Brief Summary of Minutes of Annual Meeting

1. The meeting was opened at 9:00 a.m. by Robert Godke, the W-171 chairperson. 2. Richard Denniston welcomed us to the Reproductive Biology Center, and presented a short history and background on the Embryo Biotechnology Laboratory of the LSU Reproductive Biology Center. 3. Comments from W-1171 Administrators. Dr. Mark Mirando provided us with two handouts that he went through with us. He discussed new personnel at USDA, the new CSREES website, competitive programs. He told us that electronic submission may be optional by 2006, and these submissions must be via the institution. He also mentioned that there may be opportunities for proposals on gamete and embryo physiology in the nanotechnology section. Animal Biosecurity could encompass germplasm preservation and distribution, disease prevention and animal tracking. For next year, the RFA will likely come out in August or early September, and be due November 1st or 15th. Plan for November 1st. Mini-sabbatical funding (1-3 months) is now available. Not many sabbatical grants are submitted, and there is a high percentage of proposals funded. There is a new requirement for post award management. Include funds in budget to travel to an annual meeting of awardees. Integrated proposals were discussed, and the uniqueness required for these proposals. Post-doctoral awards, were discussed, and Dr. Mirando stated they frequently get funded at the second submission. Lastly, Dr. Mirando went over the CSREES budget. Dr. C.Y. Hu told us that the NRSP review is being done now. He also addressed multi-state nomenclature. NCR will be NC ERA, to reflect integrated education, extension and research activities. For this group, Dr. Hu emphasized the importance of good impact statements. Impact statements are becoming increasingly important, so it is critical to make them be as meaningful as possible. He said there is a difference in outcome versus impact. Outcome is programmatic, whereas impact is how it affects others. Impact statements should be 2-5 sentences, not results but what the results mean to the agricultural community. He also asked us to keep in mind that there is no extension FTE at all in this group. It is not a requirement but it may be a problem if there is a push towards integration of discovery, learning and engagement. It was brought up that even though some members may not have an official extension appointment they do extension activities as part of their research appointment. Dr. Hu suggested that in the next renewal we revise our Appendix Es to reflect this. Also, we will need to talk about extension in the narrative. It is difficult to evaluate the new extension model. We must be able to estimate the measurables in extension, as there is no extension national database. In our report, Dr. Hu asked that we emphasize collaboration among group members. Report individual activities too, but joint activities should be showcased. Joint publications are important. He suggested that a good way to demonstrate joint activity would be to write a proposal for a conference symposium to USDA. 4. The 2004 minutes were approved with one spelling error to be fixed. 5. The committee discussed membership. Richard Fayer-Hoskin was asked to join and was accepted. Steve Stice would also like to be considered. Both are from Georgia. We will ask them both to request that their director submit an Appendix E electronically. Pete Hansen and Karen Moore from University of Florida were suggested for membership, as well as Jorge Piedrahita, Char and Peter Farin from NCSU and Jose Cibelli from Michigan State. Frank Guazdauskas from Virginia Tech was also suggested later that day. In addition, Neal and Lanette Edwards from Tennessee have been asked and are interested in joining the project. They need to contact their administrator and file an Appendix E. Bob Godke will follow up with this. Matt Wheeler will call the Florida faculty, Ken Bondioli will call the NCSU faculty, Rebecca Krisher will contact Virginia Tech and Jerry Yang will contact Michigan State. Our administrative advisor (Dr. Hu) encouraged us to examine member participation. Participation of all members is strongly encouraged by the administration. If there are members who consistently do not