Location: Animal Biosciences & Biotechnology Laboratory
2024 Annual Report
Objectives
Objective 1: Determine biomarkers for turkey hen fertility and assess the functional aspects of cryoconserved ovarian tissue.
Sub-objective 1.A: Establish genetic and protein biomarkers associated with hen fertility rates that can be employed for selective breeding decisions.
Sub-objective 1.B: Determine global gene and protein expression differences in sperm storage tubule (SST) molecular function between low and high fertility hens.
Sub-objective 1.C: Determine the ability of a short-term in ovo culture system to predict the viability and maturation potential of immature turkey ovarian tissue in a cryopreservation program.
Objective 2. Define methods for identifying cryopreservation potential and mitigating the negative impacts associated with cryopreservation in poultry semen.
Sub-objective 2.A: Identify biomarkers associated with cryopreservation tolerance through the comparison of poultry lines with superior and poor cryopreservation tolerance.
Sub-objective 2.B: Evaluate the efficacy of straw-in-straw vitrification, non-permeable cryoprotectants, and mitochondria-targeted antioxidants for the preservation of poultry semen.
Sub-objective 2.C: Identify molecular and cellular mechanisms associated with early embryonic mortality in turkey embryos originating from insemination with frozen/thawed semen.
Objective 3: Determine biomarkers associated with superior or inferior fertility in boars and elucidate the molecular mechanisms behind reduced fertility in stored swine semen.
Sub-objective 3.A: Identify biological and functional parameters associated with fertility in fresh semen.
Sub-objective 3.B: Elucidating biomarkers of swine fertility through integrative omic analysis in fresh semen.
Sub-objective 3.C: Physiological Factors Impacting Semen Cryopreservation in Swine.
Objective 4: Rejuvenate poultry research lines from cryopreserved semen using both surgical and non-surgical approaches.
Approach
The long-term goals of this Project Plan are to improve the efficiency of reproduction and germplasm preservation in swine and poultry to meet the demands of feeding a growing human population. Reproductive traits exhibit low heritability and cannot be phenotypically measured prior to sexual maturity. Moreover, the ability to recover swine and poultry lines from frozen/thawed semen continues to be unreliable. The central focus areas of this Project Plan are to provide the poultry and swine industries with the knowledge and tools to (1) predict fertility, (2) store semen under hypothermic conditions without a substantial loss in fertility, and (3) preserve the female genetic contribution through the development of assisted reproductive technologies for complete regeneration of poultry lines. To enable the prediction of fertility, genetic and biological markers associated with sperm quality and female fertility rates will be identified through comprehensive omic profiling. Several approaches will be used to improve cryoconservation of male and female germplasm, including: 1) physiological and omic assessments of poultry and swine males with known semen cryotolerance or cryosensitivity; 2) investigation into the molecular mechanisms leading to the high incidence of early embryonic death when frozen/thawed turkey semen is used for insemination; 3) further development of technology to preserve ovary vitrification and transplantation in the turkey; 4) investigation of poultry sperm vitrification; and 5) refinement of assisted reproductive technologies to regenerate poultry lines from cryobanked germplasm. All these approaches will contribute to improving the efficacy of reproductive technologies in the poultry and swine industries, which is the single most critical challenge to efficient food animal production.
Progress Report
National Program 101, Food Animal Production and Protection, was approved in November 2022. Progress was made for most Sub-objectives, including earlier progress planned for one Sub-objective despite unanticipated delays in completion of, (1) the new poultry quarantine facility and (2) roof replacement for the main poultry building (awarded contract was disputed), as well as the loss of key Veterinary staff.
Under Sub-objective 1A, initiation of experimental trials is pending completion of the poultry quarantine facilities, expected August 2024. Meanwhile, follicle development-based gene expression was evaluated in research turkey lines that have been selected for superior egg production rates.
