Progress 07/26/02 to 07/23/07
Outputs
Progress Report Objectives (from AD-416) Determine how and when poultry sperm lose functional competence during liquid and cryogenic storage and use as a basis for developing successful sperm storage methods in vitro. Determine the influence of sperm phenotype (mobility, fertilizing capability) on liquid and cryogenic storage of turkey sperm, as well as phenotypic traits of economic importance in breeder males (feed conversion, body conformation, liveability). Increase the efficacy of oviductal sperm transport to the sperm storage tubule (SST) and the site of fertilization by elucidating the cellular and molecular mechanisms regulating sperm selection and transport in the oviduct. Determine the molecular basis of sperm subsistence in the sperm storage tubules using serial analysis of gene expression (SAGE) and developing an in vitro model (tissue explants) to elucidate molecular and cellular events regulating prolonged oviductal sperm storage and sustained fertility in poultry. Approach (from AD-416) The commercial turkey industry relies exclusively on artificial insemination (AI) for fertile egg production. Given that every breeder hen must be inseminated weekly for 24-26 wk, AI is both time and labor- intensive. The turkey industry would benefit by having the capacity to store semen at ambient temperature for 24 h without affecting fertility. Currently, semen is stored for no longer than 6 h and fertility rates slowly drop after 12 wk egg production when using stored semen. This project will address problems associated with the use of stored semen for AI for maximum fertile egg production. To achieve this goal, both in vitro and in vivo mechanisms of sperm storage will be investigated. For in vitro sperm storage, both liquid (short-term) and cryogenic (long-term) methods will be evaluated by focusing on how and why poultry sperm lose functional competence during storage. Systematic studies characterizing the biochemical and cellular changes associated with storage of poultry sperm will provide the foundation for developing novel semen storage protocols. Integral to developing such methods is a fundamental understanding of the physiology of in vivo storage mechanisms in the hen's sperm storage tubules (SST). Here, the goals are Serial Analysis of Gene Expression (SAGE) and cell biology techniques to elucidate both the mechanisms of sperm selection and storage in the hen. The former is important if we are to develop means to augment the transport of in vitro stored sperm to the SST's, especially since liquid and cryogenically stored sperm fail to populate the SST in large enough numbers to maintain fertility acceptable for commercial practice. Significant Activities that Support Special Target Populations Using micro-magnetic beads conjugated with antibody to macromolecules discovered on cells comprising the seminiferous epithelium (sperm producing cells in the testes) we were able to obtain a partially purified population of cells for culture. Discovered that neuro- endrocrine-like cells (NEC) are distributed sparsely in the hens vaginal epithelium and these NECs may exert a local control over the sperm transport and selection mechanisms. Sperm were discovered in tubular glands in the hen�s cloaca 72 hr after the last insemination suggesting that sperm storage in the hen may not be limited to the oviduct. The mucosa containing the oviductal sperm storage tubules (SST) projects into the uterus (shell forming region in the hen) when there is no egg in the uterus. However, with a uterine egg, the mucosa containing the SST is clearly withdrawn and forms the anterior end of the vagina, also known as the utero-vaginal junction. Upon gross dissection, anatomically, the hen�s vagina has a coiled appearance possessing 2-4 turns, the most anterior of which is tightly bound by connective tissue to the uterine wall. The results are not yet available on the injections of a chemo- sterilant into eggs after 38 hr incubation in order to eradicate sperm producing cells from the testes. (1,297 spaces) Using a battery of carbohydrate-specific lectins, it was shown that the glycocalyx composition of turkey and rooster sperm differed with respect to predominant carbohydrate residues, and that the glycocalyx of both species were extensively masked by sialic acid residues. Subsequent studies demonstrated that major alterations occur in the carbohydrate component of turkey sperm membranes stored for 24h at 4 �C, and that sperm from males classified as high or low mobility responded differently with respect to changes in the glycocalyx over time. It was also shown that uptake of exogenous phosphatidylcholine by turkey sperm membranes during in vitro storage at 4 �C improved the fertility of stored turkey semen. With respect to sperm cryopreservation, physiological differences (ATP, protein, lipid, carbohydrate) in sperm cryosurvival were shown among pedigreed layer lines, and species-specific differences in optimal cooling rates for turkey and crane sperm were demonstrated. Accomplishments Serotonin discovered in the turkey hen�s vaginal epithelial cells. In poultry, the fate of sperm in the oviduct after mating or artificial insemination is the subject of considerable speculation. We discovered the neurotransmitter serotonin in specialized cells lining the vaginal surface. In addition to possible regulating the peristaltic activity of the oviduct, it was speculated that serotonin impacts sperm selection and transport in the vagina by stimulating sperm tail-beat and cell surface cilia beat frequency. This is the first evidence of a possible local control mechanism responding to the presence of sperm in the turkey vagina. Our discovery of a neurotransmitter, previously shown to augment sperm motility in other species, will be useful in the development of new semen diluents for semen storage applicable to the poultry industry. Sperm storage observed in hen�s cloaca. Up to 48 hr is required to maximize sperm storage in the oviductal sperm-storage tubules (SST). Interestingly, during that 48 hr interval, the hen could have laid 2 eggs and in doing so, the movement of these eggs through the oviduct would sequester oviductal sperm. The question addressed was where the sperm reside during the 48 hr interval between the last insemination and the maximum filling of the SSTs. Tubular glands in the central compartment of the cloaca were observed containing resident sperm. We propose that sperm are released from these tubular glands and ascend the vagina, eventually contributing to the population of sperm stored in the oviductal SST. This information will be helpful to poultry scientists addressing the question of sperm selection and storage in the hen�s oviduct. Cold storage of turkey semen affects the glycocalyx composition. Commercial turkey producers would benefit from the capability of using semen that has been stored for 24 to 48 hours after collection from the male. Current methodology for storing turkey semen does not provide the fertility rates necessary for profitability. Our research is focused on learning why and how turkey sperm lose functional competence during in vitro storage. We have recently identified the carbohydrates present on the surface of the turkey sperm membrane. In this study, our objective was to determine if these carbohydrates are altered during in vitro semen storage. Additionally, we evaluated males of high quality and marginal quality semen (in terms of sperm movement) for differences in membrane surface carbohydrates during semen