attend the annual meetings, it is reflected poorly in the administrative reviews of the project. At the time of project renewal, all members must resubmit an Appendix E. Therefore, each renewal is a natural time to assess member participation. The group discussed assessment of member participation, and the potential removal of members who are no longer actively participating in the project. A non-participatory member is one who does not attend the annual meetings, does not send a representative from the institution or does not submit a report for the meeting. It is important for all members to share committee responsibilities, such as serving in an officer position, compiling the annual reports and assisting in project renewals. It was understood that not all members are provided funding to attend the annual meeting, but it was suggested that even without physically attending members could participate electronically via videoconferencing. 6. Cindy Tian was elected as our new secretary and Rebecca Krisher will be the new Chairperson. 7. The date and location of next years annual meeting was determined to be in Orlando, FL in conjunction with IETS. The IETS board would like to work with the W-1171 group to conduct a pre- or post-conference symposium in the future. We will arrive in Orlando on Thursday the 5th of January, 2006. We will hold our annual W-1171 meeting on Friday January 6th. A recommendation was made not to hold the annual meeting with IETS every year. We also discussed the possibility of holding our meeting in Hawaii, hosted by Dr. Hu, on the way to Kyoto for the IETS meetings in 2007. It was suggested that we could even organize a symposium in Hawaii. At this point we broke for lunch. 8. The remainder of the day was spent on station reports. See the calendar year 2004 annual report for more details. 9. On Saturday morning, Dr. Mirando talked about Journal of Animal Science. They have a 7.5 month submission to print average, and the reproduction section, in particular, is very good. It is currently 2 weeks better than BOR. Larry Reynolds is the new Editor- in-Chief. A new person will take over for Mark Mirando as section editor. 10. The group then discussed the stated previous focus areas to determine if there are any we should take out or add. Oocyte ageing was combined with oocyte quality. Nuclear transfer was added, including oocyte activation and stem cells. Functional genomics was discussed as another focus area, but it was decided that it is a tool that can be applied to all focus areas, and as such does not warrant its own focus area. Micro-fluidics was removed for this year because of supply problems. In vitro embryos and sex ratio distribution was also eliminated as a focus area. Our current focus areas are, therefore, Oocyte Quality/Ageing, Nuclear Transfer/Stem Cells and Production Systems. Production Systems include sexed semen applications and estrous synchronization. The group then split into three sections to discuss each focus area, led by Rebecca Krisher, Matt Wheeler and Jack Rutledge, respectively. Oocyte Quality/Ageing The first collaborative project in this category was proposed by Rebecca Krisher and Kurt Zuelke. They propose to examine oocyte quality in pigs using SAGE. This group also discussed that the absence of the embryonic disc reported by Jack Rutledge may stem back to the oocyte. It was suggested to use viability stains, size, stage or genetic markers to sort out populations of oocytes, or two sources of ovaries, or two breeds of cattle. Populations could then be cultured using the in vivo transfer method described by Jack Rutledge to determine differences related to embryonic disc presence or absence. Libraries could then be created from different populations to determine marker genes in oocytes correlating to this phenotype. A mini-sabbatical proposal to USDA could be submitted to send a PI to Kurt Zuelkes laboratory to learn the SAGE technique, although it was pointed out that this person must come from a strengthening eligible institution. Other collaborative possibilities include looking at these oocyte populations using metabolic parameters (Rebecca Krisher). In addition, Char and Peter Farin are using SAGE to look at changes in oocyte gene expression with gonadotropin