess glial cell functional status in two critical regions of the brain: the hypothalamus and forebrain. This assessment will be based upon photosensitive and photorefractory hens. Likewise, hens will be treated with thyroid hormone agonists and antagonists as well as dopaminergic agonists and antagonists. Such experiments are warranted in view of the proven effect of glial cells and the apparent effect of neurons within the premammillary nucleus upon reproductive status. Glial cell functional status will be assessed by characterizing the immunohistochemical expression of key cytoskeletal proteins (GFAP and vimentin) as well as different phosphorylated forms of dopamine- and cyclic AMP-regulated phosphoprotein (DARPP-32). OBJECTIVE 2 Participants: Arkansas, Cornell, and Penn State (Bartell, Diaz, A. Johnson, P. Johnson, Kuenzel, and Ramachandran). For this objective, the advantage of the multistate approach is that expertise with egg-type chickens can be applied to problems of importance to the broiler industry and vice-versa. Drs. P. Bartell, F. Diaz, A. Johnson and R. Ramachandran (Penn State), Dr. P. Johnson (Cornell), and Dr. W. Kuenzel (Arkansas) will collaborate on this objective. Dr. Ramachandran and Dr. Kuenzel will address the role of adiponectin on fertility in chickens. This is significant because of the feed-intake/body weight impact on ovulation, particularly evident in broiler breeder hens. Dr. Diaz is interested in oocyte signaling and culture and this will complement the work of Dr. A. Johnson and Dr. P. Johnson toward granulosa cell signaling and function. Dr. Bartells interests address the overriding role for the circadian clock(s) in ovarian function, and his input will impact on each of the remaining sub-aims. It is planned that broiler and layer hen tissue will be exchanged between Pennsylvania and New York. Moreover, particular antibodies (i.e. GDF9) and techniques (e.g., oocyte co-culture, siRNA-mediated knock-down) will be shared. Specific Aim 1: Determine the role of adiponectin in the reproductive axis relative to egg production. This effort provides a superb example of collaboration in view of: (1) the number and quality of research tools developed and validated by the Ramachandran lab (genes cloned for chicken adiponectin and two of its receptors, specific antibodies for these three proteins, and a chicken-specific enzyme immunoassay for bloodstream adiponectin), and (2) the Kuenzel labs expertise in neuroanatomy and CNS function. Adiponectin is an adipokine hormone secreted primarily by adipose tissue. Fully-fed broiler breeder pullets grow rapidly. This induces ovarian dysfunction and elevated plasma estrogen levels. Whereas adiponectin likely plays a role in ovarian function, this role is unknown. Chickens and humans express the adiponectin gene within the central nervous system and pituitary gland (Psilopanagioti et al., 2009). This feature may be of great interest due to the relationship between visceral adiposity, circulating adiponectin, and a putative role for adiponectin as a modulator of gonadotropin secretion. The primary energy reserve within the body of a hen is contained within adipocytes located within visceral adipose tissue. Plasma adiponectin will be determined in broiler breeder pullets and sexually mature hens that are feed restricted or fully-fed. Pre-hierarchial and pre-ovulatory follicles will be obtained for in vitro culture of thecal or granulosa cells. Cultured cells will be treated with recombinant chicken adiponectin or transfected with adiponectin cDNA. The amount of estradiol, estrone, and progesterone in culture media will be determined by radioimmunoassay. Protein and mRNA levels of steroid acute regulatory protein, beta-hydroxysteroid dehydrogenase, vascular endothelial growth factor, as well as mitogen-activated protein kinase will be determined using real-time quantitative PCR and Western blotting. Changes in thecal and granulosa cell proliferation in response to adiponectin will be determined by 3H-thymidine incorporation assay. Finally, the effect of orally administered thiazolidinedione (adiponectin sensitizer compound) on ovarian follicular hierarchy will be tested in broiler breeder pullets that are either fully-fed or feed-restricted. This effort will also address the effect of environment (photostimulation and feed restriction) upon an endocrine pathway (bloodstream and cerebrospinal fluid