ENHANCING REPRODUCTIVE EFFICIENCY OF POULTRY (S285)

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0223470
• Multistate No.: S-1047
• Project Start Date: Oct 1, 2010
• Project End Date: Sep 30, 2013
• Program Code: [(N/A)]- (N/A)

Project Director
Johnson, AL.

Recipient Organization
PENNSYLVANIA STATE UNIVERSITY
208 MUELLER LABORATORY
UNIVERSITY PARK,PA 16802

Performing Department
Animal Science

Non Technical Summary
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 an 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. 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. 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 will enable such scientists to build upon their accomplishments and collaborate towards a new goal: defining gene networks that enable poultry reproduction. 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.

Animal Health Component

25%

Research Effort Categories

Basic

75%

Applied

25%

Developmental

0%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
301	3210	1020	25%
301	3210	1060	25%
301	3220	1020	25%
301	3220	1060	25%

Knowledge Area
301 - Reproductive Performance of Animals;

Subject Of Investigation
3210 - Egg-type chicken, live animal; 3220 - Meat-type chicken, live animal;

Field Of Science
1060 - Biology (whole systems); 1020 - Physiology;

Keywords

poultry

reproduction

behavior

hypothalamus

ovary

testes

photoperiod

circadian clock

granulosa

oocyte

adiponectin

cell signaling

Goals / Objectives
Characterize mechanisms enabling rhythmic regulation of dopaminergic-melatoninergic neurons within the hypothalamus of photosensitive turkey hens. Characterize molecular mechanisms affecting egg production in chickens.

Project Methods
Specific Aim 1: Determine the role of adiponectin in the reproductive axis relative to egg production. 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. 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. 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.

Progress 10/01/10 to 09/30/13

Outputs
Target Audience: Knowledge derived from this project has been widely disseminated to scientists in academic institutions via scientific meetings and publications, to scientists and producers within the commercial broiler breeder industry, and via formal classroom teaching and informal interactions with students. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Five Ph.D. candidate students and four undergraduate students have participated in conducting these studies during the past year. How have the results been disseminated to communities of interest? Results have been disseminated primarily via book chapter and journal publications, and presentations at annual meetings. What do you plan to do during the next reporting period to accomplish the goals? The present studies will be continued under a different non-capacity project, without major modifications, until completion.