treatment as well as a transcriptional inhibitor. Collaborations were discussed between Matt Wheeler and Rebecca Krisher in which mouse oocytes cultured in the micro-fluidics chambers could be an additional treatment in a mouse oocyte array experiment that Rebecca has planned. Kurt is looking at changes in gene expression in pig oocytes and embryos as they develop through the blastocyst stage. Differences in parthenogenetic, androgenetic and gynogenetic embryo development, potentially related to methylation differences in an important gene, were discussed as a way to determine oocyte genes critical to preimplantation development. Also, oocyte ageing as a way to sort out oocyte competence was discussed. Use of the 7-14 day in vivo culture system would be a good way to examine loss of competence. Oocyte quality as it relates to nuclear transfer is also an area of interest, in which Jerry Yang, Cindy Tian and Carol Keefer will likely be interested. Nuclear Transfer/Stem Cells This group focused on clinical models of embryonic stem cells and adult derived stem cells in the pig, cow and goat. They plan to determine markers of differentiation. These cells will be characterized and compared. They discussed the different approaches to germ line transmission in animals versus clinical therapies. They felt that therapies will have the bigger impact. This group proposes to develop animal models for human clinical applications. Dr. Mirando then discussed the application of this technology to animal agriculture. USDA will be involved if it is applicable. They are considering an interagency program. This would preserve resources in animal science departments, so we do not lose the ability to use large animal models. This interagency program could be a model development program in collaboration with NIH. May or may not develop by 2006 or 2007. The priority is animal agriculture, but if it has a biomedical application that helps. Production Systems. This group came up with a plan for two collaborative experiments. Thanks to Jack Rutledge for providing the following summary of their discussion. Experiment 1. Treatment Embryos Production A Fresh, sexed, in vitro B Vitrified, sexed, in vitro C Vitrified, not sexed, in vitro D Frozen or vitrified, not sexed in vivo All Holstein embryos. In vitro embryos supplied by Wisconsin, In vivo embryos supplied by Iowa. Recipient cows in Louisiana, Arkansas and Wisconsin. The same bull(s) will be used for Treatments 1 & 2; the same bull(s) will be used for Treatments 3 & 4. Basic plan is to receive embryos from Wisconsin and transferred at 7 days in bulk to the ipsilateral horn. Conceptus recoveries will be done at 9, 11 or 13 days. Recovered embryos will be examined, counted and measured for length. Presence or absence of a visible embryonic disc will be recorded for the day-13 recovered embryos. The embryos may be retransferred to recipients for gestation, if desired. If not retransferred, they should be frozen for potential SAGE analysis, individually in cryovials (possibly frozen in PBS). Recipients cows will be reused as often as possible to evaluate the repeatability and competency to nurture post-hatched embryos. Random recovery sequences will be assigned to each cow (i.e., embryos will be recovered from cow #3 on day 9, 13 and 11 in successive trials. DAPI stain can be used to localize site of the embryonic disc, if none is visible under light microscope. Experiment 2 Objective: Using sexed semen (Wisconsin) to prepare fresh IVP and vitrified IVP embryos (produced with Jerry Yangs method) for a field trial comparison. One culture system will be used prior to embryo shipment, and single or twin embryo transfers will executed by Illinois, Louisiana and Arkansas. All embryos will be Holstein and supplied by Wisconsin. Illinois will supply ~400 cows, Arkansas 150 cows and Louisiana ~150 cows. Calving data will be collected. The option to use observed-estrus recipients for transfer of vitrified IVP embryos can be exercised on up to half the cows designated to receive vitrified embryos. The overall plan is to harvest Holstein oocytes from ovaries collected on Wednesday making the fresh-transfer embryos on Thursday. Stations needing embryos for not less than 25 recipients should inform Wisconsin of their needs at least 3 weeks in advance of date of intended transfer. The discussion focus groups summarized their collaborative efforts with the combined group and the meeting was closed at 11:55 a.m. on Saturday February 5, 2005.

