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United States Department of Agriculture

Agricultural Research Service

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2008 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
This project is part of Component 1 of the NP 101 Action Plan Understanding, Improving, and Effectively Using Animal Genetic and Genomic Resources, part c. Preserve and Curate Livestock and Poultry Genetic Resources. Progress on project objectives: 1.A. Protein extraction, separation and staining methods were modified to obtain the most accurate profiles for poultry sperm. An average of 179 and 86 spots were excised from 7 turkey and 15 chicken sperm gels, respectively. To date, the identity of 56 sperm proteins have been confirmed. 1.B. Metabolic inhibitors depressed porcine sperm motility rapidly in vitro showing that motion could be blocked by three different mechanisms in the cells without causing cell death: reduction of mitochondrial transmembrane potential, inhibition of mitochondrial ATP production, and inhibition of ATP utilization in the sperm tail. 1.C. The effects of diluent composition and osmolality on striped bass sperm motility, ATP production, and mitochondrial membrane potential were evaluated using testicular sperm from 6 captive males. 2.A. RNA was extracted from the sperm storage tubules and vaginal epithelium of hens either sham (n=10) or artificially (n=10) inseminated at 38 wks of age. Macroarray and PCR validation confirmed differential gene expression among the tissues from each hen group. Microarray analysis currently is underway. 2.B. Uterovaginal junction (UVJ) and vaginal epithelia from 10 turkey hens were fixed, embedded and sectioned for immunocytochemical localization of neuropeptides. Gallanin was confirmed in the UVJ epithelial cells while vaso-intestinal peptide was associated with SST; however, synaptophsyin could not be localized in paraffin-embedded section. The next step is to evaluate frozen tissue sections for these peptides. 3.A. A total of 216 semen samples from MARC boars were frozen at BARC as part of a study to determine the genetic component of the variation in boar sperm cryosurvival. In a separate experiment, a new protocol using a single, fixed-time insemination of frozen-thawed boar semen yielded an 80% farrowing rate, equal to that obtained with double inseminations of non-frozen semen. 4.A. A recombinant plasmid encoding H6 was sequenced to verify the nucleotide sequence and encoded amino acids. Oligonucleotide primers were designed and will be used to amplify the gene from the recombinant plasmid for subsequent cloning into bacterial and plant expression vectors to produce H6. The next steps will be to evaluate sperm viability after incorporation of H6 into the plasma membrane and to determine the ability of H6 to permit intracellular transport of cryoprotectants. 4.C. Freshly-laid eggs were injected with bulsulfan (1.5, 1.8 or 2.1 µg/µl) to optimize the dose for chemical sterilization of turkeys. Embryonic testes were fixed at Day 17 of incubation and evaluated for the presence of spermatogonia. The lowest dose caused a 75-80% reduction, but not eradication of spermatogonia without degrading supportive cells. Higher dosages of busulfan caused the degradation of Sertoli and Leydig cells.

4. Accomplishments
1. Characterization of the Sperm Proteome in Poultry. It is likely that proteins 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 protein complement of poultry sperm before and after semen storage is important for understanding how and why sperm lose functional competence during semen storage. The protein complement of sperm from several mammalian species has been characterized, yet the protein complement of poultry sperm remains undefined. Our objective was to characterize the protein complement of chicken and turkey sperm prior to cryogenic storage. We used mass spectrophotometry to characterize the insoluble protein fraction of freshly collected spermatozoa from both species, and identified 56 putative proteins including ATP synthase, outer dense fiber protein, dihydropyrimidinase, tektins 1 and 2, enolase, creatin kinase, peroxiredoxin-6, dynein, voltage-dependent anion selective porin 2, triosephosphate isomerase 1, and tubulin. Additionally, we found significant amino acid sequence homology with 21 hypothetical proteins from Gallus gallus that have been isolated by other scientists, but not yet identified. 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. This accomplishment directly addresses NP 101 Action Plan Component I “Understanding, Improving, and Effectively Using Animal Genetic and Genomic Resources”, part c “Preserve and Curate Livestock and Poultry Genetic Resources”.

2. Effects of Sham Versus Artificial Insemination on Differential 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 genes that were differentially expressed in the SST of sham or artificially inseminated hens. Of the total 480 putative transcripts, 9% were differentially expressed by the SST, with a 40 -fold increase in avidin expression in the SST compared to the vaginal epithelium of artificially inseminated hens. Also, there was a 9-fold increase in avidin mRNA expression in the SST of artificially inseminated hens in comparison to sham inseminated turkey hens. Avidin protein was found to be expressed in the surface epithelium of both control (non-inseminated) and artificially inseminated hens, with protein expression higher in the control hens. Interestingly, immunohistochemical analysis showed that while control hen SST were avidin positive, the SST of inseminated hens were avidin negative. Finally, a total of 62 turkey (Meleagris gallopavo) ESTs were submitted to GenBank (Accession #s EX809858 through EX809919) and released October 29, 2007. These data provide the foundation for further study of the molecular mechanisms controlling the SST micro-environment. This accomplishment directly addresses NP 101 Action Plan Component I “Understanding, Improving, and Effectively Using Animal Genetic and Genomic Resources”, part c “Preserve and Curate Livestock and Poultry Genetic Resources”.