storage. We found several differences that have implications for the reduced fertility of stored turkey semen. Novel semen extender developed for turkey semen. Optimization of semen storage methods would greatly improve the efficiency of commercial turkey production. Traditional semen storage methods do not promote the high fertility rates required by the producers during the entire period of egg production. We have previously shown that turkey sperm are damaged by lipid peroxidation during semen storage. Our objective here was to develop a method for preventing the loss of lipid from the plasma membrane during semen storage. We first demonstrated that turkey sperm are able to incorporate supplemental phosphatidylcholine from the semen extender. We then found that the fertility rates of semen stored for 24h in the presence of exogenous phosphatidylcholine are higher than control semen stored without phosphatidylcholine. We conclude that supplemental phosphatidylcholine appears to counteract the damaging effects of lipid peroxidation during in vitro storage by providing exogenous phospholipids for incorporation into the turkey sperm plasma membrane. Comparative cooling/thawing rates for avian sperm. We have used a comparative approach to understand the physiology of sperm survival after cryopreservation. In general, crane sperm survive the freeze/thaw process relatively well, and produce 50-90% fertile eggs after artificial insemination with thawed semen. Conversely, the fertilizing of frozen/thawed turkey sperm is severely compromised after cryopreservation. As a continuation of ongoing comparative research, our objective here was to evaluate the impact of different cooling and thawing rates on the cryosurvival of turkey and crane sperm. We tested rapid, moderate and slow cooling rates, as well as moderate and slow thawing rates. Moderate and slow cooling rates were remarkably superior in preserving sperm viability for both species when compared to rapid cooling; however, slow thawing did not improve viability for either species included in this study. These data are important for developing effective semen cryopreservation protocols for avian species. This research relates to National Program 101, Animal Genomes, Germplasm, Reproduction, and Development 100% and contributes to the following 3 NP 101 research goals: 1) Improving reproductive efficiency in livestock; 2) Conservation, characterization, and utilization of genetic resources, and; 3) Developing and applying genomic tools and resources. Technology Transfer Number of Patent Applications filed: 1 Number of Non-Peer Reviewed Presentations and Proceedings: 2 Number of Newspaper Articles,Presentations for NonScience Audiences: 2
Impacts
(N/A)
Publications
- Bakst, M.R., Akuffo, V.G., Trefil, P., Brillard, J.P. 2007. Morphological and histochemical characterization of the seminiferous epithelial and leydig cells of the turkey. Animal Reproduction Sciences. 97:303-313.
- Trefil, P., Micakova, A., Mucksova, J., Hejnar, J., Brillard, J.P., Bakst, M.R., Kalina, J., Poplstein, M. 2006. Restoration of spermatogenesis and male fertility by transplantation of dispersed testicular cells in the chicken. Biology of Reproduction. 75:575-581.
- Pelaez, J., Long, J.A. 2007. Characterizing the glycocalyx of poultry spermatozoa: i. identification and distribution of carbohydrate residues using flow cytometry and epifluorescence microscopy. Journal of Andrology. 28(2):342-352.
- Stepinska, U., Bakst, M.R. 2007. Fertilization. In:Jamieson, B.G.M.,editor. Reproductive Biology and Phylogeny of Aves (Birds). Volume A. Enfield, NH: Science Publishers. p. 149-179.
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Progress 10/01/05 to 09/30/06
Outputs
Progress Report 1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? The commercial turkey industry, including both primary breeder and multiplier operations, relies exclusively on artificial insemination (AI) for production. Additionally, commercial broiler operations may soon have to incorporate AI methods as standard husbandry practice because of increasing problems in sustaining acceptable fertility levels associated with natural mating of the heavier strains. Current AI practices almost exclusively use freshly collected semen and weekly inseminations to achieve high fertility rates. In large commercial operations, 4-6 hen houses contain 2,000-4,000 turkeys each. Considering that breeder hens are in egg production for at least 24 wk and that each hen must be inseminated weekly to assure optimal fertility, it is obvious that AI is both time and
labor-intensive. It is estimated that 10-15% of turkey hens in North America are inseminated with semen from males kept on stud farms, whereas 80% are inseminated with semen from males housed in a barn with hens and subjected to hen management protocols. Objective 1: Determine how and when poultry sperm lose functional competence during liquid and cryogenic storage and use as a basis for developing successful sperm storage methods in vitro. Objective 2: Determine the influence of sperm phenotype (mobility, fertilizing capability) on liquid and cryogenic storage of turkey sperm, as well as phenotypic traits of economic importance in breeder males (feed conversion, body conformation, liveability). Objective 3: Increase the efficacy of oviductal sperm transport to the sperm storage tubule (SST) and the site of fertilization by elucidating the cellular and molecular mechanisms regulating sperm selection and transport in the oviduct. Objective 4: Determine the molecular basis of sperm
subsistence in the sperm storage tubules using serial analysis of gene expression (SAGE) and developing an in vitro model (tissue explants) to elucidate molecular and cellular events regulating prolonged oviductal sperm storage and sustained fertility in poultry. Objective 5: Develop techniques for the isolation and propagation of testicular stem cells ultimately to be the targets of gene insertion and transfer to recipient males. (This work has been approved by the NPL and is part of a multinational CRADA currently under review.) This research relates to National Program 101, Animal Genomes, Germplasm, Reproduction, and Development 100% and contributes to the following 3 NP 101 research goals: 1) Improving reproductive efficiency in livestock; 2) Conservation, characterization, and utilization of genetic resources, and; 3) Developing and applying genomic tools and resources. This work is relevant to the commercial poultry industry, those interested in germplasm preservation, and
the breeding of captive bird populations. Fundamental research is of interest to developmental, molecular and cell biologists using the bird as a model. 2. List by year the currently approved milestones (indicators of research progress) 2. List the milestones (indicators of progress) from the project plan Year 1 (2003) Begin oviductal sperm storage and sperm transport studies. Begin sperm biochemical alterations during in vitro storage. Identify differential gene expression in oviduct epithelium using SAGE. Develop chicken sperm cryogenic protocol. Year 2 (FY 2004) Test sperm cryogenic storage protocol. Determine basis for sperm loss after cryogenic storage. Characterize the surface glycoproteins associated with oviduct epithelia. Isolate and enrich population of testicular stem cells. Identify sperm subpopulations at surface of ovum. Year 3 (FY 2005) Complete sperm biochemical alterations during in vitro storage. Develop alternative methods for liquid and cryogenic storage of semen.