Impacts
What was accomplished under these goals? 1. Characterize mechanisms of G Protein-Coupled Receptor desensitization in undifferentiated granulosa cells. 2. Establish how cell signaling mechanisms regulate follicle selection, growth and differentiation. 3. Discover how the level of feed and energy alters ovarian follicular development. 4. Establish the role of biological clocks in regulating reproductive performance. Broiler breeder hens have the poorest reproductive efficiency of all commercial avian species primarily due to excessive feed intake and adiposity that results in aberrant follicle growth and selection. We have reported that levels of plasma adiponectin, an adipokine hormone, were significantly lower in broiler breeder hens that were fed ad libitum. Our results indicated that both recombinant adiponectin and metformin increased the abundance of pErk 1/2 in cultured thecal cells in a dose-dependent manner. Long-term oral administration of metformin (0, 100, and 200 mg/kg body weight/day) to broiler breeder hens from 20 weeks-of-age to 55 weeks-of-age was found to be well tolerated by the animals resulting in a significantly higher egg production in chickens fed 100 mg/kg body weight daily compared with 0 or 200 mg/kg body weight doses. Changes in blood metabolites and hormone levels in response to oral metformin treatment are being determined. To investigate the biochemical changes caused by level of feed (Full-fed, (FF) vs Restricted (RF)) in broiler-breeder hens, the immunostaining intensity of PCNA protein and various histone modifications were determined. PCNA protein immunostaining was more intense in the granulosa cell layer in full-fed hens compared to restricted-fed hens suggesting increased proliferation. Global serine and tyrosine phosphorylation were determined as a broad indicator of changes in kinase activity in full-fed and restricted-fed broiler hens. Phosphorylaton of serine was found in vesicle-like structures in oocytes of FF and RF hens, but was decreased in the stroma of RF hens. In contrast, tyrosine phosphorylation was higher on or near the plasma membrane of restricted–fed hens. Additional efforts have been focused on developing a mechanistic model for follicle selection in the hen. Recent studies investigated cellular events that: 1) maintain ovarian follicle granulosa cells (GCs) from prehierarchal (unselected) follicles in an undifferentiated state prior to selection; and 2) subsequently initiate GC differentiation at follicle selection. Specifically, efforts focused on the regulation of cell signaling via the FSH receptor (FSHR), a G-Protein Coupled Receptor, by βARRESTIN-mediated desensitization. Despite the finding that undifferentiated GCs express the highest levels of FSHR mRNA and membrane-localized protein during follicle development, freshly collected cells from prehierarchal follicles fail to initiate cAMP signaling following a 1 h challenge with FSH. Consequently, these cells remain in an undifferentiated state and demonstrate virtually no capacity for steroidogenesis due to the absence of cAMP-dependent STAR protein and CYP11A1 gene expression. Knockdown of βARRESTIN1 mRNA in cultured, undifferentiated GCs using small interfering RNA (siRNA) facilitated FSH-induced cAMP, STAR expression and progesterone production. Furthermore, over-expression of bovine βARRESTIN1 and G PROTEIN-COUPLED RECEPTOR KINASE 2 proteins in actively differentiating GCs significantly decreased cAMP accumulation and progesterone production following a challenge with FSH. These findings indicate that a βARRESTIN-mediated mechanism is both sufficient and necessary to maintain FSHR desensitization in undifferentiated GCs from prehierarchal follicles. We conclude that prior to follicle selection FSHR desensitization within the GC layer of prehierarchal follicles prevents premature GC differentiation and propose that this desensitization is maintained, at least in part, by MAPK signaling. Finally, efforts were focused on the role of biological clock regulation of reproductive performance, focusing on the role of interacting circadian clocks in brain and ovary, and specifically at the time of follicle selection, to influence egg production. To these aims, we have explored how so called “clock genes” can influence the transcription of genes encoding for enzymes involved in reproductive function. Furthermore, research has investigated the role that melatonin, the hormone most closely associated with circadian regulation, plays in modulating multiple parameters of reproductive output.

Publications

• Type: Journal Articles Status: Published Year Published: 2013 Citation: Kim, D., Oc�n-Grove, O.M., and Johnson, A.L. Bone Morphogenetic Protein 4 (BMP4) Supports the Early Differentiation of Hen (Gallus gallus) Granulosa Cells. Biol. Reprod. 88:161 1-7.
• Type: Journal Articles Status: Submitted Year Published: 2013 Citation: Kim, D., Oc�n-Grove, O.M., and Johnson, A.L. FSH Receptor-Responsiveness in Undifferentiated Hen Granulosa Cells is Precluded by a beta-ARRESTIN-Mediated Mechanism. Submitted to Endocrinology
• Type: Book Chapters Status: Awaiting Publication Year Published: 2014 Citation: Johnson, A.L. Chapter 23. Reproduction in the female. In: Sturkies Avian Physiology, 6th Ed., C.G. Scanes, Ed., New York: Elsevier.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Taylor A, Horvat-Gordon M, Moore AF, Bartell, PA. 2013. The effects of melatonin on the physical properties of bones and egg shells in the laying hen. PLoS One. (2):e55663. doi: 10.1371/journal.pone.0055663.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Ebert-Zavos E, Horvat-Gordon M, Taylor A, Bartell PA. 2013. Biological clocks in the duodenum and the diurnal regulation of duodenal and plasma serotonin. PLoS One. May 30;8(5):e58477. doi:10.1371/journal.pone.0058477.