We have improved the efficiency of bovine in vitro embryo production using sexed semen. Perhaps the most economically important reproductive technology that can be achieved is the production of offspring of pre-determined sex. We have improved estrus synchronization schemes to provide good synchrony between embryo donors and recipients, while eliminating estrus detection. This will improve the efficiency and success of embryo transfer programs. Successful artificial insemination programs are dependent on good estrus detection. We have improved management of cattle to maximize expression of estrus, and thus efficiency of artificial insemination. Endophyte-infected fescue is known to contain ergot alkaloids that adversely effect female reproduction. We have provided insight into the effects of ergot alkaloids on sperm function and should lead to a better understanding of overall sperm physiology. Reproductive efficiency is a major contributor to economically sustainable animal agriculture, since a large proportion of the resources used for animal production are used to maintain the breeding herd. Past work indicates males within a species exhibit significant variation in sperm fertilizing potential, which can affect reproductive efficiency of the breeding herd if herd management attempts to make efficient use of sperm numbers. Undergoing the acrosome reaction is an essential step to the fertilization process and a more detailed analysis of this process may contribute to diagnosis of variation in male fertility. This work evaluates a series of molecules in a potential intermediate stage of signal transduction in the acrosome reaction. Results suggest these molecules are involved in induction of this critical event in fertilization. This work, in turn, could lead to improved reproductive efficiency and improved production efficiency. Our cloning results catalog developmental anomalies in somatic-cell clones. Cloning procedures have been advanced as a productive means to improve efficiency of transgenic animal productions via use of transgenic cells as nuclear donor. Several visible examples are available in the scientific literature, but embryonic, fetal and neonatal losses in clones continue to limit the full exploitation of cloning technology, either for transgenic animal production or more generally for duplication of desirable genotypes. Full knowledge of developmental anomalies is essential for their ultimate elimination from cloning experiments. Improved methods of producing bovine embryos in vitro will result in both improved pregnancy rates and fewer abnormalities in resulting calves compared to current procedures. Our simple procedure developed for vitrifying bovine embryos will greatly decrease costs and increase flexibility of cryopreserving embryos, in addition to decreasing chances of spreading viruses because no animal-derived products are used. Studying the expression profiles of cloned embryo is the first step in identifying the cause of the low efficiency of the cloning technology and will provide insights into how NT can be improved through gene expression regulations. Regulating the epigenetic status of donor cells will significantly improve nuclear transfer efficiency and improve the health of cloned animals, if all genes can be fully reactivated by NT. Improving activation of oocytes has improved the efficiency of generating cloned embryos which will help improve the overall cloning efficiency. Behavior of cloned animals is indicative of their genetic age. This is another way of studying whether or not cloned animals from aged donors will suffer from pre-mature aging. Embryo culture and cryopreservation are important associated technologies for NT and will be necessary for large-scale cloning efforts when applied to small and large farms alike in the entire country and in the world. The conservation of important genetics materials with superior reproduction/production performance will make them available for future large scale cloning use for farmers to increase productivity. The