Identify phenotypic markers to be used for sire selection. Complete studies on oviductal sperm transport and storage studies. Complete studies on sperm competition. Continue to localize gene products based on SAGE in oviduct. Develop cell culture system for the propagation of testicular stem cells. Year 4 (FY 2006) Identify mechanisms by which sperm loose integrity after liquid storage. Identify mechanisms by which sperm loose integrity after cryogenic storage. Identify differential gene expression of sperm storage sites in oviduct. Continue lectin characterization of oviductal sperm storage sites and resident sperm. Develop technique to sterile sterilize male embryo. Transfect and propagate stem cells and transfer to recipient sterilized males. Year 5 (FY 2007) Complete SAGE analysis of oviduct sperm storage sites. Complete lectin characterization of oviductal sperm storage sites and resident sperm. Developed alternative method for liquid storage of semen. Developed alternative
method for cryogenic storage of semen. Identified phenotypic for sire selection. Identified cell products that augment sperm survival in vitro. Evaluate recipient males sperm derived from transfected stem cells. 4a List the single most significant research accomplishment during FY 2006. Using dispersed testicular cells before and after 3-10 days of culture we confirmed that Leydig cells can be localized by immunocytochemical localization of alkaline phosphatase, and the four types of spermatogonia can be identified by c-kit and PNA. One subtype of integrin that is used to localized spermatogonial stem cells in dispersed mammalian testicular cells has been localized to the basement membrane of the seminiferous tubule of the turkey and will be investigated as a probe specific for stem cell spermatogonia in dispersed turkey testicular preparations. 4b List other significant research accomplishment(s), if any. We established dispersed turkey testicular cell cultures for up to 3-10 days.
However, the mixed cell types were present. In collaboration with scientists at the University of Chicago, we have described structures associated with ejaculation in the fairy-wren (feral bird native to Australia). This specie produces ejaculates with the highest known concentration of sperm in aves. Using a battery of lectins, the glycocalyx of poultry sperm was characterized during semen storage, and it was determined that several key carbohydrate residues change after 8h of liquid turkey semen storage and after cryogenic storage of rooster semen; this is the first report of this data for avian species. Significant differences in sperm binding ability, sperm mobility and ATP production, occurred among frozen/thawed semen from different genetic lines of chickens. 5. Describe the major accomplishments to date and their predicted or actual impact. A. A novel procedure for removing the cryoprotectant glycerol from frozen-thawed chicken semen was developed to enable the immediate
banking of semen from genetically-valuable and/or unique research chicken lines. The research was conducted as collaboration between the Biotechnology and Gamete Laboratory (Beltsville, MD) and the Avian Disease and Oncology Laboratory (East Lansing, MI). The specific accomplishment during the reporting period was a four-fold improvement in the fertility of glycerol- exposed semen. Development of this novel protocol is significant for two reasons: 1) glycerol is the most effective cryoprotectant for poultry semen but is contraceptive in the hen, and must be removed prior to insemination to achieve fertility; 2) semen from at risk research populations can be cryopreserved and used to re-generate lines until new cryopreservation protocols are developed that dramatically improve the fertility of frozen/thawed poultry sperm (a major objective of this CRIS project). B. Work conducted in the Biotechnology and Gamete Laboratory (Beltsville, MD) was the first to demonstrate aquaporins
involved in sperm maturation and oviductal sperm storage in aves. This is significant because it provides a physiological explanation for the re-absorption of water during sperm maturation, a possible mechanism of sperm release from the sperm and provides an immunological procedure to be used for localization of aquaporins in other avian tissues. The localization of the neurotransmitter serotonin in the vaginal epithelium is important and unexpected observation in that it may have a significant impact on the local control sperm selection and storage. The impact is that our fundamental knowledge regarding oviductal sperm transport and storage is increased. This will stimulate further novel approaches to the question of how to increase the shelve life of poultry semen for artificial insemination. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)?
What are the constraints, if known, to the adoption and durability of the technology products? Technology Transfer: -Collaborated with following scientists: J.P. Brillard, INRA, Nouzilly, France; P. Trevil, BioPharm, Czech Republic; M.A. Ottinger, University Maryland , College Park MD; M. Rowe and S. Pruett-Jones, University Chicago, Chicago, IL. -Collaborated with the following stakeholders: Hy-Line International, Dallas Center IA; Hybrid Turkey, Ontario, Canada; Gobblers Inc, MN; Ohio State University, Wooster, OH.
Impacts
(N/A)
Publications
- Long, J.A. 2006. Avian semen cryopreservation: what are the biological challenges? Poultry Science. 85:232-236.
- Bakst, M.R., Long, J.A., Kramer, M.H. 2006. Impact of fenbendazole on turkey semen quality. Journal of Applied Poultry Research. 15:307-311.
- Noirault, J., Brillard, J.P., Bakst, M.R. 2005. Spermatogenesis in the turkey (meleagris gallopavo): quantitative approach in immature and adult males subjected to various photoperiods. Theriogenology. 65:845-859.
- Dupuy, V., Sellier, N., Brillard, J., Bakst, M.R. 2006. Comparative staging of embryo development in chicken, turkey, duck, goose, guinea fowl, and quail assessed from 5 hours after fertilization to 72 hours of incubation. Journal of Applied Poultry Research. 15:219-228.