adiponectin) that modulates the central integration (hypothalamic neurons) that determines reproductive performance. The distribution of the two adiponectin receptors (AdipoR1 and AdipoR2) throughout the brain of the chicken will be determined by immunocytochemistry. Specific Aim 2: Conduct a comprehensive assessment of the effect of diet, signal molecule profile, and gene expression upon follicle selection. Participants intend to use laying hens as well as broiler breeder hens in this effort. In vivo and in vitro experiments will be performed to evaluate factors which affect ovarian function. This approach will permit comparisons between laying and broiler breeder hens as well as between full-fed and restricted-fed hens. Nutritional and hormonal treatments will be administered to hens and RNA expression as well as protein production (i.e., AMH, GDF9, BMPs, EGF receptor ligands) will be evaluated in follicles of different sizes. As such, collaboration should afford an unprecedented view of ovarian mechanisms affecting female reproductive efficiency. We will also evaluate ovulation rate and body weight. Factors related to yolk uptake and incorporation will be evaluated in oocytes. In separate experiments, treatments will be administered to cultured oocytes in vitro and oocyte parameters examined. In vitro experiments utilizing cultured follicle cells will be conducted to evaluate hormonal and nutritional effects on follicle cell function (i.e., AMH, gonadotropin receptors). Hormones produced by follicle cells that impact yolk production will be evaluated with respect to genetic and nutritional condition of the hen. Most of the techniques necessary for this project are operational in the New York and Pennsylvania labs. Specific Aim 3: Determine the role of oocyte signaling pathways in follicle activation and early growth. This effort will be based upon a series of experiments. The first will identify granulosa cell transcripts regulated by the oocyte. Small follicles (<1 mm in diameter) will be collected from the ovarian cortex. Leghorn hens will be used as donors. Intact follicles, isolated granulosa cells or granulosa cells co-cultured with oocytes will be cultured for 1 to 2 days. Total RNA will be isolated when culture is terminated. Granulosa cell transcriptomes, i.e. from granulosa cells cultured with or without oocytes, will be analyzed using a chicken microarray. Results will be verified using qPCR techniques. The next experiment will clarify the role of a signaling pathway by use of a specific inhibitor of the pSMAD2/3 signaling pathway. Once again, small follicles will be collected and cultured with or without the inhibitor (SB431542) for 24 hours. Treatment effect upon the transcriptome will be determined by microarray. In this manner, a set of granulosa cell genes will be identified that are regulated by SMAD2/3 signaling and in response to the presence of an oocyte. Whereas it is generally accepted that the oocyte drives the function and differentiation of the follicle, particularly during the early stages of development, the precise means whereby this happens within the hens ovary is unknown. Once this set of genes is identified, their expression will be evaluated in ovarian cells from egg-type and meat-type hens. OBJECTIVE 3 Participants: Arkansas, Oregon State, Washington State (Rhoads, Froman, and McLean). Experimental lines of chickens will be maintained at Oregon State University. Previous work has enabled the collaborators to link sperm mobility phenotype with genotype. The sperm mobility assay was developed at Oregon State by Dr. Froman. The commercial version of this assay has been used in the field to phenotype pedigree-line meat-type chickens. Dr. Rhoads has assumed responsibility for genotyping males of known phenotype. This effort began with a SNP-based, genome wide association study. Specifically, SNPlotyping was conducted using replicate males of known phenotype from non-related flocks. Thereafter, primers were developed for VNTR-based genetic analysis and have been used to confirm several regions containing QTLs for sperm mobility. Whereas the focal point of the proposed work is clarifying the relationship between male phenotype  at the level of the sperm cell -- and sire genotype, a third dimension will be provided by Dr. McLean at Washington State. Specifically, microarray analysis of testicular gene expression will be performed. This