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.
This is the fourth year of the five-year project. For Subobjective 1A, evaluations of seminal plasma and sperm fraction proteins previously obtained from high and low sperm mobility males were continued under the maximum telework policy. Of the more than 1,000 proteins identified in each fraction, four could be potential biomarkers for high mobility males, including gallinacin, mannose-6-phosphate isomerase, calcium binding protein 39 and 6-phosphogluconate dehydrogenase. Ongoing pathway analyses suggest these proteins are involved with important metabolic and antimicrobial functions. In vitro sperm experiments to further explore these possibilities were postponed during maximal telework. For Subobjective 1B, a genotyping SNP array was conducted on blood samples from high and low sperm mobility males representing three different turkey lines to determine potential markers associated with sperm mobility. A GWAS analysis using the most conservative corrections was performed for each individual turkey line and yielded no significant SNPs associated with the sperm mobility phenotype. Currently, less conservative cutoffs are being evaluated to account for potential linkage disequilibrium between markers while limiting the occurrence of false positives. Additionally, SNP analysis across the three turkey lines is being conducted correlate line specific markers with sperm mobility phenotype. For Subobjective 2C, IACUC protocols received final approval in February 2021. A pilot experiment was conducted in May 2021 to determine the most ideal methods for embryo isolation. The main experiment is planned for September 2021 to determine the precise timing of the first and second wave of embryonic activation in the developing turkey and to determine how embryonic activation is disrupted when frozen thawed semen is used. For Subobjective 3A, analysis of RNA sequencing data identified a total of 592 genes differentially expressed due to treatment in sperm storage tubules isolated from virgin, sham-inseminated, or single insemination hens. In addition, gene expression changes over the duration of laying period in each treatment group were analyzed, revealing a total of 2704 differentially expressed genes. Current bioinformatic analysis is being performed on a second set of sperm storage tubule samples to determine the impact of incorporating a pre-lay insemination on gene expression. For Subobjective 3B, differentially expressed genes identified in Subobjective 3A were subjected to pathway analysis using Ingenuity Pathway Analysis software. Pathway analysis identified key roles for immune related pathways at the time of insemination and at the end of the fertile period, indicating a potential link to the duration of fertility. Additionally, upregulation of carbohydrate metabolism pathways was seen in all treatment groups, but the timing was delayed by the presence of sperm, indicating sperm regulation of sperm storage tubule function. Currently, an expansion of the pathway analysis is being conducted to identify potential upstream and master regulators of sperm storage tubule gene expression. For Subobjective 4B, three immunosuppressants, mycophenolate mofetil (MFM), cyclophosphamide (CY), and cyclosporin A (CsA) were evaluated in concert with ovarian transplantation in turkey poults. Treatment groups included: (1) no immunosuppressant ‘control’; (2-3) MFM 100 or 150 mg/kg/day via crop gavage; (4) MFM 100 mg/kg/day via subcutaneous injection; (5) CY 200 mg/kg the day before surgery only; (6) CsA 50 mg/kg/day beginning the day before surgery; (7-9) CsA 12.5, 25 or 50 mg/kg/day starting the day after surgery. At 14 days post-surgery, white blood cell count, bursa and thymus weights and ovary graft structure were evaluated. Birds receiving CsA had the lowest number of circulating lymphocytes and total leukocytes compared to the controls and grafts in the CsA 25 and 50 mg groups were all normal in appearance with no lymphocytic infiltrates. These data have been submitted for publication. Additional studies: 1) No progress was made with the CRADA regarding development of short-term, hypothermic storage methods for broiler semen. The delivery of a new group of males for FY21 was postponed indefinitely until normal work resumes. A second no-cost extension may be required to continue the research. 2) Some progress was made with the joint ARS/University of Maryland NIFA project ‘Molecular Basis for Egg Production Rates in Turkey Hens’. Ovary and follicle samples were collected from mature turkey hens and are being stored until further laboratory analyses can be conducted.
1. Isolation and transcriptome analysis of turkey sperm storage tubules. Sperm storage tubules are specialized invaginations of the oviductal epithelium and allow avian species to store spermatozoa for extended periods of time, without compromising sperm capacity for fertilization. The molecular and physiological mechanisms behind sperm storage tubule function remain largely unknown and have potential implications for drastically improving fertility of cryopreserved sperm for commercial poultry species. ARS scientists at Beltsville, Maryland, were the first to isolate sperm storage tubules from the oviductal epithelium using laser capture microdissection, allowing for the unique transcriptome of sperm storage tubules to be characterized. Characterization and pathway analysis of 745 differentially expressed genes revealed previously unknown immune pathways associated with the duration of fertility, as well as cholesterol and lipid metabolism associated with sperm protection. These data provide promising avenues for ARS scientists to develop industry-based protocols for semen insemination and cryopreservation that exploit the sperm preservation capabilities of sperm storage tubules.
2. New immunosuppressant for recovering turkey lines from ovary transplants. For most livestock species, sperm cryopreservation effectively captures the entire genome; however, in birds, it is not possible. An alternative approach is to freeze immature ovarian tissue, which fully captures the complete female genetic materials; however, surgical transplantation of this tissue into unrelated recipient birds results in graft rejection caused by a vigorous immune response. ARS scientists, in collaboration with scientists in Canada, have determined that cyclosporin A prevents ovarian transplant rejection during the first two weeks post-surgery in the domestic turkey. Grafts from 100% of birds treated with cyclosporin A were normal in appearance with no lymphocytic infiltrates, compared to other immunosuppressants where 80% of grafts were rejected. This result represents a major advancement for turkeys, an agriculturally important species with historically poor cryopreservation success.
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