Under Sub-objective 1B, initiation of experimental trials is pending completion of the poultry quarantine facilities, expected August 2024. In the meantime, the radioactive isotope permit required to complete proposal experimental trials received approval in January 2024. Radioactive radioimmunoassays that will be used in experimental trials are currently being validated. In addition, sperm storage tubule global gene expression was evaluated through RNA sequencing in virgin, sham-inseminated, and semen-inseminated turkeys, resulting in manuscript publication.
Under Sub-objective 1C, data from the in-ovo experimental trial using fresh tissue and the in-ovo experimental trail using frozen-thawed tissue (conducted January-February 2023), including gene expression analysis and histology, resulted in manuscript publication. The experimental trials and analysis for this sub-objective are complete.
Under Sub-objective 2C, initiation of experimental trials is pending completion of the poultry quarantine facilities, expected August 2024. Meanwhile, yolk samples from embryos exhibiting normal development and from embryos that had succumbed to early embryonic mortality are currently being assessed for lipid composition using multiple reaction monitoring (MRM) profiling methods. Results from this pilot study will identify lipids associated with early embryonic mortality that can be targeted in experimental trials. In addition a Research Support Agreement was initiated with University of Maryland in June 2024 to support a graduate student involvement in the data analysis.
Under Sub-objective 3A, validation of the sperm mobility assay for boars was fully completed. The ideal sample volume, reagent concentration, time and temperature for the assay to run correctly was determined. Following assay validation, semen samples from a commercial boar stud from boars with known fertility were shipped to ARS researchers in Beltsville, Maryland, to begin the fertility prediction component of the assay. Boars with high conception rate (>90%, HF) versus low (<75%, LF) were enrolled in the study (P<0.01). These numbers were selected based on industry cull standards where boars with less than 75% conception rate are culled from the breeding herd. Interestingly, boars with a low conception rate had a notably higher absorbance when compared to the high fertility boars which is the opposite case for poultry mobility phenotype. Following zinc signature assay completion, a statistical model is being created to compare current on-farm indicators of boar fertility, including zinc signature status, and determining which indicator or indicators are the best predictive measure of fertility. We are also utilizing a machine learning approach to hopefully identify subfertile boars based on these parameters.
Under Sub-objective 3B, boars from the same training cohort have been collected over the last two years. When their ejaculate met quality parameters for the first time, (i.e. they can enter the breeding herd) an aliquot of whole semen processed in methanol was sent to ARS researchers in Beltsville, Maryland, and stored at -80C until ready for lipid extraction. Fertility data from these boars are being recorded and will be ready for fertility phenotype characterization following 50 single-sire matings.
Under Sub-objective 3C, thanks to sample processing developments made in 2023, we were able to make earlier progress for this sub-objective towards the Milestone for year 4. Commercial boars with known fertility and semen quality parameters post-thaw were collected. An aliquot of fresh semen was processed in Methanol and shipped overnight to ARS researchers in Beltsville, Maryland, and stored at -80C until ready for lipid and metabolite extraction. Six straws from the same ejaculate were frozen in liquid nitrogen to be used for future lipidomic and metabolomic analysis, measurements of viability and acrosomal integrity, and in-vitro fertilization. Boars were then characterized by their post-thaw “survivability”, a term we coined based on their motility following thawing. Boars were characterized as high or low survivability based on the direction they deviated from the mean change in post-thaw motility. The change in motility post-thaw between high versus low survivability were different, with the greater reduction in motility belonging to the low survivability group (P<0.01). Acrosomal integrity, viability, lipidome/metabolome, and embryological differences following in-vitro fertilization will be evaluated amongst high versus low survivability to greater characterize cryotolerance in boars and identify biomarkers associated with cryotolerance beyond post-thaw motility. Lipid/metabolite extraction and detection has been completed.
A group of 280 female broiler breeders and 80 male broiler breeders were delivered in September 2023 to Auburn University for a 65-week study. To date, sample collection has been completed for 9 of the 12 timepoints, with the study continuing until December 2024.