Progress 10/01/11 to 09/30/12

Outputs
OUTPUTS: Broiler breeder hens have the poorest reproductive efficiency of all commercial avian species primarily due to excessive feed intake and adiposity that results in aberrant follicle growth and selection. We have reported that levels of plasma adiponectin, an adipokine hormone, were significantly lower in broiler breeder hens that were fed ad libitum. Both recombinant chicken adiponectin (rcADN) and metformin (a biguanide anti-diabetic drug,) increased the abundance of pErk1/2 in cultured theca cells dispersed from prehierarchial follicles in a dose-dependent manner. Furthermore, metformin fed to broiler breeder hens for 21 days increased adiponectin and adipoR2 gene expression in subcutaneous adipose tissue. Adiponectin produced locally in thecal tissue may act as a paracrine factor to improve metabolism of follicular cells. To investigate the biochemical changes caused by level of feed (Full-fed, (FF) vs Restricted (RF)) in broiler-breeder hens, the immunostaining intensity of PCNA protein and various histone modifications were determined. PCNA protein immunostaining was more intense in the granulosa cell layer in full-fed hens compared to restricted-fed hens suggesting increased proliferation. Global serine and tyrosine phosphorylation were determined as a broad indicator of changes in kinase activity in full-fed and restricted-fed broiler hens. Phosphorylaton of serine was found in vesicle-like structures in oocytes of FF and RF hens, but was decreased in the stroma of RF hens. In contrast, tyrosine phosphorylation was higher on or near the plasma membrane of restricted-fed hens. A growing body of literature provides evidence of a prominent role for bone morphogenetic proteins (BMPs) in regulating granulosa cell differentiation at the time of follicle selection. The hypothesis tested was that BMPs increase Follicle Stimulating Hormone receptor (FSHR) mRNA expression and enhance FSHR-responsiveness within granulosa cells from prehierarchal (6-8 mm) follicles (6-8GC). BMP4, -6 and -15 each promoted FSHR mRNA expression in a dose-related fashion. Enhanced FSH-responsiveness was indicated by increased Steroidogenic Acute regulatory (StAR) protein expression and progesterone production following a challenge with FSH. We conclude that at the time of follicle selection, BMPs promote granulosa cell differentiation and initiate the final gowth of follicles prior to ovulation. Interestingly, rhBMP6 also increased expression of Anti-Mullerian Hormone (AMH) mRNA in cultured 6-8GC. This related BMP family member has previously been implicated in negatively regulating FSH-responsiveness during follicle development. We speculate that prior to selection one action of AMH within 6-8GC may be to suppress FSHR signaling and prevent premature granulosa cell differentiation. Finally, efforts were focused on the role of biological clock regulation of reproductive performance, in particular focusing on the role of interacting circadian clocks in brain and ovary (particularly at the time of follicle selection) to regulate egg production. To these aims, we have evaluated clock regulated hormonal output and the phase dependent responses in diverse tissues. PARTICIPANTS: Dr. Paul Bartell, Assistant Professor, Department of Poultry Science, Principal Investigator; Dr. Francisco Diaz, Assistant Professor, Department of Poultry Science, Principal Investigator; Dr. Alan L. Johnson, Professor, Department of Poultry Science, Principal Investigator; Dr. Ramesh Ramachandran, Associate Professor, Department of Poultry Science, Principal Investigator ;This project has provided graduate research training for the following students: Mr. Dongwon Kim, Ph.D. candidate; Ms. Xi Tian, Ph.D. candidate; Ms. Anupama Tiwari, Ph.D. candidate; Mr. Alexander Taylor, Ph.D. candidate. This project has provided undergraduate research training for the following students: Ms. Elizabeth Ebert-Zavos, Ms. Amy Niedbala, Mr. Adam Howell and Ms. Jennifer Liscinsky. TARGET AUDIENCES: Knowledge derived from this project has been widely disseminated to scientists in academic institutions via scientific meetings and publications, to scientists and producers within the commercial broiler breeder industry, and via formal classroom teaching and informal interactions with students. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Data derived from the studies of adiponectin indicate that this adipose tissue secreted hormone can affect reproductive efficiency in broiler breeder hens. We have also identified metformin as a compound that mimics some of the beneficial effects of adiponectin signaling and therefore provides a practical solution for ameliorating excess lipid accretion in commercial broiler breeder parents. Further characterization and advancement of fundamental knowledge on the effect of adipose tissue hormones may lead to devising methods for manipulating metabolic processes to improve reproductive efficiency in broiler breeder hens. These studies also provide novel insight as to how cell signaling mechanisms regulate follicle growth and differentiation, and begin to uncover how the level of feed and energy alters ovarian follicular development. This knowledge can be used to more effectively manage broiler-breeders during the rearing period to subsequently reduce the incidence of ovarian dysfunction and increase fertility. At maturity, broiler breeder hens are subject to having more than one ovarian follicle selected into the preovulatory hierarchy per day, which results in dysfunctional follicle final growth and maturation. In turn, this results in more than one ovulation per day, multiple eggs simultaneously present within the reproductive tract, and causes significantly reduced egg production. Studies of FSH responsiveness and the initiation of granulosa cell differentiation at follicle selection have enabled us to identify the most proximal signals associated with follicle selection. These results, combined with an understanding of the ovarian circadian clock, will eventually allow us to understand the mechanisms by which excessive food intake and lipid accretion negatively impacts reproductive efficiency.

