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ARS Home Northeast Area Beltsville, Maryland (BARC) Beltsville Agricultural Research Center Animal Biosciences & Biotechnology Laboratory Research





- Long, Julie
- Mills, Kayla
- Brady, Kristen
- 2023
- Publications
- Food Animal Production (101)

Research Project: Development of New Technologies and Methods to Enhance the Fertility, Utilization, and Long-Term Storage of Poultry and Swine Germplasm **Location:
                            Animal Biosciences & Biotechnology Laboratory** 2023 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**
                                

This is the first annual report for project 8042-535-111-000D, which supports National Program 101, Food Animal Production and Protection, and was approved in November 2022. Progress was made for several sub-objectives, including earlier progress than was planned (e.g., milestones for Years 2 and 3 under Sub-objective 1.C.) despite an unanticipated delay in the completion of the new poultry quarantine facility under the auspices of the Bureau of Engraving and Printing as part of the transfer of USDA land to that Agency. 

For Sub-objective 1A, an Institutional Animal Care and Use Committee (IACUC) animal use protocol regarding the experimental trials for this sub-objective was submitted in June 2023. Existing turkey reproductive RNA sequencing datasets were mined to identify genes exhibiting differential expression in response to the preovulatory surge that triggers follicle ovulation, which is the first step of generating a fertile egg. In addition, a RSA was initiated with the University of Minnesota to determine genes associated with follicle development rates in poultry. The identified genes associated with follicle ovulation and/or follicle development will be further examined following identification of SNPs associated with fertile egg production to determine if these genes are directly or indirectly regulated by the genetic variation among hens with differing fertility rates. Initiation of experimental trials is pending IACUC approval and completion of the poultry quarantine facilities, expected August 2023.

For Sub-objective 1B, an IACUC animal use protocol regarding the experimental trials for this sub-objective was submitted in June 2023. Purchase of a gamma counter to measure steroid hormone levels for this sub-objective was submitted to procurement. In addition, a radioactive isotope permit application was submitted to allow for purchase and use of radioactive materials necessary for radioimmunoassay assessment of plasma steroid hormones in this sub-objective. The global gene expression in the vagina was examined in sham and semen inseminated turkey hens at early, peak, and late timepoints in the laying cycle. The vaginal transcriptome was compared to existing sperm storage tubule datasets (also exposed to sham and semen inseminations) to determine sperm storage tubule specific genes for further analysis in sperm storage tubules isolated from low and high fertility turkey hens. Initiation of experimental trials is pending IACUC approval and completion of the poultry quarantine facilities, expected August 2023.  

For Sub-objective 1C, the IACUC animal use protocol received final approval in January 2023. The in-ovo experimental trial using fresh tissue was conducted in January 2023 and the in-ovo experimental trail using frozen-thawed tissue was conducted in February 2023. For both trials, gene expression analysis has been completed on 10 genes of interest and 3 housekeeping genes for all samples. Resulting gene expression data was statistically analyzed to determine 6, 3, and 5 genes with mRNA levels impacted by timepoint, vitrification, and vitrification method, respectively. Most significantly, 3 novel gene biomarkers (GDF9, FSHR, and HSPA2) were identified from the gene expression analysis exhibited clear differences between poorly vitrified and optimally vitrified ovarian samples. These biomarkers are associated with ovarian development potential and will allow for screening of suboptimal ovarian samples post-vitrification. Histology analysis of all experimental samples is currently in progress. Further a collaboration was initiated with University of Guelph to evaluate germ cell nest breakdown in turkey ovarian samples from two specialized research lines housed at Beltsville, Maryland, which is the first step to determining if the donor age and in-ovo culture system developed for commercial line turkeys can also be extended to include other turkey lines, such as research lines and/or heritage lines.

For Sub-objective 2C, an IACUC animal use protocol amendment was submitted based on results from preliminary trials to ensure sufficient animal numbers for planned RNA sequencing efforts for this sub-objective. Several projects have been initiated to elucidate gene regulatory mechanisms leading to early embryonic mortality that occur when cryopreserved semen is used for insemination: (1) Transcriptome analysis was performed on deteriorating embryonic blastoderms to identify key genes and pathways involved during early embryonic mortality, (2) A MTA was recently initiated with Maple Lodge Farms Ltd. in Canada to examine blastoderm gene expression in flocks with low and high early embryonic mortality rates, and (3) Targeted gene expression analysis is currenting in progress to examine shifts in sperm storage tubule gene expression resulting from cryopreserved semen usage. Initiation of experimental trials is pending IACUC approval and completion of the poultry quarantine facilities, expected August 2023.

For Sub-objective 3A, semen samples from a commercial boar stud were collected and picked up at a local sow farm. Progress towards assay validation was performed in several steps: 1) identify the ideal temperature at which to run the assay, 2) identify the appropriate length of time to run the assay to completion 3) identify ideal concentration of sperm cells to perform the assay, 4) identify the ideal concentration of accudenz 5) identify whether sperm mobility buffer is necessary to complete the assay. During this time, we also re-calibrated our computer assisted sperm analysis (CASA) machine to provide objective measurements of sperm quality to run alongside the sperm mobility assay to enhance our results. Our final step in assay validation is to run the sperm mobility assay with its ideal parameters alongside the newly calibrated CASA prior to incorporating it into a fertility trial, which is a milestone for year two of the project plan. In addition, the scientist team for this sub-objective traveled to Iowa State University to learn how to run the zinc signature assay which is an anticipated milestone for year two of the project plan. 

