2010 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.
Significant progress has been made during the past year on all 4 project objectives, which focused on improving the ability to preserve valuable germplasm (e.g., sperm, eggs) from agriculturally-important livestock, poultry and aquatic species. For Objective 1A, we made significant progress in identifying the effects of cold-temperature storage on the surface carbohydrates of turkey and chicken sperm. Based on these data, we now have a strategy to test for improving the fertility of frozen/thawed poultry sperm. For Objectives 1B and 1C, stark contrasts were discovered in the ability of boar and fish sperm to maintain internal levels of calcium during cold-temperature storage. Boar sperm were capable of maintaining low cytosolic calcium levels for up to five days, an important factor for effective semen storage which also may assist in identifying boar semen samples that cannot be effectively stored in non-frozen form. For the striped bass, it was discovered that poor sperm survival and motility following cold-temperature storage may be related to their loss of the ability to maintain low intracellular calcium levels during storage. Substantial progress was made for Objective 2A when 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. For Objective 2B, it was determined that the neuro-endocrine hormones serotonin, glutamate and acetylcholine had no significant impact on turkey sperm motility. Significant progress was made for Objective 3B, where amino acid sequences were identified from sperm proteins that were differentially expressed among roosters. The progress here will enable the identification of genetic markers to assist in determining which males are superior and/or inferior with respect to the survival of frozen/thawed sperm. Also for Objective 3B, progress was made to determine if DNA markers can be identified that are associated with the trait of sperm cryosurvival in boars. For Objective 4A, the genetically modified gene for the a-hemolysin pore-forming protein from Staphylococcus aureus was cloned into an E. coli expression vector to produce the modified protein. Subsequent experiments conducted with the modified protein show that it cannot form pores in boar sperm, and thus is not a candidate for transport of sugars or antifreeze-proteins through the sperm membrane. Significant progress was made for Objective 4B as the use of modified diets improved the fertility of frozen/thawed turkey semen from genetically valuable lines. The next steps will be to test these diets with other poultry lines. For Objective 4C, significant progress was made when spermatogenesis was restored in sterile recipient males after transfer of spermatogonia isolated from donor male testes. Finally, it has been concluded that busulfan is not a reliable chemosterilant. Other methods to produce sterile males will now be considered.
Embryonic death is due to cold egg storage. Cold egg storage is a common practice prior to incubation in the broiler industry. However, cold storage longer than 10 days is associated with an increase in early embryo mortality. An ARS-Beltsville scientist in collaboration with scientists from Aviagen, Ltd. found that after prolonged egg storage in a 16 C room, embryonic deaths within the broiler lines examined were at different stages of development. The time of embryonic death from the broiler line with lower overall fertility was predominately before the onset of blood formation. In contrast, embryonic death in the higher fertility line was after the onset of blood formation. We will be investigating the cell and molecular mechanisms regulating the onset of embryonic blood formation and the impact of cool egg storage on these mechanisms to determine if this specific stage of embryonic development is associated with the biological basis of early embryonic mortality in broilers.
Sperm storage is not useful as a selection trait in breeder hens. ARS researchers at Beltsville, MD and Fayetteville, AK, investigated the biological basis of sustained fertility in broiler and turkey hens, specifically, the hen’s capacity to store sperm in the oviductal sperm storage tubules (SST). The objective of this study was to determine if sustained fertility in different strains of broiler hens is a function of the average number of SSTs in a given strain. No statistical differences were observed in SST numbers in the 4 strains of broilers examined or in turkey hens before and after the onset of egg production. The mean numbers of SST for broilers and turkeys were 4,893 and 30,566, respectively. We conclude that any differences between the fertility of the four broiler breeder strains examined cannot be explained by differences in SST numbers. However, differences in the duration of fertility between broilers and turkeys are, in part, related to their respective numbers of number of SST. Poultry geneticists cannot rely on numbers of SST in different strains of breeders as a selection trait for sustained fertility in their breeder hens.