effort should be expedited by the library developed by Dr. Rhoads and described by Froman et al. (2006). Specific Aim 1: Estimate allelic frequencies at select chromosomal regions using DNA from males of known sperm mobility phenotype within pedigree lines of meat-type chickens. Sperm mobility is a quantitative trait discovered in the 1990s and is a primary determinant of male fertility. In essence, this effort reflects an on-going collaboration between two S-1020 participants funded by Cobb-Vantress (Rhoads and Froman). Details, however, are currently proprietary. Nonetheless, parallel work will be performed with lines of New Hampshire chickens selected for low or high sperm mobility. This work will be based upon a genome-wide SNP analysis performed in previous work using replicate males per line. VNTR assays were developed in order to further examine SNPlotypes that differed between lines. To date, regions of interest have been identified and confirmed for chromosomes 6 and Z. DNA differences within the Z chromosome are of particular interest because this is one of two sex chromosomes, males are homozygous for this chromosome, and an exclusively maternal additive genetic effect was observed when heritability was estimated (Froman et al., 2002). The basis for maternal inheritance was initially attributed to the mitochondrial genome. Therefore, the mitochondrial genome was sequenced in its entirety for replicate males within lines. Though a heritable SNP was discovered within the gene for tRNAArg, this difference was not observed within the mitochondrial genomes of pedigree line broiler breeders with distinct sperm mobility phenotypes. Therefore, the maternal effect upon sperm mobility phenotype appears to be mediated by a region of the Z chromosome. Consequently, the role of the two loci of interest identified on the Z chromosome is under investigation. Specific Aim 2: Divergent selection of low and high sperm mobility phenotypes based upon dam and sire genotype. The OSU low sperm mobility line is characterized by a pronounced skewed phenotypic distribution. In contrast, the shape of the high lines distribution is normal and has remained stable over a decade. The mean and standard deviation of this population is comparable to that of the random bred population from which lines were generated. In retrospect, genetic selection did indeed produce a uniform population of high sperm mobility males. However, it could be argued that selection produced high mobility males even though there exists a pronounced difference in phenotype between lines. The variability in the high line affords an opportunity to conduct divergent selection with a random bred population serving as a control. Divergent selection will be performed by first categorizing predominant allelic combinations at two loci of interest upon the Z chromosome using test subjects within the lower and upper tails of the phenotypic distribution. Next, allelic combinations will be categorized for females within the flock (two loci per Z chromosome). Then, dams will be bred with semen from non-related sires of common genotype. Progeny will be reproduced in this manner for 3 generations (n = 100 male progeny per generation). As explained above, a random bred population will serve as a control. The following variables will be measured per male once progeny have been produced: body weight, sperm mobility, ejaculate volume, sperm concentration, and testis weight. A subsample of males from each distributions central tendency will be used for microarray analysis. OBJECTIVE 4 Participants: Arkansas and Penn State (Bramwell, Jurkevich, Kuenzel, and R.Ramachandran). The overall goal is to examine reproductive behavior that occurs in broiler breeder males, ascertain neural structures and neuroendocrine pathways involved, and determine hatchability of chicks under experimental and commercial conditions. Drs. Bramwell, Jurkevich and Kuenzel will conduct behavioral studies of broilers with an emphasis upon male reproductive behavior. In particular, Kuenzel and Jurkevich will address a possible mechanism by which light activates reproduction. Photoperiodic conditions will be varied while other key environmental factors will be controlled. Dr. Ramachandran will provide molecular probes required for certain aspects of this as well as continue to develop additional research tools suitable for broiler breeder research. Specific Aim 