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United States Department of Agriculture

Agricultural Research Service

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2009 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.

3. Progress Report
Significant progress has been made during the past year and work continues in the following areas. 1) In the area of sperm physiology during hypothermic storage, a flow cytometry procedure for measurement of intracellular free calcium in boar sperm was developed for evaluation of sperm function after low temperature storage or freeze-thawing, and high-pressure liquid chromatography was used to delineate the phospholipid content of poultry sperm. Striped bass sperm survival during hypothermic storage was increased from 43 to 98% in a simple Tris-base NaCl storage medium by adding EGTA to eliminate the accumulation of intracellular calcium. 2) A total of 216 semen samples from 72 boars were frozen, completing the first part of multi-year study to determine the genetic component of the variation in boar sperm cryosurvival. 3) Differences in gene expression of hen reproductive tracts after artificial insemination of sperm were evaluated and avidin was identified as the most highly-differentially expressed gene in the SST of sham or artificially inseminated hens. 4) To develop alternative methods for sperm cryopreservation, the genetically modified gene for a-hemolysin pore-forming protein from Staphylococcus aureus was cloned into E. coli and plant expression vectors to produce the modified protein, H6. Only the H6 gene cloned into an E. coli expression vector was successful in producing H6; experiments conducted with a-hemolysin show that it can form pores in rooster sperm and pig fibroblasts, but not in boar sperm. A revised protocol for the in ovo injections of the chemosterilant busulfan into turkeys eggs after 38 hr incubation has resulted in variable numbers of germ cells being eradicated from the seminiferous epithelium but not 100% sterility. Currently we are evaluating increased injection volume and concentration of busulfan in egg injections. The progress made in each of these areas contributes to developing more efficient hypothermic storage methods for poultry, swine and fish sperm.

4. Accomplishments
1. Characterization of the Lipid Complement in Poultry Sperm. It is likely that lipids which are essential for poultry sperm functions, such as sperm/egg recognition, sperm energetics and sperm motility, are disrupted during cryogenic storage and are partly responsible for the low fertility rates of frozen/thawed semen. Characterizing the lipid complement of poultry sperm before and after semen storage is important for understanding how and why sperm lose functional competence during semen storage. The lipid complement of sperm from several mammalian species has been characterized, yet the lipid complement of poultry sperm remains undefined. Our objective was to characterize the lipid complement of chicken and turkey sperm prior to cryogenic storage. We used high-pressure liquid chromatography to characterize lipid fraction of freshly collected spermatozoa from both species, as well as classify the proportion of phospholipid to cholesterol ratio. This baseline data provides the foundation necessary for delineating the effect of hypothermic storage on poultry sperm, and is critical for developing reliable methods for poultry semen storage and long-term germplasm preservation.

2. Effects of Sham Versus Artificial Insemination on Avidin Gene Expression in the Turkey Hen Reproductive Tract. Turkey sperm maintained in the hen’s sperm storage tubules (SST) are capable of fertilization for up to 10 weeks after a single insemination; whereas the fertility rates of turkey semen stored in vitro decline dramatically after 6-8 h of storage. The overall objective is to delineate the physiological and molecular mechanisms involved in regulating long-term sperm storage in vivo in order to improve in vitro sperm storage methodology. Using suppressive subtractive hybridization methods, we characterized genes expressed in the SST and vaginal epithelium, and identified avidin as the most highly-differentially expressed gene in the SST of sham or artificially inseminated hens. Real-time polymerase chain reaction was utilized to analyze the expression of the avidin, avidin-related protein 2 (AVR2) and progesterone receptor (PR) gene expression. The up-regulation of avidin and AVR2 within the sperm storage region indicates these factors may be involved in the sustained storage of sperm in the SST; however, there is no known metabolic role for avidin in the oviduct. The possibility that avidin in and around the SSTs may inhibit infiltration of bacteria should be considered. Greater insight of the regulatory mechanisms involved in prolonged sperm storage may improve in vitro semen storage conditions.

3. Identification of spermatogonial stem cell population. The capacities to isolate, propagate, freeze and, upon thawing, transfer testicular stem cells into sterilized recipient males’ is a novel approach to germplasm preservation. This same technique can be adapted into programs aimed at the integration of exogenous genetic material into individual testicular stem cells prior to freezing. While spermatogonia can be identified in histological sections with specific stains, their identification in dispersed cell suspension is more challenging. To identify and collect spermatogonia that include a subpopulation of stem cells, the cells in the testes of immature and mature chickens dispersed and stained with a nuclear fluorescent dye. The nuclear DNA content and the differential expression of two genes unique to stem cells were detected by fluorescence-activated cell sorting. A small but specific subpopulation of testicular cells was identified at the junction between the haploid and diploid cell populations. These cells accounted for 4.1% and 1.3% of the total cell in pubertal and adult cockerels, respectively and expressed Dazl and Stra8, genes known to be expressed in premeiotic cells including stem spermatogonia. We concluded that these cells spermatogonia including the subpopulation of spermatogonial. Utilizing these techniques we will be able to provide enriched cell populations of spermatogonial stem cells capable of colonizing recipient seminiferous tubules in larger proportions than in our previous work.

4. Improved low temperature storage of Atlantic sturgeon spermatozoa. Atlantic sturgeon are not endangered, but a species of concern. In order to replenish this species methods of improved short-term, non-frozen storage and/or cryopreservation of semen are required to maximize availability of viable spermatozoa whenever a rare fertile female is found. Methods of short-term, non-frozen storage for 7 days at 4 degrees C were investigated for the maintenance of sperm viability, motility, and ATP content under the following conditions: oxygen or nitrogen atmosphere, undiluted semen, and semen diluted (1:3) in two different extenders in collaboration with the University of Maryland. The Atlantic sturgeon spermatozoa stored in an oxygen atmosphere and diluted in the new Park and Chapman medium maintained sperm for seven days with no significant loss of viability, motility, and ATP content. This discovery provides the basis for studies to determine if the stored sperm are capable of in vitro fertilization.

Review Publications
Guthrie, H.D., Welch, G., Long, J.A. 2008. Mitochondrial function and reactive oxygen species action in relation to boar motility. Theriogenology. 70:1209-1215.

Bakst, M.R., Akuffo, V.G. 2009. Morphology of the Turkey Vagina and Uterus With and Without an Egg Mass. Poultry Science. 88:631-635.

Last Modified: 07/24/2017
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