analyses of food products from cloned animals are of vital importance for regulatory agencies to make their decision on whether or not products from cloned animals are safe for human consumption. The development of more efficient oocyte and embryo culture systems will allow the genetic improvement livestock via embryo transfer and associated technologies. The production of ±-lactalbumin and IGF transgenic swine will allow for improvement of piglet growth and health in swine production systems. These observations may have profound effects on milk and meat production. Conducting in vitro maturation in defined conditions permits better control over experimental conditions, the ability to better compare experimental results between laboratories, and reduced risk of disease transmission. Determining the rate of oocyte meiosis is critical for developing systems for in vitro embryo production, as the time of completion of maturation determines the appropriate time for in vitro insemination. Development of IVP systems in more common antelope species may then be applied to endangered species for conservation management programs. Optimal ionic composition of the maturation and culture medium is critical to produce normal, viable in vitro porcine embryos for agricultural and biomedical applications. Determining embryo metabolic profiles in domestic cats will allow us to develop an optimized culture medium for the in vitro production of cat embryos. This medium can then be used in assisted reproductive technologies applied to the conservation of endangered cat species. Determining the control mechanisms of meiotic and cytoplasmic maturation in porcine oocytes is critical in developing maturation systems that support developmentally competent oocytes for use in transgenic animal production, in addition to contributing to the knowledge base of oocyte physiology. Lamin C, although present, can not be used in porcine embryos to assess the efficacy of genetic reprogramming after nuclear transfer. Being able to produce pregnancies from epididymal sperm from testes after 24 hours of storage at 4º would allow the livestock producer to save germplasm from injured or deceased breeding males. Being able to improve the efficiency of laboratory embryo production by increasing the number and the quality of bovine embryos produced from test tube fertilization with the addition of progesterone to the culture medium, will help make this new assisted reproductive technology more acceptable to the cattle producers for on-farm use in the future. The ability to produce cloned transgenic goats that express the MSP1-42 transgene for malaria antigen in their milk has implications for future human malaria vaccine production. Identification of regulatory mechanisms controlling trophoectoderm lineage differentiation will provide a better understanding of placental development and insights into the etiology of early embryonic loss and implantation failure. Furthermore, knowledge gained will aid in the improvement of in vitro procedures and will ultimately result in improved fertility especially in procedures involving advanced biotechnological approaches (e.g. cloning and transgenics). The pluripotency-related transcription factor Nanog was identified in caprine and bovine preimplantation embryos. Unlike other embryonic stem cell markers used in mouse and humans, this transcription factor can be used as a specific marker of pluripotency in ruminant embryos. This will aid greatly in the derivation of embryonic stem cells in cattle, goats and other livestock. ESC in domestic species will provide an invaluable tool in genetic engineering of transgenic animals for improved production traits, disease resistance and production of biopharmaceuticals. The number of live piglets farrowed per litter is perhaps the most important component of sow productivity. Prenatal mortality in the pig ranges from 20% to 46% by term, the majority of which occurs during two key developmental stages, peri-implantation (15-30%) in early gestation and placental development in late gestation (5-10%). We have shown a 3-piglet-increase in litter size selected on the basis of placental efficiency, suggesting the possibility of increasing litter size by selection. We believe that, by taking a