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Progress 10/01/04 to 09/30/05
Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? The commercial turkey industry, including both primary breeder and multiplier operations, relies exclusively on artificial insemination (AI) for production. Additionally, commercial broiler operations may soon have to incorporate AI methods as standard husbandry practice because of increasing problems in sustaining acceptable fertility levels associated with natural mating of the heavier strains. Current AI practices almost exclusively use freshly collected semen and weekly inseminations to achieve high fertility rates. In large commercial operations, 4-6 hen houses contain 2,000-4,000 turkeys each. Considering that breeder hens are in egg production for at least 24 wk and that each hen must be inseminated weekly to assure optimal fertility, it is obvious that AI is both time and labor-intensive. It
is estimated that 10-15% of turkey hens in North America are inseminated with semen from males kept on stud farms, whereas 80% are inseminated with semen from males housed in a barn with hens and subjected to hen management protocols. Objective 1: Determine how and when poultry sperm lose functional competence during liquid and cryogenic storage and use as a basis for developing successful sperm storage methods in vitro. Objective 2: Determine the influence of sperm phenotype (mobility, fertilizing capability) on liquid and cryogenic storage of turkey sperm, as well as phenotypic traits of economic importance in breeder males (feed conversion, body conformation, viability). Objective 3: Increase the efficacy of oviductal sperm transport to the sperm storage tubule (SST) and the site of fertilization by elucidating the cellular and molecular mechanisms regulating sperm selection and transport in the oviduct. Objective 4: Determine the molecular basis of sperm subsistence in the sperm
storage tubules using serial analysis of gene expression (SAGE) and developing an in vitro model (tissue explants) to elucidate molecular and cellular events regulating prolonged oviductal sperm storage and sustained fertility in poultry Objective 5: Develop techniques for the isolation and propagation of testicular stem cells ultimately to be the targets of gene insertion and transfer to recipient males. (This work has been approved by the NPL and is part of a multinational CRADA currently under review.) This research relates to National Program 101, Animal Genomes, Germplasm, Reproduction, and Development 100% and contributes to the following 3 NP 101 research goals: 1) Improving reproductive efficiency in livestock; 2) Conservation, characterization, and utilization of genetic resources, and; 3) Developing and applying genomic tools and resources. This work is relevant to the commercial poultry industry, those interested in germplasm preservation, and the breeding of captive
bird populations. Fundamental research is of interest to developmental, molecular and cell biologists using the bird as a model. 2. List the milestones (indicators of progress) from your Project Plan. Year 1 (2003) 1) Mechanisms by which turkey sperm lose functional competence during in vitro liquid storage will be identified. 2) Begin oviductal sperm storage and sperm transport studies 3) Begin preliminary work on isolation and purification of testicular stem cells (addendum) Year 2 (FY 2004) 1) Continue to investigate mechanism by which turkey sperm lose functional competence during in vitro liquid storage 2) Alternative methods for liquid storage of turkey sperm that maintain high fertility rates will be developed 3) Characterize the oviduct epithelia using lectins, alkaline phosphatase and probes to localize enterochromaffin cells in relation to sperm transport and storage 4) Continue isolation and purification of testicular stem cells (addendum) Year 3 (FY 2005) 1) Continue to
investigations on mechanisms by which turkey sperm lose functional competence during in vitro liquid storage and continue to develop new methods for the liquid storage of turkey sperm 2) Lipid enrichment of sperm plasmalemma 3) Complete studies on oviductal sperm transport and storage studies, write manuscripts 4) Complete studies on sperm competition, write manuscript 5) Continue to localize gene products based on SAGE in oviduct 6) Lectin characterization of oviductal sperm storage sites and resident sperm 7) Develop cell culture system for the propagation of testicular stem cells Year 4 (FY 2006) 1) Alternative methods for cryogenic storage of poultry sperm will be developed 2) Continue lipid enrichment of sperm plasmalemma 3) Continue to localize gene products based on SAGE in oviduct 4) Continue lectin characterization of oviductal sperm storage sites and resident sperm 5) Transfect and propagate stem cells and transfer to recipient sterilized males (addendum) 6) Evaluate
efficacy of recipient males to produce sperm derived from transfected stem cells (addendum) Year 5 (FY 2007) 1) Complete development of improved methods for cryogenic storage of poultry sperm 2) Complete lipid enrichment of sperm plasmalemma 3) Complete localization of gene products based on SAGE in oviduct and write manuscripts 4) Complete stem cell transfers and write manuscripts 5) Complete lectin characterization of oviductal sperm storage sites and resident sperm 6) Transfer donor stem cell procedure technology to industry (addendum) 3a List the milestones that were scheduled to be addressed in FY 2005. For each milestone, indicate the status: fully met, substantially met, or not met. If not met, why. 1. Surface glycoproteins of both turkey and rooster sperm have been characterized using fluorescent-labeled lectins as markers. Milestone Fully Met 2. Mitochondrial function during liquid storage of turkey semen and cryogenic storage of rooster semen has been assessed by
monitoring ATP production. Milestone Fully Met 3. A new extender supplement to improve fertility of 24-h stored turkey semen has been tested and found to be effective. Milestone Fully Met 4. Oviduct epithelial proteins and glycoproteins that may impact on sperm transport and storage (alkaline phosphatase, different lectin-specific glycoproteins and the presumptive localization of enterochromaffin cells) are either fully or substantially completed and corresponding manuscripts will be written in 05. Milestone Fully Met 5. Localize gene products based on SAGE in oviduct: The distribution of SAGE identified gene products is continuing as new SAGE data becomes available. Milestone Fully Met 6. Identify surface markers to be used in isolation of stem cells (addendum) : Two markers have been used with limited success to identify the testicular stem cells. We have identified 4 subtypes of spermatogonia (one of which is the stem cell). Milestone Fully Met 7. Develop cell culture system for
the propagation of testicular stem cells (addendum): The dispersion of the testicular cells have been successful but culture of the isolates gives rise to mixed populations of cells. Milestone Fully Met 3b List the milestones that you expect to address over the next 3 years (FY 2006, 2007, and 2008). What do you expect to accomplish, year by year, over the next 3 years under each milestone? Year 4 (FY 2006) Complete analysis of sperm function loss during liquid and cryogenic storage and anticipate development of alternative storage methods for both turkey and chicken sperm. Studies began in FY 05 on oviductal sperm storage and transport using tissue explants and expect to be completed by end of FY 06. We expect to determine how sperm enter and then egress from the sperm storage sites and provide insight into why some sperm are more successful than others in reaching the sperm storage sites. Continue lectin characterization of oviductal sperm storage sites and resident sperm and