1: To determine the productivity of broiler breeders brought into the reproductive state at different ages by manipulating photoperiod and examine the mechanism of photoperiodic activation of the neuroendocrine reproductive system. Photoperiod remains one of the key environmental variables used by researchers and the poultry industry to bring birds into reproduction at the appropriate time. In order to maintain experimental and commercial lines of poultry, one must maintain acceptable fertility and hatchability throughout a production cycle within each generation. However, broiler breeder fertility and hatchability decline prematurely, and it is likely that male reproductive behavior contributes to this problem. Broiler breeder flocks are photostimulated at 21 weeks of age. However, this practice is based upon convention as opposed to an empirical optimal age. Therefore, the latter will be sought using males photostimulated at 12, 15, 18, 21 (controls) and 24 weeks of age. Variables to be measured will include reproductive behavior, fertility and hatchability. Hens will be uniformly photostimulated at 21 weeks of age. Likewise, hens will be uniformly maintained until weeks of age. Drs. Kuenzel and Jurkevich will examinie the most likely means by which light stimulates the hypothalamo-pituitary-gonadal system within breeder males. A group of neurons in the septal region of the brain may function as photoreceptors. What is unclear at this time is how the photoreceptors interact with the primary set of neurons, gonadotropin-releasing hormone neurons, as well as a secondary group, gonadotropin inhibitory neurons (GnIH) and their respective receptors (GnIHR). Each set activates and inhibits, respectively, the release gonadotropins, luteinizing hormone and follicle stimulating hormone, from the pituitary gland. The Kuenzel lab will complete the immunocytochemistry of brain sections and anterior pituitary using antibodies produced by the Ramachandran lab. The spatial distribution of key groups of CNS neurons and their ultimate target cells within pituitary gland will be defined. Specific Aim 2: To define neural sites in the brain activated by reproductive behavior and outline central neural networks affecting reproductive behavior. As mentioned above, the reproductive potential of broiler breeder flocks has declined within the past few decades. Compromised fitness has been attributed to selection for growth rate, improved feed conversion and yield. However, mating behavior has emerged as a contributing variable. In economic terms, male fertility is critical in naturally mated flocks because one male copulates with many hens. Thus, the effectiveness of a single sire and the quality of his semen affects the quality of hatching eggs from many hens. Surprisingly, neural structures not only control reproduction but can be affected by the reproductive act. And in this regard, it is noteworthy that reproductive behavior comes with a biological cost to a sire. Therefore, the identification of structures in the brain activated by mating behavior and in response to a particular stress are warranted. Members of the Kuenzel lab will utilize Fos protein, a product of immediate early gene, cfos, that has been shown to be an effective marker of groups of activated neurons. Individual broiler breeder males will therefore be exposed to individual females or another male within a typical floor pen (open field test). Thereafter the brain of each test subject will be examined and brain sections quantified for the number of neurons showing Fos protein expressed within the cell nucleus. Male-male interactions are particularly stressful. Consequently, two major groups of neurons involved in the hypothalamo-pituitary-adrenal axis and their respective receptors will be examined to determine possible co-localization with Fos protein. These neurons include those that secrete arginine vasotocin and corticotropin releasing hormone. Brain regions that will be examined include the subpallium (structures located in the basal forebrain) and diencephalon (preoptic area, hypothalamus and thalamus). Examination of CNS tissue from test subjects will determine which brain nucleus or set of nuclei are activated following either sexual or agonistic behavior. Differential activation will allow the development of neural networks for each behavior and identify neural structures where both behaviors overlap.

