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 second Annual Report for Project 8042-31000-110-00D, which was delayed by one year and initiated August 2018. Progress was made on all objectives and sub objectives, which directly support National Program 101 and relate to multiple Problem Statements under Component 1, Increasing Production and Production Efficiencies while Enhancing Animal Well-being across Diverse Food Animal Production Systems and Component 2, Understanding, Improving, and Effectively Using Animal Genetic and Genomic Resources. Some of the progress described here relates to work planned for FY19 that was delayed by the furlough that occurred within the first six months of the project and subsequently either partially or fully completed in FY20. For Sub objective 1A, immature males arrived in May 2019 and reached maturity in July 2019. Males were phenotyped for sperm mobility in August 2019. Repeated samples were obtained for males from the high and low sperm mobility groups for the next three months for proteomic analyses to discover potential biomarkers. More than 1,000 proteins were identified in each fraction (e.g. sperm; seminal plasma) of semen from high and low mobility males. Ongoing analyses reveal that seminal plasma proteins from males with the high mobility phenotype are enriched with extracellular exosomal proteins such as mannose-6-phosphate isomerase, calcium binding protein 39 and 6-phosphogluconate dehydrogenase. Based on spectral counts, seminal plasma from high mobility males had a 7-fold difference in abundance of gallinacin, an anti-microbial peptide. Seminal plasma from low mobility males had a higher abundance of serum proteins compared to high mobility males. For example, serum albumin was 1.5-fold higher in low mobility males. For Sub objective 1B, blood samples were collected from high and low sperm mobility males representing three different turkey lines. Genomic DNA was genotyped using the Affymetrix SNP array and is being evaluated for potential markers associated with the two phenotypes. For Sub objective 1C, a preliminary trial with six boars demonstrated that the sperm mobility assay developed for poultry, with some modifications (e.g. diluent; sperm ratio; accudenz concentration) is suitable for use with boar sperm. For Sub objective 4B, IACUC protocols received final approval in January 2020 and experiments were planned for March 2020. The experimental design is to evaluate three immunosuppressants, alone and in combination, administering one of two concentrations weekly during a two-month period after surgery. These studies have not yet been initiated due to restrictions imposed by COVID-19. Some progress was made with the CRADA regarding development of short-term, hypothermic storage methods for broiler semen. Forty immature males arrived in January 2020 and reached maturity in late February. Males were phenotyped for sperm mobility in early March. Further work has been postponed while under maximal telework status. A no-cost extension has been approved to continue the research. Additionally, 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 inseminated hens at the initiation, peak, and cessation of egg lay. Through the identification and pathway analysis of differentially expressed genes during early, peak, and late egg production, new insights into the role of collagen in the formation and regression of sperm storage tubules and of cholesterol and lipid metabolism in sperm protection, can be exploited to advance insemination and semen cryopreservation protocols. 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.
1. Optimal donor age for transplantation of ovarian tissue in the turkey. 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. ARS scientists in Beltsville, Maryland, in collaboration with scientists in Canada, have determined that seven days post-hatch is the optimal age to recover and transplant ovarian tissue. Ninety-one percent of grafts will attach if ovarian tissue from 7-day old donors is transplanted into 2-day old recipients. This result represents a major advancement for chickens and turkeys, an agriculturally important species with historically poor cryopreservation success.
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