develop from such information a diluent containing dispersed lipids for their incorporation into the sperm plasmalemma. The distribution of SAGE identified gene products continues as new SAGE data becomes available. These observations will allow us to see if gene products are being produced only by the sperm storage site epithelium or throughout the oviduct mucosa. Lipid enrichment of sperm plasmalemma. Successful incorporation of lipid into the sperm plasmalemma will modify the sperm thereby affording possible longer in vitro semen storage times. Transfect and propagate stem cells and transfer to recipient sterilized males and evaluate efficacy of recipient males to produce sperm derived from transfected stem cells (addendum). Year 5 (FY 2007) Complete analysis of sperm function loss during liquid and cryogenic storage and anticipate development of alternative storage methods for both turkey and chicken sperm. Complete localization of gene products based on SAGE in oviduct and
write manuscripts. These data will provide insight into the molecular mechanisms successful sperm storage by the oviduct. Complete lectin characterization of oviductal sperm storage sites and resident sperm and the lipid enrichment of sperm plasmalemma. Field trials will be initiated with genetics companies, universities, and commercial poultry producers to test new semen storage methods. The results of this work should lead to wider use of longer semen storage periods and lead to the sale of semen for hatching egg production. Transfer donor stem cell procedure technology to industry (addendum). This will be a novel approach to modifying the genome of birds and ultimately can be used to alter the sex ratio of hatching eggs. Field trials will be initiated with genetics companies, universities, and commercial poultry producers to test transgenic bird production. The results of this work should lead to insertion of genes of economic importance into poultry. Year 6 (2008) Complete
analysis of sperm function loss during liquid and cryogenic storage and anticipate development of alternative storage methods for both turkey and chicken sperm. Complete localization of gene products based on SAGE in oviduct and write manuscripts. These data will provide insight into the molecular mechanisms successful sperm storage by the oviduct. 4a What was the single most significant accomplishment this past year? The finding of enterochromaffin-like cells in the vagina has implications for a local control of sperm transport and selection by the oviduct. Serotonin, a neurotransmitter that is synthesized by the enterochromaffin cells in the gut epithelium, was also localized to the vaginal epithelium and not the sperm storage tubule epithelium. Serotonin increases cell membrane (sperm?) permeability to calcium and could influence sperm transport and selection by modulating sperm calcium uptake. 4b List other significant accomplishments, if any. In conjunction with French
scientists, we determined that the dispersed testicular cells can be cultured for up to 2 wk. However, the mixed cell types were present. Histologically, we identified 4-subtypes of the spermatogonia which will be useful in their identification following cell dispersals. The sperm storage sites in the oviduct are alkaline phosphatase positive indicating continued cell proliferation and differentiation. The testosterone producing cells in the testes (Leydig cells) likewise are strongly alkaline positive, which permits us to positively identify this cell type in cell dispersions. Successfully used newly devised whole mount procedure for localization of alkaline phosphatase in vagina mucosa. A patent application was submitted for a new turkey semen storage extender formulation. Using a battery of lectins, the glycocalyx of both turkey and rooster sperm was characterized in fresh, non-stored semen; this is the first report of this data for avian species. Significant differences in ATP
production were discovered between fresh and stored poultry semen. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. A. A novel procedure for removing the cryoprotectant glycerol from frozen-thawed chicken semen was developed to enable the immediate banking of semen from genetically-valuable and/or unique research chicken lines. The research was conducted as collaboration between the Biotechnology and Gamete Laboratory (Beltsville, MD) and the Avian Disease and Oncology Laboratory (East Lansing, MI). The specific accomplishment during the reporting period was a four-fold improvement in the fertility of glycerol- exposed semen. Development of this novel protocol is significant for two reasons: 1) glycerol is the most effective cryoprotectant for poultry semen but is contraceptive in the hen, and must be removed prior to insemination to achieve fertility; 2) semen from "at risk" research populations can be cryopreserved
and used to re-generate lines until new cryopreservation protocols are developed that dramatically improve the fertility of frozen/thawed poultry sperm (a major objective of this project). B. We discovered the presence of a class of molecules, aquaporins, which regulate water transport in the poultry male reproductive tract. The research was conducted in the Biotechnology and Gamete Laboratory (Beltsville, MD). The specific accomplishment during the reporting period was the first demonstration of aquaporins in avian male reproduction. This is significant because it provides a physiological explanation for the resorption of water during sperm maturation and provides an immunological procedure to be used for localization of aquaporins in other avian tissues. The localization of serotonin in the vaginal epithelium is important and unexpected observation in that it may have a significant impact on sperm selection and storage. The impact is that our fundamental knowledge regarding
oviductal sperm storage is increased. This will stimulate further novel approaches to the question of how to increase the shelve life of poultry semen for artificial insemination. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Technology Transfer -Submitted patent application for new turkey semen storage extender; initiated contacts with industry to field test and mass produce the extender -Collaborated with following scientists: J.P. Brillard, INRA, Nouzilly, France (stem cell dispersal, isolation and culture); G. Fasenko, U. Alberta, Canada (localization of apoptotic cells in poultry blastoderms). -Represented ARS in meeting with the 10-member, Iraqi Chochran Fellows, and spoke about poultry research in ARS and academia. -Demonstrated how to
isolate sperm storage tubule containing mucosa from feral birds to interested graduate students and faculty, Department of Ecology & Evolution, University of Chicago and instructed on how to prepare reproductive tissues and gonads for microscopy. -Submitted CRADA (12/04) with INRA-Nouzilly (France) and BioPharm (Czech Republic) which is presently under review in Czech Republic. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Bakst, M.R. and Long, J.A. Oviducal sperm storage: model for in vitro sperm storage Proceedings - XII World's Poultry Congress, Istambul, Turkey, June 8-13, 2004, CD - Full-text and participant list; Category, M-3-Poultry Reproductive Physiology Approved by Institute: 4/6/04 Submitted to Journal: 4/1/04 Bakst, M.R., Long, J.A., Zuelke, K.A Sperm Storage in the Hen's Oviduct Feedinfo News/Feedinfo.com
http://www.feedinfo.com/console/PageViewer.aspx?page=76347 Approved by Institute: 2/11/04 Submitted to Journal: 2/25/04 Accepted by Journal: 3/10/04 Published by Journal: 3/10/04 Invited seminars: Invited speaker at the International Plant and Animal Genome Conference in San Diego, Jan 2005; "Addressing the Biological Challenges of Germplasm Preservation in Poultry: New Approaches for Semen Cryopreservation". Invited speaker for the Poultry Science Association Ancillary Scientists Symposium in July 2005; "Avian Semen Cryopreservation: What Are the Biological Challenges?" Invited to give talk "Avian testicular stem cells", at the Incubation and Fertility Research Group, WPSA Working Group 6 - Reproduction, University of Lincoln, Lincoln,UK. September 2005. Invited to give talk "The why's and how's of staging avian embryos" at the The 2nd Combined Workshop of Fundamental Physiology and Perinatal Development in Poultry, WPSA European Working Group of Physiology and the Working Group
Perinatal Adaptation, Humboldt-University of Berlin, Germany. September 2005.