Publications

• Diaz, F. J. and K. Anthony. 2012. Feed restriction inhibits early follicular development in broiler-breeder hens. Animal Reproduction (Submitted).
• Ocon-Grove, O. M., D. H. Poole, and A. L. Johnson. 2011. Bone morphogenetic protein 6 promotes FSH receptor and anti-Mullerian Hormone mRNA expression in granulosa cells from hen prehierarchal follicles. Reproduction 143:825-833.
• Johnson, A. L., O. M. Ocon-Grove, and D. Kim. 2012. Bone morphogenetic proteins 4 and 15 promote FSH-responsiveness in granulose cells at follicle selection. Biology of Reproduction (Submitted).
• Diaz, F. J., K. Anthony, and A. N. Halfhill. 2010. Effect of feed restriction on early avian follicular development. Proceedings of the 43th Annual Meeting of the Society for Study of Reproduction. Milwaukee, WI. (Abstract #598).

Progress 10/01/10 to 09/30/11

Outputs
OUTPUTS: Data derived from the studies of adiponectin indicate that this adipose tissue-secreted hormone can affect reproductive efficiency in broiler breeder hens. Metformin is a compound that mimics some of the beneficial effects of adiponectin signaling and therefore provides a potential practical solution for ameliorating excess lipid accretion in this important poultry species. Further characterization and advancement of fundamental knowledge on the effect of adipose tissue hormones may lead to devising methods for manipulating metabolic processes for improving reproductive efficiency in broiler breeder hens. Overall, the studies described provide novel insight as to how cell signaling mechanisms regulate follicle growth and differentiation, and also begin to uncover how various feeding regimens can alter ovarian follicular development. This knowledge can be used to more effectively manage broiler-breeder reproduction to decrease ovarian dysfunction and increase fertility. PARTICIPANTS: Dr. Paul Bartell, Assistant Professor, Department of Poultry Science, Principal Investigator. Dr. Francisco Diaz, Assistant Professor, Department of Poultry Science, Principal Investigator. Dr. Alan L. Johnson, Professor, Department of Poultry Science, Principal Investigator. Dr. Ramesh Ramachandran, Associate Professor, Department of Poultry Science, Principal Investigator. This project has provided graduate research training for the following students: Dongwon Kim, Ph.D. candidate; Xi Tian, Ph.D. candidate; Anupama Tiwari, Ph.D. candidate; Alexander Taylor, Ph.D candidate. TARGET AUDIENCES: Scientists in academic institutions together with scientists and producers within the commercial broiler breeder industry. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Broiler breeder hens have the poorest reproductive efficiency of all poultry species, due primarily to aberrant follicle growth and selection resulting from excessive feed intake and adiposity. Levels of plasma adiponectin, an adipokine hormone, were found to be significantly lower in ad libitum fed broiler breeder hens vs. feed-restricted hens. Phosphorylation of mitogen-activated protein kinase (Erk1/2) exerts a critical inhibitory effect on chicken prehierarchial follicular granulosa cells thereby preventing granulosa cell differentiation and follicle selection. Both recombinant chicken adiponectin and metformin (a biguanide anti-diabetic drug) increased the abundance of pErk 1/2 in cultured theca cells dispersed from prehierarchial follicles in a dose-dependent manner. Furthermore, metformin fed to broiler breeder hens for 21 days increased adiponectin and adipoR2 gene expression in subcutaneous adipose