functional genomic approach that includes using multiple, tissue-specific, high-density, high- fidelity annotated microarrays to address the problem of embryo loss at these two critical stages of gestation, our studies will provide insight into molecular genetic mechanisms that control litter size. An improved understanding of these mechanisms should lead to novel strategies for litter size selection, and provide significant potential towards long-term genetic improvement of U.S. pig breeds and the sustainability of this industry. Research from the Oregon station has added new information regarding the developmental biology of the early bovine embryo and the establishment of extraembryonic endoderm. We demonstrate that SAGE enables simultaneous gene expression analyses at both the metabolic pathway and single gene levels, in embryos from any stage of development, and is thus ideally suited for establishing a novel systems biology approach to investigating swine embryo development. These libraries have been placed in the public domain for accession by researchers around the world. Rapid methods for analyzing boar sperm physiology are important because they can be used to distinguish between physiological activities and non-physiological damage in spermatozoa that are found during low temperature liquid storage and after cryopreservation. Utilization of these techniques is valuable in study of sperm physiology, but also more practically can be used monitor improvement boar sperm storage and cryopreservation. Mastitis costs the US dairy industry an estimated $2B annually. Results of our transgenic dairy cow experiments establish positive proof of principle that a transgenic based strategy can be employed to establish and convey a heritable, intrinsic enhanced resistance to mastitis in dairy cattle. Cell lines derived from bovine embryos of various origins provide potential cell culture models for investigating embryonic gene expression (e.g. via RNAi analyses), improving nuclear transfer efficiencies, and identifying potential markers indicative of embryo development and cellular reprogramming. Using the technique of clustering analysis of gene expression patterns in tissues derived from cattle has the potential of tracing the developmental origin of tissues and organs. IVP of cattle embryos allows for cattle production systems that are not possible with natural mating or artificial insemination. Induced twinning in beef production, production of dairy heifers using beef surrogate mothers and continuous F1 hybrid systems are all made feasible with IVP.

ARKANSAS Nihsen M.E., E.L. Piper, C.P. West, R.J. Crawford, Jr, T.M. Denard , Z.B. Johnson, C.A. Roberts, D.A. Spiers, C.F. Rosenkrans, Jr. 2004. Growth rate and physiology of steers grazing tall fescue inoculated with novel endophytes. J Anim Sci. 82:878-83. Post, N.M., D.L. Kreider and R.W. Rorie. 2004. Timed insemination in beef heifers after synchronization of estrus with controlled intravaginal drug releasing (CIDR) device and mesengesterol acetate (MGA). Prof. Anim. Sci (accepted). Post, N.M., D.L. Kreider, R.W. Rorie and T.D. Lester. 2004. Timed AI in postpartum beef cows using melengestrol acetate, PGF2alpha and GnRH. Prof. Anim. Sci (in press). CALIFORNIA Bertolini M., A.L.Moyer, J.B. Mason, C.A. Batchelder, K.A. Hoffert, L.R. Bertolini, G.F. Carneiro, S.L. Cargill, T.F. Famula, C.C. Calvert, R.D. Sainz and G.B. Anderson. 2004. Evidence of increased substrate availability in invitro-derived bovine foetuses and association with accelerated conceptus growth. Reproduction 128:341-354. DuTeaux, S.B., T. Berger, R. A. Hess, B.L. Sartini, and M.G. Miller. 2004. Male reproductive toxicity of trichloroethylene: sperm protein oxidation and decreased fertilizing ability. Biol. Reprod. 70:1518-1526. Maga, E.A., G. Sargent, H. Zeng, S. Pati, D. Zarling, S. M. Oppenheim, N.M.B. Collette, A.L. Moyer, J.S. Conrad-Brink, J. D. Rowe, R.H. BonDurant, G.B. Anderson, and J.D. Murray. Increased efficiency of transgenic livestock production. Transgenic Research (in press). Reh, W. A., Maga, E. A., Collette, N. M. B., Moyer, A., Conrad-Brink, J. S., Taylor, S. J., DePeters, E. J., Oppenheim, S., Rowe, J. D., BonDurant, R. H., Anderson, G. B. and Murray, J. D. 2004. Using a stearoyl-CoA desaturase transgene to alter milk fatty acid composition. J. Dairy Sci. 87:3510-3514. COLORADO Bass, L.D., D.J. Denniston, L.J. Maclellan, P.M. McCue, G.E. Seidel, Jr. and E.L. Squires. 