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ARS Home » Northeast Area » Beltsville, Maryland (BARC) » Beltsville Agricultural Research Center » Animal Biosciences & Biotechnology Laboratory » Research » Research Project #430411

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

2022 Annual Report

Objective 1: Identify genetic markers and semen biological markers that can effectively predict fertility traits in poultry and swine. • Sub-objective 1.A. Discover biomarkers associated with the high and low sperm mobility phenotypes in adult male poultry. • Sub-objective 1.B. Delineate genetic markers associated with the high and low sperm mobility phenotypes in poultry and use to predict the phenotype of sexually immature males. • Sub-objective 1.C. Identify biological and/or functional parameters associated with fertility in boars. • Sub-objective 1.D. Elucidate genetic markers associated with high and low fertility in boars. Objective 2: Determine the contribution of genetics and other factors towards the survival and fertility of frozen/thawed semen in poultry. • Sub-objective 2.A. Develop a turkey line with superior sperm function by selecting for the duration of fertility of frozen/thawed semen. • Sub-objective 2.B. Characterize sperm function and protein expression of males from superior cryosurvival lines and compare with unselected lines. • 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: Delineate the molecular and physiological mechanisms associated with in vivo sperm storage and duration of fertility in poultry. • Sub-objective 3.A. Characterize gene expression of sperm storage tubules from virgin, high-fertility and low-fertility hens and identify genetic markers associated with duration of fertility. • Sub-objective 3.B. Identify biological pathways associated with the function of sperm storage tubules. Objective 4: Develop ovarian cryoconservation for the turkey to fully capture the female genetic contribution and augment germplasm cryopreservation efforts. • Sub-objective 4.A. Characterize the ovarian morphology of young female poults and determine the optimal age for ovary vitrification. • Sub-objective 4.B. Identify the optimal recipient age and develop an immunosuppressant protocol to prevent rejection of donor tissue. • Sub-objective 4.C. Use optimized methods to recreate a unique research line from vitrified ovaries.

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 phenotypically cannot be measured prior to sexual maturity. Moreover, the ability to recover poultry lines from frozen/thawed semen continues to be unreliable. Central focus areas of this Project Plan are to provide the swine and/or poultry industries with the knowledge and tools to (1) predict male fertility, (2) store semen under hypothermic conditions without a substantial loss in fertility, and (3) preserve the female genetic contribution for complete line regeneration. To enable prediction of male fertility, males with known fertility will be evaluated for genetic and biological markers associated with the sperm mobility phenotype and the sperm zinc signature. Several approaches will be used to improve hypothermic semen storage, including: 1) development of a cryoresistant turkey line (e.g. selected for superior sperm cryosurvival) to elucidate biological attributes associated with superior sperm cryosurvival; 2) an investigation of why there is such a high incidence of early embryonic death when frozen/thawed turkey semen is used for insemination; and 3) identifying biological pathways associated with sperm storage tubules in the hen to better mimic the in vivo semen storage environment and improve in vitro storage conditions. Finally, cryopreserved semen alone is not adequate for complete line regeneration in birds because the female gamete determines gender and the biology of the avian egg prevents traditional cryopreservation of this gamete. Transplanting frozen/thawed immature ovarian tissue will be investigated as a means to preserve female germplasm for the turkey. All these approaches will contribute to improving reproductive efficiency.

