1a. Objectives (from AD-416):
Objective 1: Identify the physiological impacts of hypothermic storage on the carbohydrate, lipid, protein, calcium homeostasis and residual mRNA components of poultry, swine and striped bass sperm. Objective 2: Elucidate the cellular and molecular mechanisms controlling sperm selection and storage in the female reproductive tract of poultry. Objective 3: Determine the genetic component of post-thaw sperm survivability by identifying genetic markers and residual mRNA profiles associated with poultry and swine lines demonstrating superior and inferior sperm cryosurvival. Objective 4: Elucidate the cellular and molecular mechanisms impacted by cold storage in the fertile egg blastoderm in poultry.
1b. Approach (from AD-416):
In the food animal industries, generating offspring that possess economically important traits is most effectively accomplished by artificial insemination (AI) in poultry and swine or in vitro fertilization (IVF) in striped bass. The efficiency of AI and IVF for swine, poultry and striped bass is limited by the inability to store male gametes at refrigerated or frozen temperatures without a significant loss in sperm function. Improving hypothermic semen storage methods for these species is dependent upon a fundamental understanding of the cellular and molecular biology of the sperm cell and how prolonged exposure to hypothermic storage impacts sperm physiology. Previously, we identified physiological components of sperm cells that are negatively impacted by hypothermic storage, and also have demonstrated the impact of genetics on sperm cryosurvival. The current plan will expand on this knowledge by investigating methods to prevent sperm membrane alterations and maintain calcium homeostasis during hypothermic storage, and exploring the feasibility of using genetic markers to predict sperm cryosurvival. We will use transciptome analysis to study the physiological phenomenon of prolonged semen storage in the hen’s oviduct. We also will test the possibility that residual mRNA in sperm can serve as a predictor of sperm cryosurvival and fertility. A new objective important for the poultry industry is evaluating the impact of storing eggs at cool temperatures for long time periods on embryonic development. This comprehensive research approach will permit development of more efficient methods of preserving poultry, swine, and fish semen, and increase hatchability following egg storage under hypothermic conditions.
3. Progress Report:
Objective 1A: It has been shown that hypothermic conditions that are used during storage of poultry semen alter the sugars on the sperm surface, or glycocalyx, which are important for sperm-egg interaction. Experiments conclusively demonstrated that, when diluted with extender containing the sugar sialic acid, the glycocalyx of chicken and turkey sperm bound this sugar in a dose and time-dependent manner, with a minimum of 30 min required for the uptake of sialic acid. Turkey sperm exhibited better uptake of sialic acid at 4C than 25C; while in chicken sperm uptake of sialic acid was not affected by temperature. Objective 1C: Methods for extracting RNA from swine sperm and testicular tissue were compared. In testis RNA, the two most abundant forms of ribosomal RNA were detected at similar levels regardless of the method used. The quantity of RNA isolated from sperm was much less; only the smaller ribosomal RNA maybe present. Progress was made in developing an RNA extraction method for striped bass sperm, specifically in isolating sperm from other cell types present in the semen. A flow cytometry procedure was developed to determine the percentage of contaminating somatic cells in semen. Boar sperm contained 0.008 to 0.125% somatic cells; however, somatic cells could not be detected in striped bass semen. Objective 1D: Calcium homeostasis, or maintaining low levels of intracellular calcium in the presence of extracellular calcium that is 1000 times greater, is vital for cell function and sperm expend considerable energy to maintain this imbalance. Experimental results showed that most striped bass sperm stored in cooled or frozen form were unable to maintain calcium homeostasis, which was hypothesized to contribute to their reduced survival and fertility following hypothermic storage. Objective 2A: Discrete tubular invaginations in the lower part of the hen’s reproductive tract, collectively referred to as sperm storage tubules (SSTs), can store sperm for up to 70 days in the turkey after artificial insemination and still fertilize an egg. Yet sperm from freshly collected semen left at room temperature lose viability within 90 min. To better understand the cellular basis of sperm storage in the hen’s SSTs, methods were investigated to culture reproductive tissue containing the SSTs and sperm to determine the impact of various biologically active compounds on SST function. Objective 4A: To address the basis of early embryonic death in poultry eggs that have been stored at 18C for 7 or more days, short periods of incubation (SPI) were evaluated, where eggs were exposed to 37.5C for 4-hour periods. Using this SPI treatment at 4-6 day intervals during a 21-day storage period increased chicken embryo survival by 10% compared to the stored non-SPI eggs. The biological basis for this increase in embryo survival included recovery of metabolic activity during and after the SPIs and an increase number of viable embryonic cells in the incubated eggs. A similar study was conducted with turkey eggs and the result was a 6% increase in embryo survival in the SPI eggs when compared to non-SPI eggs.
Aslam, M.L., Bastiaansen, J.W., Elferink, M., Megens, H., Crooijmas, R.P., Blomberg, L., Fleischer, R.C., Van Tassell, C.P., Sonstegard, T.S., Schroeder, S.G., Groenen, M.A., Long, J.A. 2012. Whole genome snp discovery and analysis of genetic diversity in turkey (meleagris gallopavo). Biomed Central (BMC) Genomics. 13:391.