Impacts
(N/A)
Publications
- Zaniboni, L., Bakst, M.R. 2004. Localization of aquaporins in the sperm storage tubules in the turkey oviduct. Poultry Science. 83:1209-1212.
- Long, J.A., Zuelke, K.A. 2005. Addressing the biological challenges of germplasm preservation in poultry: new approaches for semen cryopreservation [abstract]. Plant and Animal Genome Abstracts. 6(43):68.
- Pelaez, J., Long, J.A. 2005. Lectin characterization of membrane surface carbohydrates in poultry spermatozoa [abstract]. American Society of Andrology Meeting. 26(2) Suppl. 1:58.
- Zaniboni, L., Bakst, M.R. 2004. Aquaporins are observed in the duct epithelia of the epididymal region of the large white turkey. Poultry Science. 83:1917-1920.
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Progress 10/01/03 to 09/30/04
Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? The commercial turkey industry, including both primary breeder and multiplier operations, relies exclusively on artificial insemination (AI) for production. Additionally, commercial broiler operations may soon have to incorporate AI methods as standard husbandry practice because of increasing problems in sustaining acceptable fertility levels associated with natural mating of the heavier strains. Current AI practices almost exclusively use freshly collected semen and weekly inseminations to achieve high fertility rates. In large commercial operations, 4-6 hen houses contain 2,000-4,000 turkeys each. Considering that breeder hens are in egg production for at least 24 wk and that each hen must be inseminated weekly to assure optimal fertility, it is obvious that AI is both time and labor-intensive. It
is estimated that 10-15% of turkey hens in North America are inseminated with semen from males kept on stud farms, whereas 80% are inseminated with semen from males housed in a barn with hens and subjected to hen management protocols. Under the stud farm system, elite males are selected and managed to attain maximum reproductive fitness. Objective 1: Determine how and when poultry sperm lose functional competence during liquid and cryogenic storage and use as a basis for developing successful sperm storage methods in vitro. Objective 2: Determine the influence of sperm phenotype (mobility, fertilizing capability) on liquid and cryogenic storage of turkey sperm, as well as phenotypic traits of economic importance in breeder males (feed conversion, body conformation, liveability). Objective 3: Increase the efficacy of oviductal sperm transport to the sperm storage tubule (SST) and the site of fertilization by elucidating the cellular and molecular mechanisms regulating sperm selection
and transport in the oviduct. Objective 4: Determine the molecular basis of sperm subsistence in the sperm storage tubules using serial analysis of gene expression (SAGE) and developing an in vitro model (tissue explants) to elucidate molecular and cellular events regulating prolonged oviductal sperm storage and sustained fertility in poultry Addendum: An additional objective addresses the isolation and propagation of testicular stem cells ultimately to be the targets of gene insertion and transfer to recipient males. This work has been approved by the NPL and is part of an anticipated multinational cooperative agreement. This research relates to National Program 101, Animal Genomes, Germplasm, Reproduction, and Development 100% and contributes to the following 3 NP 101 research goals: 1) Improving reproductive efficiency in livestock; 2) Conservation, characterization, and utilization of genetic resources, and; 3) Developing and applying genomic tools and resources. 2. List the
milestones (indicators of progress) from your Project Plan. Year 1 (2003) Begin sperm biochemistry studies and development of glycerol removal methods Begin oviductal sperm storage and sperm transport studies Begin preliminary work on isolation and purification of testicular stem cells (addendum) Year 2 (FY 2004) Complete glycerol removal methodology study Continue sperm biochemistry studies Begin loss of sperm function studies Establish explant culture system for turkey SST's to enable in vitro analyses of SST function. SAGE analysis of sperm storage tubule gene expression. Year 3 (FY 2005) Complete sperm biochemistry studies Continue loss of sperm function studies Begin phenotype evaluations and development of alternative semen storage methods Complete studies on oviductal sperm storage and transport studies, write manuscripts Complete studies on sperm competition, write manuscript Continue to localize gene products based on SAGE in oviduct Lectin characterization of oviductal sperm
storage sites and resident sperm Lipid enrichment of sperm plasmalemma Transfect and propagate stem cells and transfer to recipient sterilized males (addendum) Year 4 (FY 2006) Complete loss of sperm function studies Continue phenotype evaluations and development of alternative semen storage methods Continue to localize gene products based on SAGE in oviduct Continue lectin characterization of oviductal sperm storage sites and resident sperm Continue lipid enrichment of sperm plasmalemma Evaluate efficacy of recipient males to produce sperm derived from transfected stem cells (addendum) Year 5 (FY 2007) Complete phenotype evaluations and development of alternative semen storage methods Complete localization of gene products based on SAGE in oviduct and write manuscripts Complete stem cell transfers and write manuscripts Complete lectin characterization of oviductal sperm storage sites and resident sperm Complete lipid enrichment of sperm plasmalemma Transfer donor stem cell procedure
technology to industry (addendum) 3. Milestones: A. The milestones fully or substantially met in FY 2004 are listed below. Experiments were initiated to investigate the loss of turkey sperm function occurring during liquid storage of semen. Specifically, we have begun to identify the surface glycoproteins of the turkey sperm plasma membrane using a series of fluorescent-labeled lectins. A batch analysis method for determination of ATP concentration was validated for poultry sperm to assess the mitochondrial function of sperm during semen storage. Proteomic analysis of turkey seminal plasma and sperm cells has been initiated to detect changes in protein occurring during in vitro storage of semen. We are conducting the final fertility trial involving efficacy of glycerol reduction method for frozen/thawed chicken semen and anticipate this work to be completed by October 2004. We have begun to assess the biochemical changes in chicken sperm caused by the cryogenic process, using the
methodology from turkey sperm evaluations and initiated a collaborative project with a primary chicken breeder company to determine the effects of genetics on the ability of chicken semen to survive the freeze/thaw process. Begin study on sperm competition, complete data anaylsis: Sperm competition studies were initiated and data was not consistent or difficult to collect due to methodology problems. Study will not be continued. Localize gene products based on SAGE in oviduct: The distribution of SAGE identified gene products was initiated and will continue as new SAGE data becomes available. Initiated proteomic analyses of SST proteins to enable investigation of SST gene expression at both the mRNA (SAGE) and protein levels. Identify surface markers to be used in isolation of stem cells (addendum) : Two markers can be used to identify the testicular stem cells. Overall morphology including the nuclear:cytoplasmic ratios are also being developed as markers of stem cells Establish
