Recipient Organization
UNIV OF MINNESOTA
(N/A)
ST PAUL,MN 55108
Performing Department
Animal Science
Non Technical Summary
The turkey industry relies on parent breeders to lay fertile eggs that will be hatched and raised for meat. In 2018 alone, 466 million meat turkeys were produced globally. This requires a large number of hens as each hen only produces up to 60 poults. Further, turkey hens do not start laying until they reach 32 to 35 weeks of age. By this age, a flock of 1000 hens would have consumed approximately 8,275 gallons of water, 121,600 pounds of feed, and required up to 6,018 Kwh of electricity. After only 10 to 14 weeks of lay (WOL), the fertility of the breeders declines by up to 1% each week. By week 26-28 WOL, the flock is turned over. Determining ways to improve turkey fertility and extend laying time, offers an opportunity to increase production while conserving resources, and reducing the number of breeders needed to produce the same number of progeny. Even a 1% increase in fertility will lead to 1000 more poults per 1000 breeder flock.Here, we propose to explore the genetic factors that may be responsible for the decline in turkey breeder fertility. In addition, we propose to look at the role of inflammation, and if reducing it in older turkey breeders will lead to improved fertility. The goal of this study is gain a deeper understand of the root causes of turkey breeder decline in fertility and help, at least partially, reverse it. The knowledge gained in this study can serve to solve similar issues encountered in the boiler breeder industry.The avian ovary is unique in that it simultaneously contains follicles at all stages of development. These stages of development are not necessarily regulated by the same mechanisms; yet they coexist in the ovary and their development is coordinated so that the end product is one egg a day for the duration of a clutch. Many problems encountered when ovarian function is dysregulated, as is the case in broiler breeders, reside in the dysfunction of important regulatory steps in early follicle development. Laying hens have served as excellent model organisms to study avian and vertebrate reproduction. They are particularly well suited to study ovarian follicle development. Because they lay eggs all year around, their ovarian function is extremely well regulated. Their ovulatory cycle is ~24 hours, and their one functional ovary contains follicles at all stages of development at any given time. Understanding the reproductive function of avian species, using layers as a model organism, and specifically the mechanisms of follicle development, will help address many issue pertaining to the dysregulation of follicle development in the broiler breeders, and maximize ovarian function in endangered avian species.Here we propose to use the laying hen ovary as a model to investigate the role of the oocyte during early follicle development by looking at the changes in gene expression that occur as the follicle develops. In addition, we are proposing to explore the role of relaxin 3, which is a neuropeptide that we recently discovered to be differentially expressed in avian ovarian follicle. Understanding the role of relaxin 3 will help us understand how different compartment of the avian ovarian follicle communicate and coordinate to grow in a timely fashion. Results from this study will allows us to initiate future studies that will look at follicle dysregulation in broiler breeders as well as how to maximize ovarian function in endangered avian species in breeding programs.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Goals / Objectives
Goal #1:Prolonging Fertility in Turkey Breeders a Sustainable Model for Poultry ProductionObjective 1:Determine the age-related changes in gene expression of the vagina and the uterovaginal junction of turkey hens.Objective 2:Determine if inflammation leads to decreased sperm retentionGoal #2:Investigate the role of follicle compartments and their interactions in ovarian follicle developmentObjective 1: Determine the changes in localization and gene expression of the oocyte nucleus during early stages of follicle development.Objective 2: To determine the role of relaxin 3 during early follicle development.
Project Methods
Efforts: To address objective 1 two experiments have been designed.Experiment 1. Determine the age-related changes in gene expression of the vagina and the uterovaginal junction of turkey hens. Biopsies from the vagina and UVJ will be taken from both inseminated (n=100) and virgin (n=100) at four time points: (1) after photo-stimulation/before insemination (2) after insemination/before onset of lay (3) after onset of lay/at peak fertility, week 40 of age, and (4) at end of lay/low fertility, week 55 of age. Based on their fertility record, the turkeys will be retrospectively divided into two groups based on fertility level. Total RNA will be extracted from vaginal and UVJ tissue for virgin, low-fertility, and high-fertility groups from experiment 1 at all four time-points (n=6/group). The RNA samples will be sent to the University of Minnesota Genomic Center for Illumina TruSeq Stranded mRNA sequencing at standard scale. Quality analysis and pre-processing of RNAseq data will be conducted by the UMGC. Sequence alignment to the reference genome assembly 5.0 (GCA_000146605.3), measure of transcript abundance, and differential expression analysis will be performed by using the Bioconductor package in R.Experiment 2. Determine if inflammation leads to decreased sperm retention. Turkeys retrospectively designated as either high fertility or low fertility in Experiment 1 will be treated with injection doses of Carprofen, 22 weeks after onset of lay. Within each fertility level, turkeys will be assigned to either: (1) a no Carprofen control or (2), Carprofen treatment. Sperm retention in SST will be assessed via histology, and fertility will be assessed via percent egg hatchability. The expression of inflammatory markers in the