Poor sperm survival and motility following cold-temperature storage may be related to the inability of striped bass sperm to maintain low levels of intracellular calcium. The use of striped bass semen for fertilizing of white bass eggs in vitro to produce hybrid striped bass for meat production has been limiting for meat production because semen must be diluted with a buffering medium to retain sperm motility for shipment to fertilize eggs collected at a different location. Unfortunately current buffering media are not satisfactory for storage or transport of semen for 24 to 48 hours. Studies to address the cause of this problem by ARS and University of Maryland scientists revealed that reduced survival and poor motility of striped bass sperm was associated with a failure of the sperm cells to maintain low intracellular calcium concentrations that are required for calcium ions to act as secondary messengers in regulation of cell function. The conclusion from this research is that the buffering medium should be modified to prevent or compensate for the striped bass sperm cell’s poor ability to maintain control of calcium levels during storage. This information is useful for hybrid striped bass producers and researchers trying to improve hybrid striped bass meat production.
A draft turkey genome assembly was completed using a novel combination of sequencing and computational technologies. The research was a grass-roots partnership among scientists from ARS-Beltsville, Virginia Tech’s Bioinformatics Institute and the University of Maryland’s Center for Bioinformatics and Computational Biology. Americans consume about 17.6 pounds of turkey per capita every year, and the U.S. produces nearly 6 billion pounds of turkey meat annually. The nearly complete first-ever genomic map of turkey could help growers produce more efficient, healthier birds. The information gleaned from these studies will help breeders develop improved commercial turkey breeds to meet consumers’ demands in the United States and worldwide. The turkey genome assembly was further strengthened when physical, comparative and genetic maps built by researchers from Michigan State University and the University of Minnesota were used to match the DNA sequences to the turkey chromosomes. By the end of the project, the original partnership expanded to include 68 scientists affiliated with 28 national and international research institutions. This project underscores how rapidly the field of genome sequencing has changed. We sequenced the turkey genome in less than a year at a fraction of the cost of sequencing the chicken and cow genomes. The turkey industry and the consumer both benefit from this research.
Oviduct tissue type, the presence of sperm, and genetic line affect the expression of avidin, avidin-related protein-2 and progesterone receptor in turkey hens. Turkey sperm maintained in the hen’s sperm storage tubules (SST) are capable of fertilization for up to ten weeks after a single insemination; whereas the fertility rates of turkey semen stored in vitro decline dramatically after six to eight hours of storage. The genetic basis of SST sperm storage activity is unknown. An ARS Research Associate at Beltsville found that differences existed between lines of turkeys with respect to expression of avidin and avidin-related proteins, and that these differences correlated with the known fertility of the turkey lines. Moreover, the presence sperm in the oviduct increased avidin mRNA expression in the SST. The present study was somewhat limited by the lack of a complete turkey genome sequence. The availability of the first draft turkey genome sequence earlier this year provides an opportunity for a more thorough investigation of the genetic basis of sperm storage in the turkey hen.
Replenishment of essential sperm surface carbohydrate improves the fertility of turkey sperm held at 4 degrees Celsius for 24 hours. An ARS scientist at Beltsville previously has shown that carbohydrates essential for poultry sperm function (sperm/egg recognition, sperm energetics and sperm motility) are lost during cold storage, and that this loss is partly responsible for the low fertility rates of stored poultry semen. This basic data served as the foundation for a strategy to improve the fertility of stored poultry semen by supplying exogenous carbohydrates in the semen extender. The scientist first demonstrated that turkey sperm can bind the carbohydrates in a time- and dose-dependent manner, followed by selection of the optimal dose that substantially improved the fertility of stored semen from 40% to 80%. While commercial breeders require fertility rates of 96-98% to maintain economic profitability, the results here provide significant progress towards the goal of using stored semen instead of freshly-collected semen for artificial insemination of turkeys.