Accomplishments
1. Differentiating sperm storage tubule expression due to artificial insemination and the presence of semen. The mechanisms that allow for sperm survival in oviductal sperm storage tubules (SST) and how poultry industry practices, such as artificial insemination, impact the biological functions of these specialized structures are currently unknown. SST function directly relates to hen fertility rates, which is important for poultry industry efficiency. USDA-ARS researchers in Beltsville, Maryland, utilized laser capture microdissection coupled with RNA sequencing technologies to define SST-specific gene expression in virgin, sham-inseminated, and semen-inseminated turkeys to interrogate changes due to the process of insemination and to the presence of resident sperm. In the sham-inseminated SSTs, increased expression of immune pathways and nonreproductive endocrine hormones were observed, while in the semen-inseminated SSTs, increased expression of reproductive pathways, cellular structure and metabolism, and pH regulation of the oviduct were observed. Results from this study will help to outline industry practices regarding turkey artificial insemination to maximize SST function, ultimately driving fertility outcomes.
2. Poultry semen lipidome characterization and identification of sperm mobility associated lipids. Semen lipids are essential for sperm function, regulating sperm membrane integrity, signaling processes, and sperm movement. Reliable fertility biomarkers are needed in the poultry industry, with lipid biomarkers emerging as potential predictors of the fertility correlated sperm mobility phenotype. USDA-ARS researchers in Beltsville, Maryland, were the first to characterize the global lipid profile (lipidome) of poultry sperm cells, seminal plasma, and whole semen using multiple reaction monitoring (MRM) profiling, detecting a total of 926 unique lipid species. Low and high sperm mobility samples were compared to identify a total of 74 sperm mobility associated lipid species, with biological functions in sperm cell membrane lateral diffusion and lipid bilayer thickness. Of the sperm mobility associated lipid species, 23 lipid species were identified as excellent biomarkers of the sperm mobility phenotype and were detectable in whole semen, lending to feasibility for screening in an industry setting. Identified lipid biomarkers of sperm mobility in poultry semen will assist with making accurate predictions of and selection for male fertility, a desirable trait in the primary breeder sector of the poultry industry.
Review Publications
Yadav, S., Widmar, N.O., Lay Jr., D.C., Croney, C., Weng, H. 2016. Evaluation of movement restriction zone sizes in controlling classical swine fever outbreaks. Frontiers in Veterinary Science. 3:124. doi: 10.3389/fvets.2016.00124.
Mills, K.M., Sheets, J., Teeple, K., Mann, A., Suarez-Trujillo, A., Stewart, K., Ferreira, C., Casey, T. 2023. Low colostrum intake results in accumulation of peroxisome lipid substrates in stratified epithelial vaginal tissue of 3 week old gilts. Biology Open. https://doi.org/10.1242/bio.060044.
Mills, K.M., Mahoney, J.A., Duttlinger, A.W., Elefson, S.H., Radcliffe, J.S., Rambo, Z.J., Richert, B.T. 2024. Effect of chronic cyclic heat stress and supplemented inorganic and organic zinc source levels on grow-finish pig growth performance and estimated body composition. Translational Animal Science. 8. Article etxae029. https://doi.org/10.1093/tas/txae029.
Diehl, K.M., Liu, H., Hicks, J., Long, J.A., Porter, T.E. 2023. Global gene expression of the turkey hen reproductive axis during the preovulatory hormonal surge. Poultry Science. 102(4). Article e102547. https://doi.org/10.1016/j.psj.2023.102547.
Diehl, K.M., Hanlon, C., Krasnec, K., Long, J.A. 2024. Turkey hen sperm storage tubule transcriptome response to artificial insemination and the presence of semen. Frontiers in Physiology. 14(1). Article e1305168. https://doi.org/10.3389/fphys.2023.1305168.