3. New Protocol Improves Fertility of Frozen-thawed Boar Semen. Artificial insemination with frozen-thawed semen results in farrowing rates of 55% and 8 piglets/litter, which is 20-30 percentage points lower and 2-3 fewer pigs per litter than expected with non-frozen semen. The fertility of thawed boar sperm in the female reproductive tract is limited to approximately 4 hours compared to 34 hours with non-frozen semen. In order to obtain better fertility with cryopreserved boar semen, we developed a new protocol where females were treated with altrenogest and hCG to artificially control the time of ovulation, and a single insemination of thawed boar semen was timed to have fertile sperm at the site of fertilization 0-4 hours before the expected time of ovulation. With our protocol litter size was about 96% of that using non-frozen semen; while the farrowing rate (80%) was equal to that using non-frozen semen in conjunction with standard industry breeding procedures. These results show that, with proper treatment of recipient females, frozen-thawed boar semen can be used by swine producers to obtain fertility that is almost as good as non-frozen semen This accomplishment directly addresses NP 101 Action Plan Component I “Understanding, Improving, and Effectively Using Animal Genetic and Genomic Resources”, part c “Preserve and Curate Livestock and Poultry Genetic Resources”.

4. Striped Bass (Morone saxatilis) Sperm Viability and Energy Status. Striped bass sperm collected from captive or wild fish are used for in vitro fertilization of eggs from white bass to produce a striped bass hybrid for aquaculture. Aquaculture of the bass hybrid is difficult because striped bass sperm lose the ability to fertilize eggs after being held for more than 45 minutes in liquid form after collection. We developed a unique approach to assess the energetic status and viability of striped bass sperm to investigate factors that might enhance the shelf life of semen stored in liquid form. We found that water activation of sperm, a standard technique for in vitro fertilization, quickly killed the sperm. The storage of sperm in a hypertonic, TRIS-free base-NaCl medium, designed to inhibit premature induction of motility, reduced sperm viability, mitochondria energy potential and ATP production compared to storage in an isotonic medium. These results demonstrate that diluent composition and osmolality must be considered when designing activation and storage extenders to maintain striped bass sperm motility, viability, and fertility in vitro. This accomplishment directly addresses NP 101 Action Plan Component I “Understanding, Improving, and Effectively Using Animal Genetic and Genomic Resources”, part c “Preserve and Curate Livestock and Poultry Genetic Resources”.

5. Significant Activities that Support Special Target Populations

Review Publications
Bakst, M.R., Akuffo, V. 2007. Alkaline phosphatase reactivity in the vagina and uterovaginal junction sperm-storage tubules of turkeys in egg production: implications for sperm storage. British Poultry Science. 48:515-8.

Bakst, M.R., Akuffo, V. 2008. Turkey sperm reside in the tubular glands in the urodeum following artificial insemination. Poultry Science. 87(4):790-2.

Bakst, M.R., Akuffo, V. 2008. Serotonin localization in the turkey vaginal but not sperm storage tubule epithelia. Poultry Science. 87(2):356-61.

Blanco, J.M., Long, J.A., Gee, G., Donoghue, A.M., Wildt, D.E. 2008. Osmotic tolerance of avian spermatozoa: influence of time, temperature, cryoprotectant and membrane ion pump function on sperm viability. Cryobiology. 56:8-14.

Guthrie, H.D., Woods, L.C., III, Long, J.A., Welch, G.R. 2008. Effects of osmolality on inner mitochondrial transmembrane potential and ATP content in spermatozoa recovered from the testes of striped bass (Morone saxatilis). Theriogenology. 69:1007-12.

Long, J.A. 2008. Reproductive Biotechnology and Gene Mapping: Tools for Conserving Rare Breeds of Livestock. Reproduction of Domestic Animals. 43 Suppl 2:83-8.

Pelaez, J. Long, J.A. 2008. Characterizing the glycocalyx of poultry spermatozoa: II. In vitro storage of Turkey semen and mobility phenotype affects the carbohydrate component of sperm membrane glycoconjugates. Journal of Andrology. 29:431-9.

Rowe, M., Bakst, M.R., and Pruett-Jones, S. 2008. Good vibrations? Structure and function of the cloacal tip of male Australian Maluridae. Journal of Avian Biology. 39(3):348-354.

Last Modified: 2/23/2016
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