2004. Methanol as a cryoprotectant for equine embryos. Theriogenology 62:1153-1159. Carnevale, E.M., M.A. Coutinho da Silva, L.J. Maclellan, G.E. Seidel, Jr. and E.L. Squires. 2004. Use of parentage testing to determine optimum insemination time and culture media for oocyte transfer in mares. Reproduction 128:623-628. Coutinho da Silva, M.A., E.M. Carnevale, L.J. Maclellan, K.A. Preis, G.E. Seidel, Jr. and E.L. Squires. 2004. Oocyte transfer in mares with intrauterine or intraoviductal insemination using fresh, cooled and frozen stallion semen. Theriogenology 61:705-713. Eldridge-Panuska, W.D., V. Caracciolo di Brienza, G.E. Seidel, Jr., E.L. Squires and E.M. Carnevale. 2005. Development of equine embryos in vivo post vitrification and recovery rates of embryos 6.5 days after ovulation. Theriogenology. In Press. Garner, D.L. and G.E. Seidel, Jr. 2004. Applications of flow cytometry in animal reproduction. In: Flow Cytometry in Biotechnology, L. Sklar (ed). Oxford University Press (USA). In Press. Kimura, K., L.D. Spete, M.P. Green, C.N. Murphy, G.E. Seidel, Jr. and R.M. Roberts. 2004. Sexual dimorphism and interferon-tau production by in vivo-derived bovine embryos. Molec. Reprod. Develop. 67:193-199. Li, G.-P., G.E. Seidel, Jr. and E.L. Squires. 2004. Improved cleavage of bovine ICSI ova cultured in heparin-containing medium. Theriogenology 61:1077-1084. Lindsey, A.C., D.D. Varner, G.E. Seidel, Jr., J.E. Bruemmer and E.L. Squires. 2005. Hysteroscopic or rectally guided, deep-uterine insemination of mares with spermatozoa stored at 18 h at either 5°C or 15°C prior to flow-cytometric sorting. Anim. Reprod. Sci. 85:125-130. Lu, K.H. and G.E. Seidel, Jr. 2004. Effects of heparin and sperm concentration on cleavage and blastocyst development rates of bovine oocytes inseminated with flow cytometrically-sorted sperm. Theriogenology 62:819-830. Seidel, G.E., Jr. 2004. Sexing of spermatozoa. In: Bovine Theriogenology, G. Bo and M. De La Soto (eds). Inter-Media, Buenos Aires. In Press. Suh, T.K., J.L. Schenk and G.E. Seidel, Jr. 2005. High pressure damages sperm during flow sorting. Theriogenology. In Press. Tubman, L.M., Z. Brink, T.K. Suh and G.E. Seidel, Jr. 2004. Characteristics of calves produced with sperm sexed by flow cytometry/cell sorting. J. Anim. Sci. 82:1029-1036. CONNECTICUT Liu JL, Sung LY, Du FL, Julian M, Jiang S, Barber M, Xu J, Tian X and Yang X. Differential Development of Rabbit Embryos Derived from Parthenogenesis and Nuclear Transfer. Mol Reprod Dev 68:58-64, 2004 Nedambale TL, Dinnyés A, Groen W, Dobrinsky JR, Tian X and Yang X . Comparison on in vitro fertilized bovine embryos cultured in KSOM or SOF and cryopreserved by slow freezing or vitrification. Theriogenology 62:437-449, 2004. Lee J, Tian X and Yang X. Optimization of Parthenogenetic Activation Protocol in Porcine. Mol Reprod Dev 68:51-57. 2004 Ji-Long Liu, Hirokazu Kusakabe, Ching-Chien Chang, Hiroyuki Suzuki, David W Schmidt, Marina Julian, Robert Pfeffer, Charles L Bormann, X. Cindy Tian, R. Yanagimachi, and X. Yang. Freeze-dried sperm fertilization led to term development in rabbits. Biol Reprod 70:1776-1781. 2004. Jiang L, Carter DB, Xu J, Yang X, Prather RS, Tian XC. Telomere Lengths in Cloned Transgenic Pigs. Biol Reprod 2004 70:1589-1593. Chang CC, Nagy P, Abdelmassih R, Yang X, Tian XC. Morphology of the G2/M nuclei during somatic cell haploidization by GV stage mouse oocytes. Biol Reprod 70:752-758 2004. X Yang. Therapeutic Cloning and Embryo-based Research on Biotechnology and Medicine: Opportunities and Challenges. Nature 2004; 428:210-212. Kubota C, Tian XC, Yang X. Differential fertility in second and third generation clones of a prize breeding bull. Nature Biotechnology 2004; 22: 693-694. Tian XC. Reprogramming of epigenetic inheritance by somatic cell nuclear transfer. RBMonline 2004; 8:501-508. Zhang, S, Kubota C, Yang L, Zhang Y, Page R, ONeill M, Yang X, Tian XC. Genomic imprinting of H19 in naturally reproduced and cloned cattle. Biol Reprod 2004 (accepted). Tian, XC, Kubota C, Sakashita K, Izaike Y, Okano R, Tabara N, Curchoe C, Jacob L, Zhang Y, Smith S, Bormann C, Andrew S, Yang X. Meat and Milk Compositions of Bovine Clones Compared with Matched Controls. Science, 2004 (full length research article, accepted). Yang, X, XC Tian, W Fodor. 