Measurement of Progress and Results

Outputs

• The most readily measured output of the proposed project is the peer-reviewed journal article. While this means of information dissemination has limitations, the peer-reviewed research article remains a primary means by which the effectiveness of a research scientist is measured. Therefore, peer-reviewed journal articles published by participants will be itemized annually in a project report. However, other forms of information sharing include books and book chapters, university-sponsored seminars, professional meetings and symposia, as well as industry-sponsored meetings. Likewise, grantsmanship and technology transfer may also result from collaboration.
• To date, industry representatives have attended each annual meeting, and their comments have been positive and supportive. It is the chairpersons contention that the annual meeting could serve as a forum wherein common commercial problems could be identified and prioritized with the help of industry representatives. In this manner, submission of highly competitive integrated research projects from groups of participants within the project could very well be an additional form of output.

Outcomes or Projected Impacts

• The proposed work is predicated on the assumption that a mechanistic understanding of photorefractoriness is perquisite for any intervention that serves to eliminate or reduce photorefractoriness. If the correct biological means for countering photorefractoriness can be identified, then a profound increase in the reproductive efficiency of turkey hens would follow. Specific benefits to end users (primary and secondary turkey breeders) would include decreased environmental impact, increased genetic selection pressure, and decreased operating cost.
• First, the role of adiponectin as a modulator of ovarian function will be described. In doing so, it is probable that a cell network will be identified that links energy metabolism with the hens hypothalamo-pituitary-gonadal axis. Such a discovery would constitute a fundamental advance in poultry science and would lead to new strategies fro improving broiler breeder hen reproductive efficiency. Second, even though the basic mechanism of egg production holds for egg- and meat-type chickens, there is a profound difference in ovarian follicular development between these two types of commercial chickens. Experimental outcomes should improve our understanding of this critical difference in reproductive performance. Moreover, specific genes and signaling pathways affected by feeding regimen will be characterized relative to inefficient follicle growth. Consequently, it may become possible to select hens less prone to ovarian dysfunction. At present, hens characterized by short laying sequences and excessive follicle development compromise the reproductive efficiency of broiler breeder flocks. Therefore, minimizing the percentage of such hens within commercial flocks would have a profound effect upon egg production during a laying cycle. Finally, experimental outcomes will provide crucial information on the role of oocytes and occycte factors (e.g. GDF9) in promoting differentiation of granulosa cells early in follicular development. Thuis information will provide a nuanced understanding of ovarian function within egg- and meat-type chickens. Once again, such knowledge should increase the probability of finding effective strategies to remedy ovarian dysfunction in broiler breeder hens.
• Experimental outcomes should serve to further refine the relationship between phenotype and genotype of this quantitative trait. Moreover, the outcome of Specific Aim 2 will further confirm that genetic selection can be used to alter phenotype. However, in this case, the selection criterion will be genotype rather than phenotype. It is anticipated that experimental outcomes will increase the likelihood that primary breeders will be able to breed for male fertility in addition to other desirable traits.
• First, if there is an optimal age for photostimulating broiler breeder males, then this will be ascertained. The demonstration of such an optimum would have great benefit in and of itself. However, such an outcome would dovetail with either the emergence or operation of critical CNS networks that ultimately control GnRH secretion, which is a key control point within the entire reproductive process. This advance, in turn, will be understood in terms of neurons involved in sexual as well as aggressive behavior.

Milestones

(0):ective 1 (1) Confirm the relationship between each of three CNS attributes (glial cell status, thyroid hormone effect, and phosphorylation status of DARPP-32) and reproductive status, (2) describe underlying gene expression, (3) publish experimental outcomes in peer-reviewed journals, and (4) share any advance with extension faculty in key turkey producing states.

(0):ective 2 (1) basic knowledge of ovarian function will be advanced, in particular ovarian dysfunction that compromises the reproductive efficiency of broiler breeder hens, (2) publish experimental outcomes in peer-reviewed journals, and (3) share outcomes with extension faculty in key states, in part, with the help of Drs. Mike Hulet and Paul Patterson at Penn State.

(0):ective 3 (1) Clarify the relationship between Z chromosome alleles and sperm mobility phenotype, (2) demonstrate that selection for specific alleles on the sex chromosome alters phenotype, (3) publish experimental outcomes in peer-reviewed journals, and (4) share experimental outcomes with appropriate R&D personnel employed by primary breeders of poultry.

(0):ective 4 (1) Specify the age at which broiler breeder males are most responsive to photostimulation in terms of overall subsequent reproductive performance, (2) outline specific cellular networks within the brain that make this possible, (3) describe how such networks are subject to various forms of social interaction, (4) publish experimental outcomes in peer-reviewed journals, and (5) share pertinent experimental outcomes with those who manage commercial breeder flocks.

Projected Participation

View Appendix E: Participation

Outreach Plan

Annual meetings will be timed and structured to facilitate information sharing with stakeholders, e.g. representatives of the US primary breeder industry and related industries. It is noteworthy that the extension specialist within the group (Bramwell) is a faculty member in the Center of Excellence for Poultry Science and is well-known by poultry extension faculty throughout the U.S. It is also noteworthy that most participants have working relationships with key people employed by U.S poultry breeders or related companies.

Organization/Governance

Chair: David Froman, Oregon State University, David.Froman@oregonstate.edu;

Secretary: Douglas Rhoads, University of Arkansas.


Each annual meeting will conclude with a formal business meeting.

Literature Cited

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Froman, D. P., 2007. Sperm motility in birds: insights from fowl sperm. In Spermatology, E. R. S. Roldan and M. Gomendio (eds.), Nottingham University Press, pp. 293-307.

Froman, D. P., 2009. A theoretical approach to sperm preservation based upon mitochondrial energetics. J. Anim. Sci. Invited manuscript for Cell Biology Symposium entitled REDOX Regulation of Cell Function.

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Attachments

Land Grant Participating States/Institutions

AR, CA, IL, MD, MN, NY, PA

Non Land Grant Participating States/Institutions