Progress Report
This is the final year of the project. Progress was made for some of the sub-objectives. For Sub-objective 1C, a new Lead scientist, hired in August 2021 to fill a critical vacancy, established a new collaboration with a regional boar stud to further evaluate the sperm mobility assay in swine semen. Efforts to determine the optimal temperature (37°C vs 39°C vs 41°C), sperm concentration (1x108 vs 5x107 vs 1x107), and necessity of sperm mobility buffer are underway. For Sub-objective 2C, results from a pilot study completed in May 2021 indicated that changes needed to be made to the Institutional Animal Care and Use Committee (IACUC) protocol regarding embryo isolation and required animal number. Amendments have been drafted and are in the process of submission to the IACUC panel for review. Pending the state of avian influenza in the U.S., the main experiment is tentatively scheduled for September 2022. For Sub-objective 3A, read quality and read trimming have been completed for the second set of sperm storage tubule samples aimed at determining the impact on gene expression due to pre-lay inseminations. Currently, samples are mapping to the latest turkey genome in preparation for differential expression analysis. For Sub- objective 3B, potential upstream and master regulators, classical molecular pathways, and key networks of sperm storage tubule duration of fertility were identified using differential gene expression from inseminated sperm storage tubule samples isolated at various timepoints throughout the fertility window. Currently, the same extended analysis is being conducted using the differential gene expression output from the virgin, sham-inseminated, and semen-inseminated sperm storage tubule samples to determine regulators and key pathways/networks governing the changes in gene expression observed. For Sub-objective 4C, a series of studies were conducted to improve the surgical technique of ovarian transplantation in the turkey, including the amount of ovarian tissue to remove, size of the transplanted tissue, and use of the abdominal air sac as anchor for transplanted tissue. These data were published in April 2022. Additional studies: No progress was made with the CRADA regarding development of short-term, hypothermic storage methods for broiler semen because of the COVID-19 maximal telework policy and the outbreak of highly pathogenic avian influenza. The delivery of a new group of males for resumption of the project is planned for December 2022, and a no-cost extension has been implemented to continue the research. Some progress was made with the joint ARS/University of MD NIFA project ‘Molecular Basis for Egg Production Rates in Turkey Hens’. Laboratory analyses of ovary and follicle samples collected from mature turkey hens in FY21 have resumed. Project Summary: The Project Plan was a four-year plan (2018-2022) that was delayed by one year due to critical vacancies (2 of 3 SY positions were vacant in 2017). The first vacancy was filled in June 2019 and the second vacancy was filled in August 2021. The first year of the project was impacted by the furlough in 2018 and the last two years of the project were impacted by the COVID-19 maximal telework policy and the 2022 outbreak of highly pathogenic avian influenza. Nonetheless, significant advancements were made for three of the four objectives. For Objective 1, proteomic analyses of commercial roosters identified candidate biomarkers predictive of the sperm mobility phenotype, the only sperm function assay to date that is positively correlated with fertility in poultry. It was discovered that seminal plasma proteins from males with the high mobility phenotype are enriched with the extracellular exosomal proteins mannose-6-phosphate isomerase, calcium binding protein 39, and 6-phosphogluconate dehydrogenase. Additionally, seminal plasma from high mobility males had a 7-fold difference in abundance of gallinacin, an anti-microbial peptide. Validation studies that could not be conducted during the final two years of the project are still pending. Seminal plasma from low sperm mobility males consistently contained more serum albumin than high mobility males, providing previously unknown physiological factors indicating a disruption of reproductive function. These results have laid the foundation for further work in the next five-year plan to identify biomarkers predictive of fertility. For Objective 2, the planned studies to develop cryotolerant turkey lines required four full years to complete; however, ARS scientists at Beltsville, Maryland, acquired two historic turkey lines from the Ohio State University in 2018 that were scheduled to be terminated. During the remainder of the project plan, these lines have been fully established and characterized at the Beltsville location and are serving as a model to continue investigations of turkey sperm cryosensitivity in the next five-year plan. For Objective 3, ARS scientists at Beltsville, Maryland, were the first to utilize laser capture microdissection to separate sperm storage tubules from oviduct epithelial cells, allowing for the identification of transcriptome differences exclusive to sperm storage tubule function among virgin, sham-inseminated, and semen-inseminated hens at the initiation, peak, and cessation of egg lay. At onset of reproduction, differentially expressed genes were primarily involved in transport of ions (e.g., sodium, zinc, potassium), and catalytic activity, suggesting that these functions are important for the initial residence of sperm within the sperm storage tubules. During the middle of reproduction, differentially expressed genes were associated with functions such as receptor/signal transducer activity and transport, where potential functions could be to reduce or prevent cell death or immune response against sperm. At the end of reproduction, when most of the sperm storage tubules did not contain sperm, functions were largely related to catalytic activity and apoptosis. Further, pathway analysis revealed differences occurring at early (e.g., upregulation of cytokine response genes and downregulation of thyroid hormone metabolism genes), peak (e.g., upregulation of ATP and carbohydrate derivative metabolism), and late (e.g., lower expression of ion homeostasis and cell death pathways) reproduction. Understanding sperm storage tubule basal gene expression, and how basal expression is perturbed by timing in the egg laying cycle and in the presence of spermatozoa, provides a fundamental basis for ARS scientists to improve assisted reproductive technologies utilized in the poultry industry. These studies will be continued in the next five-year plan. For Objective 4, a systematic approach resulted in several important discoveries that will improve the utility of biobanked ovaries for regeneration of turkey lines. Although ovarian transplantation methods have been successful for chicken, quail and duck, those methodologies were not successful in the turkey. ARS scientists in Beltsville, Maryland, in collaboration with Canadian researchers, provided the first characterization of ovarian development in newly hatched turkeys, including the dynamics of germ cell breakdown and formation of the primordial follicle pool. These data formed the basis for the subsequent determination that the optimal donor age of immature turkey ovaries (seven days old) differs from the chicken (one day old) but is similar to the quail (seven days old). An in ovo culture system also was utilized to evaluate the viability and maturation potential of donor ovarian tissue. Because the transplantation of donor ovarian tissue into recipient birds elicits a severe immune response leading to graft rejection, an immunosuppressant protocol also was developed utilizing cyclosporin A, an immunosuppressant not previously investigated in this context. Finally, chimera formation has been a challenge with previous poultry species, as it was thought that some of the recipient ovary tissue should remain to provide an anchor for the transplanted donor tissue. It was determined that, in the turkey, 100% of the recipient tissue can be removed and 91% of the grafts will attach. The goal of the final study of Objective 4 was to regenerate a turkey line using the aforementioned methods; however, the outbreak of highly pathogenic avian influenza has temporarily halted the experiments. Line regeneration will be attempted after the outbreak of highly pathogenic avian influenza has subsided.