vagina, the uterovaginal junction, and the population of immune cells will be measured using NanoString technology for immune exhaustion panel and immune cell profiling. The effect of NSAID dosage and fertility on response variables will be tested as a 2 x 2 factorial design using a generalized linear model and preplanned contrasts will be used to compare means across all treatments.Evaluations: In experiment 1 described above will be considered successful if the gene expression profile of turkey breeders at 4 different ages are characterized. This will allow for comparisons to be made among the age-groups, and for the identification of potential target genes that may help improve and prolong fertility in turkey breeders. Experiment 2 will be considered successful if we determine if reducing inflammation in breeders results in a 1% increase or more in fertility.To address goal #2 following experiments were designedExperiment 1. Localize the nucleus of the oocyte at various stages of development Ovaries will be harvested from White Leghorn hens (n=3) with fully functioning ovaries, as indicated by an 8 to 10 egg laying sequence. Follicles between 1mm and 12mm in diameter will be dissected and fixed in 10% neutral buffered formalin solution for 24 hours. Fixed follicles will be embedded in paraffin. Cross sections will be processed and stained with hematoxylin and eosin (H&E). The distance between the nucleus and perivitelline membrane will be measured using Image J software. A simple linear regression analysis will be conducted to determine if there is a relationship between the developmental stage of the follicle and the distance between its nucleus and its vitelline membrane.Experiment 2. Develop a method to isolate oocyte nuclear and cytoplasmic content at different stages of development. The nucleus and the cytoplasm contain different yet overlapping populations of RNA transcripts. With each method we will be able to extract nuclear RNA versus total RNA. Ovaries will be harvested from four White Leghorn hens (n=3) with fully functioning ovaries, as indicated by an 8 to 10 egg laying sequence. Follicles between 1mm and 12mm in diameter will be dissected using the following methods. RNA will be extracted, quantified, analyzed for integrity.Experiment 3. Determine the changes in gene and protein expression in the oocyte during follicle development. The RNA samples from experiment 2 will be sent to the University of Minnesota Genomic Center for Illumina TruSeq Stranded mRNA sequencing at standard scale. Quality analysis and pre-processing of RNAseq data will be conducted by the UMGC. Sequence alignment to the reference genome assembly 5.0 (GCA_000146605.3), measure of transcript abundance, and differential expression analysis will be performed by using the Bioconductor package in R. Transcript abundance and differential expression will be compared among stages of follicle development.Experiment 4. Determine specific cells in the theca tissue and stroma in which the relaxin 3 receptors RXFP1 and RXFP3 are expressed: Ovaries will be harvested from White Leghorn hens (n=3) with fully functioning ovaries, as indicated by an 8 to 10 eggs per sequence. Follicles 1 mm, 2 mm, 4 mm, 6 mm, and 9 mm, F3, F1, and in the ovarian stroma will be dissected and fixed in 10% neutral buffered formalin solution. Fixed samples will be embedded in paraffin. Cross sections will be processed for immunohistochemistry to localize relaxin 3 receptors RXFP1 and RXFP3 in these tissues.Experiment 5. Generate recombinant chicken relaxin 3. In a first step, chicken recombinant relaxin 3 will be produced in a bacterial protein expression system using relaxin 3 cDNA generated from GC RNA. The cDNA will then be cloned into an expression plasmid with an N-terminal epitope tag and a protease cleavage site between these tags and the cDNA. The expression plasmid will be transformed into competent E. Coli cells and colonies that successfully incorporated the plasmid will be expanded for plasmid extraction and analysis. Plasmid that successfully incorporated the cDNA will be transformed into competent E.Coli cells that the protein expression will be induced. Necessary steps will be taken to optimize expression and protein yield. Protein will be purified using affinity column purification. The N-terminal fusion tag will be subsequently removed using the appropriate protease. In a second step, the resulting chicken recombinant relaxin 3 will be tested for bioactivity by its ability to induce cyclic AMP in cells that express the RXFP1. Production cyclic AMP will be measured using precise enzyme-linked immunosorbent assay, ELISA, assay.Experiment 6. Determine the effect of relaxin 3 on gene expression of theca and cortical cells. Theca and cortical cells that express RXFP1 and RXFP3, respectively will be sorted and isolated using Fluorescence-activated cell sorting (FACS). The cells will then be incubated with the chicken recombinant relaxin 3. The global gene expression of TH and cortical cells in response to relaxin 3 will be determine using RNA sequencing, described in objective 2. Experiment 2. Prior to sequencing, the appropriate dose of relaxin 3 and time of incubation will be determined, empirically with a dose response curve and a timeline experiments. Results from this experiment will inform future studies that will be conducted to determine the role of relaxin 3 in the development of avian ovarian follicles.Evaluation: Experiments for the second goal will be considered successful if we are able to determine the localization of the nucleus of the oocyte as it progresses in development along with the gene expression profile.Experiment designed to investigate the role of relaxin 3 during follicle development will be considered successful if we are able to synthesize a bioactive chicken relaxin 3 and localize its binding site within the follicle. In addition, we will be able to determine the gene expression profile of specific follicle compartments in response to relaxin 3.