tissue. Therefore, adiponectin produced locally in thecal tissue may act as a paracrine factor to increase pErk1/2 levels and diminish FSH-responsiveness. To investigate the biochemical changes caused by feeding regimen, the immunostaining intensity of proliferating cell nuclear antigen (PCNA) protein and various histone modifications were determined in full-fed and restricted-fed broiler breeder hens. PCNA protein immunostaining was more intense in the granulosa cell layer in full-fed hens compared to restricted-fed hens, indicating increased proliferation. Trimethylated H4K4 was uniform throughout the ovarian cortex, while trimethylated H3K27 and acetylated H4K9 were higher in the granulosa cells compared to the surrounding stroma, but did not differ between full-fed and restricted-fed hens. Thus, increased granulosa cell proliferation is not due to changes in these specific histone modifications. A growing body of literature provides evidence of a prominent role for bone morphogenetic protein 6 (BMP6) in regulating granulosa cell differentiation at the time of follicle selection. Recombinant human BMP6 promoted follicle stimulating hormone receptor (FSHR) mRNA expression in a dose-related fashion. Enhanced follicle stimulating hormone (FSH)-responsiveness after culture with BMP6 was indicated by increased steroidogenic acute regulatory protein expression and progesterone production following a challenge with FSH. We conclude that at the time of follicle selection, BMP promotes granulosa cell differentiation. BMP6 also increased expression of Anti-Mullerian Hormone (AMH) mRNA in cultured prehierarchal (6-8 mm) follicles. AMH has been implicated in negatively regulating FSH-responsiveness during follicle development. We speculate that prior to selection one action of AMH may be to suppress FSHR signaling and prevent premature granulosa cell differentiation. Finally, efforts have focused on the role of biological clock regulation of reproductive performance. Our work suggests that biological clocks determine the timing and frequency of ovulation and, ultimately, the number of eggs in a laying sequence. In addition, melatonin and "clock genes" (e.g., Period, Cryptochrome, etc.) play fundamental roles in the process of eggshell formation.

Publications

• Ocon-Grove, O. M., D. H. Poole, and A. L. Johnson. 2011. Bone morphogenetic protein 6 (BMP6) promotes FSH-responsiveness AND Anti-Mullerian Hormone (AMH) mRNA expression in undifferentiated granulosa cells from hen prehierarchal follicles. Proceedings of the 44th Annual Meeting of the Society for Study of Reproduction. Portland, OR. (Abstract #662).
• Diaz, F. J., A. N. Halfhill, and K. Anthony. 2011. Early avian follicular development is characterized by changes in transcripts involved in steroidogenesis, paracrine signaling and transcription. Molec. Reprod. Develop. 78:212-223.
• Kumar, V., J. C. Wingfield, A. Dawson, M. Ramenofsky, S. Rani, and P. Bartell. 2010. Biological clocks and regulation of seasonal reproduction and migration in birds. Physiol. Biochem. Zool. 83:827-835.
• Horvat-Gordon, M., C. Praul, R. Ramachandran, P. A. Bartell, and R. M. Leach. 2010. Use of microarray analysis to study gene expression in the avian epiphyseal growth plate. Comp. Biochem. Physiol. Part D Genomics Proteomics 5:12-23.
• Diaz, F. J., K. Anthony, and A. N. Halfhill. 2010. Effect of feed restriction on early avian follicular development (2010). Proceedings of the 43th Annual Meeting of the Society for Study of Reproduction. Milwaukee, WI. (Abstract #598).