2012 Annual Report
1a.Objectives (from AD-416):
The long-term goal of this research team is to develop efficient methods of preserving poultry, swine and fish germplasm. Over the next five years we will (1) identify the physiological and biochemical impacts of hypothermic storage on poultry, swine and fish sperm, (2) elucidate the cellular and molecular mechanisms controlling sperm selection, transport and storage in the female reproductive tract of poultry, (3) determine the impact of genetics on the success of semen storage methodology for poultry and swine, and (4) investigate alternative strategies for conserving valuable poultry and swine germplasm. Alternative strategies to be investigated include:.
1)creation of transient pores and/or use of endogenous plasma membrane transporters to deliver antioxidants, cryoprotectants and/or nutrients intracellularly;.
2)development of diets to modify the plasma membranes of sperm from congenic and/or inbred poultry lines to improve cryosurvival; and.
3)development of methods to isolate, propagate, freeze/thaw and transfer poultry spermatogonia to recipient sterilized testes.
1b.Approach (from AD-416):
In the food animal industries, production of offspring that possess economically important traits is most effectively accomplished by artificial insemination (AI) or in vitro fertilization (IVF), where semen from a few males is distributed among a large number of females. The poultry and swine industries use AI in their breeding programs to accelerate genetic advancement, while the striped bass industry relies on IVF. Because of gaps in our fundamental knowledge of sperm biology, the fate of sperm in the oviduct and impact of freezing on sperm function, there has been limited success in the long-term preservation of poultry, swine and bass germplasm, and existing methodologies are not adequate for the needs of these industries. Development of effective semen storage methodology necessitates a scientific foundation addressing the cellular and molecular biology of both the sperm cell and the female cells that interact with sperm after insemination. Experiments in this project will address these fundamental questions by focusing on (1) sperm membrane composition and energetics before and after hypothermic storage, (2) impact of sperm on oviductal epithelial cell gene expression and secretory activity, and (3) potential genetic basis of sperm cryosurvival. Included in this project are several alternative strategies for germplasm preservation: introduction of cryoprotectants intracellularly; dietary modification of sperm cell membranes; and use of cryopreserved testicular cells as an alternative means of male germplasm cryopreservation. This systematic approach will address the gaps in our knowledge and permit development of novel and/or more efficient methods of preserving poultry, swine and fish semen.
In this final year of the five-year project, significant progress was made for all four objectives. For Objective 1, hypothermic (or low temperature) storage of turkey semen was substantially improved by addition of physiological amounts of lipid (phospholipid) to the semen extender to counteract the damage caused by lipid peroxidation (degradation) during semen storage. For Objective 2, it was determined that RNA from cryopreserved samples collected at field sites were not degraded during isolation and storage, and that the procedures being used to develop a molecular profile of the individual cells composing the sperm storage sites are applicable to this work. For Objective 3, the success of turkey sperm cryopreservation was shown to be distinguished on the basis of genetics, where some turkey lines did not respond favorably to any freezing method. For Objective 4, a genetically-altered protein, that forms pores in bacteria, was successfully interpolated into the cell wall of chicken, turkey, and striped bass sperm, forming pores that we intend to use for loading sperm cells with compounds, such as the sugar trehalose, that protect sperm cells from ice formation during freezing and thawing. Very low amounts of zinc salts closed the pores so that potential damage to the sperm cells was minimized. The pore-forming protein, however, did not install itself in the cell wall of boar sperm.
This is the final report for this project. Key findings/outcome of the project are as follows. A new protocol using a single, fixed-time insemination of frozen-thawed boar semen yielded an 80% farrowing rate, equal to that obtained with double inseminations of non-frozen semen. Poultry sperm respond favorably to exogenous substrates, such as monosaccharides and phospholipids, with dramatic improvements in fertility from 30 to 85% after hypothermic semen storage. For striped bass sperm, a modified buffered-saline extender maintained high sperm viability and motility levels for 48 hours, which provides adequate semen storage prior to in vitro fertilization procedures necessary for hybrid striped bass production. In turkeys, the protein avidin was identified as the most highly-differentially expressed gene in the sperm storage tubules of artificially inseminated hens. Avidin is known for immunosuppressive properties and may be produced in order to protect sperm residing in the female reproductive tract before fertilization. It also was discovered that diet modification improves the fertilizing ability of frozen/thawed turkey sperm, where fertile embryos were obtained up to 12 weeks and hatched poults were obtained up to 5 weeks after a single insemination. This degree of success has not been demonstrated previously for frozen/thawed turkey semen.
Supplementing turkey semen extender with lipids and sugars improves the fertility of turkey sperm stored at four degrees Celsius for twenty-four hours. ARS scientists at Beltsville, MD discovered that specific components of the sperm membrane (phospholipids, carbohydrates) are altered when poultry semen is stored under hypothermic conditions. Loss of these membrane components affects the ability of stored sperm cells to fertilize eggs, resulting in only the use of freshly-collected (non-stored) semen (containing sperm) for artificial insemination. Commercial turkey operations would benefit from the ability to collect and store semen for one to two days prior to use. The semen extender formulation developed by ARS scientists improves the fertility of stored turkey semen from 30 to 85%, and represents a significant advancement in turkey artificial insemination practices.
The genetic line affects the fertilizing ability of frozen/thawed turkey sperm. ARS scientists at Beltsville, MD demonstrated that semen from different genetic lines of turkeys responds in a line-characteristic manner to different semen cryopreservation methods. Semen from four elite turkey lines from Ohio State University and one commercial line was frozen using one of eight different methods. One elite line exhibited poor fertility irrespective of the cryopreservation method used. The commercial and two other elite lines favored one cryopreservation method, while the fourth elite line yielded acceptable fertility for most of the cryopreservation protocols. Using line-specific cryopreservation methods, a single insemination of thawed sperm resulted in 10-12 weeks of fertile eggs, and live poults hatched for the first 4-6 weeks after insemination.
Henry, P.F., Chen, Y., Karouna-Renier, N., Sprague, D., Bakst, M.R., Akuffo, V.G. 2012. Effect of 17ß trenbolone on male and female reproduction in japanese quail (Coturnix japonica japonica). Poultry and Avian Biology Reviews. 5:61-68.
Blanco, J.M., Long, J.A., Gee, G., Wildt, D.E., Donoghue, A.M. 2011. Comparative Cryopreservation of Avian Spermatozoa: Benefits of Non-Permeating Osmoprotectants and ATP on Turkey and Crane Sperm Cryosurvival. Animal Reproduction Sciences. 123(3-4):242-248.
Blanco, J.M., Long, J.A., Gee, G., Donoghue, A.M., Wildt, D.E. 2012. Comparative Cryopreservation of Avian Spermatozoa: Effects of Cooling and Thawing Rates on Turkey and Crane Sandhill Sperm Cryosurvival. Animal Reproduction Sciences. 13:1-8.