Guthrie, H.D., Welch, G.R. 2010. Using fluorescence-activated flow cytometry to determine reactive oxygen species formation and membrane lipid peroxidation in viable boar spermatozoa. Methods in Molecular Biology (Advanced Protocols in Oxidative Stress II). 594:163-171.
Bakst, M.R., Donoghue, A.M., Yoho, D.E., Moyle, J.R., Whipple, S.M., Camp, M.J., Liu, G.Q., Bramwell, R.K. 2010. Comparisons of sperm storage tubule distribution and number in four strains of mature broiler breeders and in turkey hens before and after the onset of photostimulation. Poultry Science. 89:986-992.
Bakst, M.R. 2010. Chapter II. Determination of sperm concentration. Section 1. Hemocytometer procedure. In: Bakst, M.R., Long, J.A., editors. Techniques for Semen Evaluation, Semen Storage, and Fertility Determination. 2nd edition. St. Paul, MN: The Midwest Poultry Federation. p. 11-15.
Bakst, M.R. 2010. Chapter III. Sperm Viability. Section 1. Nigrosin eosin stain for determining live dead and abnormal sperm counts. In: Bakst, M.R., Long, J.A., editors. Techniques for Semen Evaluation, Semen Storage, and Fertility Determination. 2nd edition. St Paul, MN: The Midwest Poultry Federation. p. 28-44.
Bakst, M.R. 2010. Chapter IV. Sperm motility and metabolism. Section 1. Visual scoring of motility using the hanging drip method. In: Bakst, M.R., Long, J.A., editors. Techniques for Semen Evaluation, Semen Storage, and Fertility Determination. 2nd edition. St. Paul, MN: The Midwest Poultry Federation. p. 45-46.
Christensen, V.L., Bagley L., Long, J.A. 2010. Chapter I. Semen collection and dilution. In: Bakst, M.R., Long, J.A., editors. Techniques for Semen Evaluation, Semen Storage, and Fertility Determination. 2nd edition. St. Paul, MN: The Midwest Poultry Federation. p. 7-10.
Long, J.A. 2010. Chapter II. Determination of sperm concentration. Section 3. Photometer procedure. In: Bakst, M.R., Long, J.A., editors. Techniques for Semen Evaluation, Semen Storage, and Fertility Determination. 2nd edition. St. Paul, MN: The Midwest Poultry Federation. p. 23-25.
Long, J.A. 2010. Chapter VI. Semen cryopreservation. Section 1. Cryopreservation of poultry sperm using glycerol. In: Bakst, M.R., Long, J.A., editors. Techniques for Semen Evaluation, Semen Storage, and Fertility Determination. 2nd edition. St. Paul, MN: The Midwest Poultry Federation. p. 62-64.
Long, J.A. 2010. Chapter VI. Semen cryopreservation. Section 2. Cryopreservation of rooster sperm using DMA. In: Bakst, M.R., Long, J.A., editors. Techniques for Semen Evaluation, Semen Storage, and Fertility Determination. 2nd edition. St. Paul, MN: The Midwest Poultry Federation. p. 65-67.
Long, J.A. 2010. Chapter VI. Semen cryopreservation. Section 3. Glycerol removal from thawed semen. In: Bakst, M.R., Long, J.A., editors. Techniques for Semen Evaluation, Semen Storage, and Fertility Determination. 2nd edition. St. Paul, MN: The Midwest Poultry Federation. p. 68-71.
Long, J.A., Bongalhardo, D.C., Pelaéz, J., Saxena, S., Settar, P., O'Sullivan, N.P., Fulton, J.E. 2010. Rooster semen cryopreservation: Effect of pedigree line and male age on postthaw sperm function. Poultry Science. 89(5):966-973.
Long, J.A. 2010. Chapter VIII. Sperm function assessment. Section 2. ATP assay. In: Bakst, M.R., Long, J.A., editors. Techniques for Semen Evaluation, Semen Storage, and Fertility Determination. 2nd edition. St. Paul, MN: The Midwest Poultry Federation. p. 99-102.