1. Artificial insemination of turkey semen impacts sperm storage tubule dynamics in the hen. Turkey hens can produce fertile eggs for up to ten weeks after a single insemination with freshly collected semen because sperm are protected and nourished in regions known as sperm storage tubules; however, the fertilizing ability of turkey semen held outside the hen is diminished after only 6 hours. ARS scientists at Beltsville, Maryland, were the first to characterize sperm storage tubules gene expression changes due to the presence of semen, through the comparison of global gene expression from sperm storage tubules isolated from virgin, sham-inseminated, and semen-inseminated hens at three timepoints representing sperm entrance into the tubules (D1), peak fertility (D30), and declined fertility (D90). Bioinformatic analysis of RNA sequencing data revealed 798 differentially expressed genes due to insemination treatment, with pathway analysis revealing upregulation of cytokine response genes and downregulation of thyroid hormone metabolism genes at D1, upregulation of ATP and carbohydrate derivative metabolism at D30, and lower expression of ion homeostasis and cell death pathways at D90 in semen inseminated samples compared to virgin and sham-inseminated samples. These results unlock potential molecular and physiological mechanisms of how sperm storage tubules preserve sperm function in vivo and provide practical implications for mimicking these natural mechanisms in vitro to extend the shelf life of turkey semen for producers.

2. Optimized surgical methods for transplantation of ovarian tissue in the turkey. For most livestock species, sperm cryopreservation effectively captures the entire genome. In birds, the female determines gender and thus the genetics are not fully captured when only semen is biobanked; however, the biology of the egg prevents effective cryopreservation. An alternative approach is to freeze immature ovarian tissue, which fully captures the complete female genetic materials. ARS scientists in Beltsville, Maryland, in collaboration with scientists in Canada, determined that the entire recipient ovary should be removed, and the abdominal air sac is not needed to cover the transplanted tissue. Ninety-one percent of grafts will attach if ovarian tissue from 7-day old donors is transplanted into 2-day old recipients using this surgical method. This discovery represents a major advancement for industry and research institutions to preserve valuable turkey lines in frozen form for future use.

Review Publications
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.
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.
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.
Hall, G.B., Long, J.A., Susta, L., Wood, B.A., Bedecarrats, G.Y. 2022. Turkey ovarian tissue transplantation: effects of surgical technique on graft attachment and immunological status of the grafts, 6 days post-surgery. Cell and Tissue Banking. 101(3):101648.
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.