and propagate purified population of stem cells (addendum): The dispersion and semi-purification of stem cell populations have been successful and continue to be developed. B. One milestone was not completely met due to changing research priorities within the CRIS; plus the lead scientist spent a 3-month sabbatical in France. During time in France, preliminary work was done on the short term culture of oviduct explants. Unforeseen disease outbreak prevented initiation of this study in latter half of 2003. Oviductal sperm transport and storage study will commence in fall 2004 and be completed within 12 months. B. Milestones that are expected to be addressed over the next three years: Year 3 (FY 2005) Characterize turkey sperm biochemistry alterations during in vitro liquid storage; Develop alternative liquid storage protocols for turkey sperm; and investigate sire phenotype markers for storage protocols. Studies will commence in Fall '02 on oviductal sperm storage and transport
studies and expect to be completed by end of FY '05. We expect to determine how sperm enter and then egress from the sperm storage sites and provide insight into why some sperm are more successful than others in reaching the sperm storage sites. The distribution of SAGE identified gene products will continue as new SAGE data becomes available. These observations will allow us to see if gene products are being produced only by the sperm storage site epithelium or throughout the oviduct mucosa. Proteomic analysis of turkey sperm storage tubule proteins will be performed to complement the SST SAGE analyses. Lectin characterization of oviductal sperm storage sites and resident sperm will continue. Determine which glycoprotein/glycolipids on the sperm interact with the surface of the sperm storage site epithelium. Lipid enrichment of sperm plasmalemma. Successful incorporation of lipid into the sperm plasmalemma will modify the sperm thereby affording possible longer in vitro semen
storage times. Transfect and propagate stem cells and transfer to recipient sterilized males (addendum). Isolated and dispersed testicular tissue will be stained with probes and sorted by flow cytometry based in staining affinities and DNA content. Year 4 (FY 2006) Complete analysis of sperm function loss during liquid and cryogenic storage and anticipate development of alternative storage methods for both turkey and chicken sperm. The distribution of SAGE identified gene products will continue as new SAGE data becomes available. These observations will allow us to see if gene products are being produced only by the sperm storage site epithelium or throughout the oviduct mucosa. Continue lectin characterization of oviductal sperm storage sites and resident sperm and develop from such information a diluent containing dispersed lipids for their incorporation into the sperm plasmalemma. Evaluate efficacy of recipient males to produce sperm derived from transfected stem cells (addendum).
Year 5 (FY 2007) Complete analysis of sperm function loss during liquid and cryogenic storage and anticipate development of alternative storage methods for both turkey and chicken sperm. Complete localization of gene products based on SAGE in oviduct and write manuscripts. These data will provide insight into the molecular mechanisms successful sperm storage by the oviduct. Complete lectin characterization of oviductal sperm storage sites and resident sperm and the lipid enrichment of sperm plasmalemma. Field trials will be initiated with genetics companies, universities, and commercial poultry producers to test new semen storage methods. The results of this work should lead to wider use of longer semen storage periods and lead to the sale of semen for hatching egg production. Transfer donor stem cell procedure technology to industry (addendum). This will be a novel approach to modifying the genome of birds and ultimately can be used to alter the sex ratio of hatching eggs. Field
trials will be initiated with genetics companies, universities, and commercial poultry producers to test transgenic bird production. The results of this work should lead to insertion of genes of economic importance into poultry. 4. What were the most significant accomplishments this past year? A. What were the most significant accomplishments this past year? We established the protein extraction and separations methods combined with mass spectroscopy-based proteomic analysis techniques to identify proteins that are expressed in the turkey sperm storage tubule epithelium in the presence and absence of resident sperm. During FY04 we identified over 40 individual proteins from the SST epithelium. The majority of these proteins were present in both sperm and control AI SSTs. However, four proteins were found only in control AI SST and three were found only in sperm AI SST. The majority of the proteins were identified via MALDI- TOF and Ion Trap mass spectroscopy using sequence
information available from the chicken genome. These data confirm the utility of the chicken genome sequence in performing functional genomic and proteomic analyses in turkeys. These data also indicate that the turkey SST mucosal epithelium expresses a differing array of proteins depending upon whether there are sperm present or absent in the SSTs. These results represent an important first step in the process of identifying novel proteins that may play a role in maintaining sperm viability and fertility during prolonged storage. B. Other significant accomplishments Observed that specific lectins are selectively positive for spermatogonia and Sertoli cells in immature and mature turkey seminiferous tubules but not in chicken, quail or guinea fowl testes (addendum). Developed procedure for use of whole mounts to bypass sectioning which worked for some immunocytochemistry procedures and completed work on localization of aquaporin on the luminal border of the SST epithelium. Developed
an ATP assay for poultry sperm. This sperm function assay is important for assessing the adverse effects of storage on poultry sperm. What is unique here is that we modified the traditional method of processing/reading single samples to permit high throughput analysis of multiple samples by using a plate-reader with luminometer features. Developed a sample processing method that permits storage of both unextracted and extracted samples at -20C prior to conducting the ATP reaction. Both of these aspects have resulted in the ability to greatly expand experimental designs (e.g., more frequent sampling during storage intervals). Additionally, the ATP assay has proven effective with boar sperm (conducted in collaboration with Dr. David Guthrie, BGL). In conjunction with French scientists, we verified one and discovered a second testicular stem cells surface protein currently being used as molecular markers to identify stem cells. To determine whether the SAGE detected genes are being
