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.
1. New semen extender supplement improved the fertility of turkey semen. The turkey industry relies exclusively on artificial insemination, a time and labor-intensive process, to reproduce birds in commercial operations. When producers use freshly collected semen for artificial insemination, fertility rates are typically 94-98%; however, if semen is held for longer than 6 hours prior to insemination, fertility rates drop to 40%. ARS scientists have been evaluating the biological basis for this drop in fertility, and have shown that the sugar residues on the surface of the sperm membrane change when semen is held for longer than 6 hours. To address this issue, an intensive evaluation of the concentration of different sugars, as well as the effect of time and temperature on these sugars has been conducted. Results indicate that providing extra sialic acid, a sugar, in the semen extender can boost the fertility rates of semen held at a cool temperature (4C) for 24 hours from 40 to 85%. This represents a significant advance in poultry semen storage technology that should save producers time and money when reproducing flocks.
2. Development of semen cryopreservation methodology for a unique commercial turkey line. Cryopreserved turkey semen historically yields very low fertility rates (0-5%) that prevent any meaningful application of semen repositories for the long-term preservation of genetics. ARS scientists entered into an agreement with a turkey breeding company to test semen freezing and resulting fertility rates on a specialized line of turkeys that was no longer needed for production but might be required in the future. After examining 14 variations in semen freezing methods, a new approach was discovered that increased fertility rates to 26% over 3 consecutive weeks, with 100% of the fertile eggs hatching live, viable poults. This breakthrough resulted in sperm from the first commercial turkey line to be frozen and stored in the USDA’s National Germplasm Facility with real potential for line regeneration in the future.
3. Localization of unique proteins associated with the cells forming the sperm storage tubules. To better understand the interaction between sperm residing in the oviductal sperm storage tubules, the cells forming the sperm storage tubules, and the sustained fertility in laying hens, a group of plant proteins that bind to specific unique proteins on the surface of cells (lectins), were used by ARS scientists to identify possible sites associated with sperm binding within the sperm storage tubules. Lectin binding demonstrated the presence of specific unique proteins lining the sperm storage tubule cells. However, there were no differences in the lectin binding patterns in the sperm storage tubules between high and low fertility hens. This information will contribute to the relatively meager body of information on the biology of sperm storage in the sperm storage tubules.
4. Improved method to determine egg fertility. Scientists and hatchery managers want a quick and easy way to differentiate between fertilized, unfertilized and embryos that die very early during incubation. The embryo develops on the surface of the egg yolk underlying a thin fibrous investment surrounding the yolk. Sperm must penetrate this investment to fertilize the ovum. ARS scientists modified an existing procedure by simply adding an additional washing step to the procedure used to isolate unincubated embryos so the path of the sperm penetrating the fibrous investment can be made visible. This modification permits the rapid assessment of the actual number of sperm interacting with the hen’s ovum, a factor that can be used to predict the duration of fertility. This has been useful to assess eggs from ducks, quail, chickens, geese and turkeys. This technique is useful to poultry scientists and to industry personnel interested in determining the basis of hatchery losses.
5. Short periods of incubation improved the percentage of eggs that hatch. Egg storage is a common practice in the turkey and chicken industries with eggs being stored up to 3 weeks prior to incubation. However, after 7 days of 18C storage, the rate of embryonic death begins to increase and after 18-21 days of storage, there may be a 20-30% drop in the hatch of fertilized eggs. ARS scientists refined the existing protocol by pre-incubating stored eggs to 37.8C for 4 hours at 4-6 day intervals and the hatch of fertile turkey eggs was increased by 6% over non-incubated stored eggs. Considering that about 250 million turkeys are produced each year for consumption, an increase in hatchability of just 1 percent (2.5 million) of the eggs would lead to a significant financial benefit.
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.