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.
For Sub objective 1A, the furlough delayed the arrival of male poultry to the ARS location until May 2019. The males will reach maturity in July 2019 and experiments for discovery of biomarkers are scheduled for August 2019, after initial sperm mobility screening is completed. For Sub objective 1C, the furlough prevented the scheduled shipment of semen samples from the collaborator to the ARS location. Experiments have been rescheduled for August and September 2019 based on the availability of the collaborator. For Sub objective 3A, significant progress has been made with respect to the transcriptomic evaluation of sperm storage tubules in the turkey hen. To assess the effect of sperm on differentially expressed genes (DEGs) in the SSTs, non-inseminated (n=10) and inseminated (twice pre-lay and once immediately after onset of egg lay; n=22) hens were sampled at the onset (day 1), middle (day 30), and end (day 90) of reproduction. The utero-vaginal junction region containing SSTs was dissected and flash frozen. Laser capture microdissection was used to isolate SSTs from the surrounding epithelial tissue for RNA extraction. Approximately 70-80 million fragments of 150bp paired-end reads per sample were generated using an Illumina NextSeq. Sequences were aligned to the turkey reference genome (v. 5.0) and annotations were used to identify gene transcripts. At each timepoint, pairwise comparisons between hen groups were performed to identify DEGs that were significantly (±1.5-fold change, FDR p-value <0.05) up- or down-regulated, relative to inseminated hens. A total of 349 unique genes were differentially expressed from onset to end of reproduction. Most DEGs occurred during the middle of reproduction compared to onset and end; however, at each timepoint, the pattern of expression varied. At onset of reproduction, 77.7% of DEGs were up-regulated; while at the middle, 75.6% of DEGs were down-regulated. By the end of reproduction, 36.3% and 63.6% were up- and down-regulated, respectively. The DEGs were broadly categorized using Gene Ontology terms for molecular function including: activity (catalytic, structural, receptor, molecular transducer, and transporter), binding, and regulation. At onset of reproduction, DEGs 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 SSTs. During the middle of reproduction, DEGs 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 SSTs did not contain sperm, functions were largely related to catalytic activity and apoptosis. The overall pattern of gene regulation and ontology of SSTs with or without sperm provides novel insights regarding the function of this unique reproductive structure. For Sub objective 4A, significant progress was made in determining the optimal age of donor ovaries in the turkey. Ovaries were collected from poults at 1, 3, 5, 7 and 15 days post-hatch, vitrified, thawed and cultured in-ovo for 6 days, via xenografting to the chorioallantoic membrane. These were compared to ovarian development in fresh counterparts. Viability of tissue was assessed visually on whole grafts, with cell viability being measured on sections via the terminal deoxynucleotidyl (TUNEL) assay. Size and density of pre-follicular germ cells and primordial follicles were measured to assess developmental progression. Visually, all grafts re-vascularized. Analyses via TUNEL showed minimal late-stage apoptotic or necrotic cells within all treatments. Pre-follicular germ cell density and primordial follicle development form day 7 cultured ovaries were the most comparable to the fresh counterparts. Based on our results, the initial age of ovaries did not affect viability during culture; however, age did affect germ cell development. Transplantation of 1- and 15-day old turkey ovaries should be avoided, due to marginal developmental success, instead, 7 day old ovaries appear to be more suitable. Progress was made with the Cooperative Research and Development Agreement (CRADA) regarding development of short-term, hypothermic storage methods for broiler semen. Forty-eight males were phenotyped for sperm mobility, and then evaluated for sperm viability, calcium uptake, phospholipid alteration and protein complement at 0, 6, 24, 48 and 72 hours of in vitro storage at 4, 17 and 25 degrees Celsius. Viability was maintained at 4 and 17 degrees Celsius; however, the other parameters were negatively impacted at 17 degrees Celsius. Storage of broiler semen at 25 degrees Celsius was not successful for longer than 6 hours.
Manier, M.K., Welch, G.R., Van Nispen, C., Bakst, M., Long, J.A. 2019. Low mobility sperm phenotype in the turkey: impact on sperm morphometrics and early embryonic death. Theriogenology. 54(3):613-621. https://doi.org/10.1111/rda.13403.
Talbot, N.C., Krasnec, K.V., Garrett, W.M., Shannon, A.E., Long, J.A. 2018. Finite cell lines of turkey sperm storage tubule cells: ultrastructure and protein analysis. Poultry Science. https://doi.org/10.3382/ps/pey208.