expressed in the sperm storage tubule or the surface epithelia of the oviduct, immunocytochemical localizations were performed. Actin, tubulin and avidin are expressed by cells in both epithelia. Demonstrated that turkey sperm can incorporate exogenous lecithin during in vitro storage. These data are significant in that turkey sperm lose up to 30% of membrane phospholipids during the first 4 hours of in vitro storage, and are not capable of replacing the lost components through cellular synthesis. Because this work is under consideration for patent submission, further details are not being disclosed at this time. C. Significant activities that support special target populations. NONE 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. A. A novel procedure for removing the cryoprotectant glycerol from frozen-thawed chicken semen was developed to enable the immediate banking of semen from genetically-valuable and/or unique
research chicken lines. The research was conducted as collaboration between the Biotechnology and Gamete Laboratory (Beltsville, MD) and the Avian Disease and Oncology Laboratory (East Lansing, MI). The specific accomplishment during the reporting period was a four-fold improvement in the fertility of glycerol- exposed semen. Development of this novel protocol is significant for two reasons: 1) glycerol is the most effective cryoprotectant for poultry semen but is contraceptive in the hen, and must be removed prior to insemination to achieve fertility; 2) semen from 'at risk' research populations can be cryopreserved and used to re-generate lines until new cryopreservation protocols are developed that dramatically improve the fertility of frozen/thawed poultry sperm (a major objective of this CRIS project). B. We discovered the presence of a class of molecules, aquaporins, which regulate water transport in the poultry male reproductive tract. The research was conducted in the
Biotechnology and Gamete Laboratory (Beltsville, MD). The specific accomplishment during the reporting period was the first demonstration of aquaporins in avian male reproduction. This is significant because it provides a physiological explanation for the resorption of water during sperm maturation and provides an immunological procedure to be used for localization of aquaporins in other avian tissues. The impact is that our fundamental knowledge regarding oviductal sperm storage is increased. This will stimulate further novel approaches to the question of how to increase the shelve life of poultry semen for artificial insemination. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Technology Transfer -At the request of a major turkey breeder
operation experiencing sudden and dramatic fertility loss, on-site training in semen quality analysis methods was provided, with subsequent training of breeder personnel at the BARC laboratory. -The glycerol reduction protocol developed under the current Project Plan was provided to the USDA's National Germplasm Program to improve fertility from chicken semen samples previously frozen with glycerol as the cryoprotectant. -Collaboration with following scientists: I. Estevez, UMD; J.P. Brillard, INRA, Nouzilly, France; G. Fasenko, U. Alberta; S. Cerolini, U. Milan (Italy). Techniques and data exchanged have immediate application to on- going collaborative research efforts. -Consulted with European poultry scientists and industry personnel to discuss semen and fertility evaluation techniques (Netherlands, November 2003). These techniques have application to commercial poultry operations doing semen and fertility evaluation. -Instructed French scientist on methods to isolate and
identify status of germinal disc on hens egg. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. Popular press publications: Bakst, M.R., Long, J.A. 2004. Oviducal sperm storage: model for in vitro sperm storage. XXII World's Poultry Congress Books of Abstracts, Istambul, Turkey. p. 248. Bakst, M.R., Long, J.A., Zuelke, K.A. 2004. Sperm storage in the hen's oviduct. Feedinfo News Service www.feedinfo.com (electronic only). Invited seminars: Bakst, M.R. Oviducal sperm storage in birds, Meeting of the European Society of Reproduction, Dublin, Ireland, September 2003. Review of the male and female reproductive tracts and gametes in birds, Symposium on Turkey Reproduction, Raleigh, North Carolina, October 2003. Oviducal sperm storage in birds, University of Tours (France), Reproduction Group, September 2003. Oviducal sperm storage in birds, SRA/INRA (France), Poultry Research Unit, November 2003. The
turkey oviduct- sperm storage, fertilization and early embryonic development, International Symposium on Turkey Reproduction, Raleigh NC, October 2003. Impact of egg storage on early embryo mortality, Workshop on Fundamental Biology and Perinatal Development in Poultry, Berlin, Germany, October 2003. Invited to organize a session on poultry reproduction and present talk on oviductal sperm storage at the World's Poultry Science Meeting, Istambul, Turkey, June 2004. Long, J.A. Poultry semen storage, Delegation of Italian poultry farmers, Beltsville, MD, October 2003. Semen storage in poultry, Women in Andrology luncheon in conjunction with the American Society of Andrology Annual Conference, Baltimore MD, April 2004. Current and future cryopreservation poultry research, National Animal Germplasm Program's Poultry Species Committee, St. Louis, MO, July 2004. Zuelke, K.A. Serial analysis of gene expression (SAGE) in livestock models. Dept of Animal Science, North Carolina State
University, Raleigh, NC, Oct 2003. Serial analysis of gene expression (SAGE) and proteomics research in swine and poultry). Roslin Institute, Roslin (Edinburgh) Scotland, UK, Nov. 2003. Serial analysis of gene expression (SAGE) and proteomics research in swine and poultry. INRA-Tours, Nouzilly, France, April 2004. Functional genomics and proteomics research in livestock models. FDA Center for Veterinary Medicine, Rockville, MD, June 2004.
Impacts
(N/A)
Publications
- Bakst, M.R., Long, J.A. 2004. Oviducal sperm storage: model for in vitro sperm storage. Proceedings of the XXII World's Poultry Congress Book of Abstracts. p. 248.
- Long, J.A., Kramer, M.H. 2003. Effect of vitamin e on lipid peroxidation and fertility after artificial insemination with liquid-stored turkey semen. Poultry Science. 82:1802-1807.
- Long, J.A., Kulkanri, G. 2003. A new method for improving the fertility of glycerol-exposed poultry semen. [abstract]. Poultry Science. 82(1):45.
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Progress 10/01/02 to 09/30/03
Outputs
(N/A)
Impacts
(N/A)
Publications
- Long J.A., Kramer M. Effect of vitamin E on fertility after artificial insemination with liquid-stored turkey spermatozoa. Poultry Science. 2002. v. 81(Suppl 1). p. 47.
- Long J.A., Kulkarni G. A Novel Method for Improving the Fertility of Glycerol-exposed Semen. Poultry Science. 2003. v. 82 (Suppl 1). p. 45.
- Long, E.L., Sonstegard, T.S., Long, J.A., Van Tassel, C.P., and Zuelke, K. A. (2003). Serial analysis of gene expression (SAGE) in turkey sperm storage tubules in the presence and absence of sperm. Biol. Reprod. 69:469-474.
- Bakst, M.R. 2002. How to maximize fertility and early embryonic development from a biologist's point of view. Turkeys. 50:65-68, 2002.
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