For Sub-objective 3B, semen samples from a commercial boar stud were collected, processed, and sent to Purdue University for method validation on the mass spectrometer for lipidomic analysis and multiple reaction monitoring (MRM) profiling. Because MRM profiling is a novel approach to evaluating boar semen samples for fertility biomarkers, we needed to determine if the mass spectrometer could handle the high concentration of sperm cells in a boar’s ejaculate. A very high concentration of lipids that result in high ion intensity readings can mask lipids that have lower concentrations within samples. Because the objective of this study is to identify biomarkers associated with fertility, we need to ensure that we can identify as many lipids as possible. It was also important to confirm that a subsample from a raw ejaculate can be sent in for testing as this is crucial for future transfer of this technology into breeding selection programs. In addition, we were faced with a secondary challenge at the boar stud where they lacked freezing capabilities which is commonplace for industry. Therefore, we developed an alternative method to freezing samples and by arresting any biochemical processes through the addition of methanol to semen samples prior to shipping. Removing the need for a freezer will make this kind of testing more feasible to producers when the time comes to implement this assay. Following preliminary lipidomic analysis, we confirmed that preserving the samples in methanol works as an alternative to freezing in -20C and the mass spectrometer can handle the high concentration of sperm cells in a boar’s ejaculate. The MRM profiling identified a total of 341 MRMs, which is a more robust result than we have seen in the past with our previous work done in gilts. Phosphatidylcholines (PC) represented 40% of the lipids profiled. Ceramides (Cer) represented, Phosphatidylethanolamines (PE), Acylcarnitines (AC), and Phosphatidylserines (PS) represented 15%, 11%, 8%, and 7% of the sperm cell lipidome signature, respectively. Thus, the majority (73%) of the sperm cell lipidome was comprised of cell membrane lipids, which are represented by the PC, Cer, PE, and PS lipid classes. Following these validation steps, semen samples from the commercial boar stud were sent to Beltsville, Maryland, for processing and storage until fertility data is available to determine fertility phenotypes in year two of this project plan. Lastly, while we have been accumulating boar fertility data to complete our milestones for year 3, we are in the process of developing an additional screening method to detect seminolipid, which is a sulfogalactosylglycerolipid associated with subfertility in mice.  

For the CRADA (8042-31000-111-001C), a group of 40 male broiler breeders were delivered in May 2023 and began a 30-day quarantine prior to initiation of short-term semen storage studies in July 2023 for in vitro assays to assess carbohydrate, lipid, and protein complement of sperm cells in response to differing time and temperatures.

For the NIFA-AFRI award ‘Regulation of Reproduction in Turkey Hens by Thyroid Hormones’ (8042-32000-111-003I), the initiation of the 3-year grant began May 2023. The IACUC animal use protocol received final approval in May 2023. The NACA with University of Maryland was initiated June 2023 and is pending approval. Initiation of experimental trials is pending completion of the poultry quarantine facilities, expected August 2023. **Accomplishments**
                                

1. Assessing the viability and development potential of vitrified immature turkey ovarian tissue. Assessing the viability and development potential of vitrified immature turkey ovarian tissue. Ovarian cryoconservation is a critical tool for genetic resource preservation of poultry lines. The success of this effort requires optimal freeze-thaw protocols and the ability to screen for suboptimal donor samples prior to recipient transplantation of the tissue. ARS scientists in Beltsville, Maryland, utilized an in ovo culture system to compare fresh, poorly vitrified, and optimally vitrified immature turkey ovaries to identify three novel gene biomarkers associated with ovarian viability and follicle growth, both of which are indicators of ovarian development potential post-transplantation. The identified gene biomarkers are oocyte-secreted factors (GDF9), follicle cell steroid hormone receptors (FSHR), and cell stress indicators (HSPA2), providing a rounded assessment of ovarian tissue viability, function, and damage post-vitrification. This breakthrough in turkey ovarian cryoconcervation efforts, is the first report of a screening method to identify suboptimal donor ovarian samples in turkeys. The developed technology will ensure transplantation of ovarian samples with high development potential, greatly improving turkey cryopreservation success rates. **Review Publications**
                            *Potts, S., Brady, K.M., Scholte, C.M., Moyes, K.M., Sunny, N.E., Erdman, R.A. 2023. Rumen-protected choline and methionine during the periparturient period affect choline metabolites, amino acids, and hepatic expression of genes associated with one-carbon and lipid metabolism. Journal of Dairy Science. 102 (4):102547. https://doi.org/10.1016/j.psj.2023.102547.*
                                *Brady, K.M., Krasnec, K., Long, J.A. 2022. Transcriptome analysis of inseminated sperm storage tubules throughout the duration of fertility. Poultry Science. 101:101704-101722. https://doi.org/10.1016/j.psj.2022.101704.*
                                *Brady, K.M., Talbot, C.C., Long, J.A., Welch, G., French, N., Dinah, N., Bakst, M. 2022. Transcriptome analysis of blastoderms exposed to prolonged egg storage and short periods of incubation during egg storage. BMC Genomics. 23:262-286. https://doi.org/10.1186/s12864-022-08463-2.*
                                *Vonderohe, C., Mills, K.M., Liu, S., Asmus, M.D., Otto-Tice, E., Richert, B.T., Ni, J., Radcliffe, J. 2022. The effect of reduced cp, synthetic amino acid supplemented diets on growth performance and nutrient excretion in wean to finish swine. Journal of Animal Science. https://doi.org/10.1093/jas/skac075.*
                                *Hall, G.B., Long, J.A., Beeler, M.J., Wood, B.A., Bedecarrats, G.Y. 2022. Cyclosporin A prevents ovarian graft rejection, and permits normal germ cell maturation within the first 5 weeks post-transplantation, in the domestic turkey (Meleagris gallopavo). Frontiers in Veterinary Science. 9:855164. https://doi.org/10.3389/fvets.2022.855164.*


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