2004. Cattle call for gene targeting. Nature Genetic (invited News and Views, accepted). Enright BP, Sung L-Y, Chang C-C, Yang X, Tian XC. Methylation and Acetylation Characteristics of Cloned Bovine Embryos from Donor Cells Treated with 5-aza-2-deoxycytidine. Biol Reprod 2004 (accepted). Sung L-Y, Du F, Xu J, Chang W, Jiang S, Tian XC, Yang X. The effect of albumin and sodium citrate on the development of in vitro produced bovine embryos under different oxygen tension. Reprod Nutr Dev 2004 (accepted). Nedambale TL, Dinnyés A, Yang X, Tian XC. Bovine blastocyst development in vitro: Timing, sex and viability following vitrification. Biol Reprod (accepted), 2004. ILLINOIS Wheeler, M.B., Clark, S.G., Beebe, D.J. 2004. Developments in in vitro technologies for swine embryo production. Reprod. Fertil. Devel. 16:15-25. Raty, S., Walters, E.M., Davis, J., Zeringue, H. C., Beebe, D. J., Rodriguez-Zas, S.L., M. B.Wheeler. 2004. Embryonic Development in the Mouse Enhanced by via Microchannel Culture. Lab on a Chip 4:186-190. Fischer-Brown, A.E., Lindsey, B.R., Ireland, F.A., Northey, D.L., Monson, R.L., Clark, S.G., Wheeler, M.B., Kesler, D.J., Lane, S.J., Weigel, K.A., Rutledge, J.J. 2004. Day 14 bovine embryo morphology: evaluation tool and potential marker of viability following in vitro production and in vivo culture. Reprod. Fert. Devel. 16:787-793. Zeringue, H.C., Wheeler, M.B., Beebe, D.J. 2004. A Microfluidic Method for Removal of the Zona Pellucida from Mammalian Embryos. Lab on a Chip, 5(advance web article October 14th, 2004) 108-110. Reeder, A. L., Ruiz, M.O., Pessier, A., Brown, L.E., Levengood, J.M., Phillips, C.A., Wheeler, M.B., Warner, R.E., Beasley, V.R. 2004. Intersexuality and the cricket frog decline: Historic and geographic trends. Environmental Health Perspectives (in press). Hartke, J.L., Monaco, M.H., Wheeler, M.B, and Donovan, S.M. 2004. Effect of a short-term fast on intestinal disaccharidase activity and villus morphology in piglets suckling insulin-like growth factor-I (IGF-I) transgenic sows. J.Animal Sci. (in press). Donovan, S.M., J.L. Hartke, M.H. Monaco and M.B. Wheeler. 2004. Over-expression of IGF-I: Effects on piglet intestinal growth. J. Dairy. Sci 87:(E. Suppl.): E47-E54. INDIANA Herrick, J.R., P. Bartels, and R.L. Krisher. 2004. Cryopreservation and post-thaw evaluation of in vitro function in epididymal spermatozoa from four species of free-ranging African bovids. Biol. Reprod. 71:948-958. Herrick, J.R., E. Behboodi, E. Memili, S. Blash, Y. Echelard, and R.L. Krisher. 2004. Effect of macromolecule supplementation during in vitro maturation of goat oocytes on developmental potential. Mol. Reprod. Dev. 69:338-346. Krisher, R.L.* (2004) The Impact of Oocyte Quality on Development. J. Anim. Sci. 82(E. Suppl.):E14-E23. Durkin, R.E., Herrick, J.R., Bartels, P., Krisher, R.L. Preliminary observations on the development of in vitro embryo production systems for South African ungulates. Zoo Biol. Submitted. Swain, J.E., Bormann, C.L., Krisher, R.L. Metabolism of glucose, pyruvate and glutamine by porcine oocytes matured in vitro and in vivo. Mol. Reprod. Dev. Submitted. LOUISIANA Behboodi, E., S.L. Ayres, E. Memili, B.C. Reggio, M. O'Coin, L. H. Chen, A. Landry, H. M. Meade, R.A. Godke and Y. Echelard. Health and reproductive profiles of transfected somatic cell nuclear transfer derived goats producing malaria antigen. Cloning & Stem Cells (Submitted). Collins II, M.G., J.W. Lynn, K. Bondioli and R.A. Godke. Co-culture of mammalian embryos with cells having increased HPRT activity. Molec. Reprod. Develop. (Under Review). Devireddy,R.V., B. Fahrig, R.A. Godke and S.P. Leibo. 2004. The effect of extracellular ice and glycerol on boar sperm water transport parameters: convergence of theoretical and empirical rates of optimal freezing. Molec. Reprod. Develop. 67:446-457. Godke, R.A., A. James, K. Hebert, R.S. Denniston, D. Paccamonti. 2004. The potential use of epididymal sperm from bulls and stallions for assisted fertilization. Havemeyer Fdn. Mono. 12:55-60. Godke, R.A. and C. R. Youngs. 2004. Biotechnology: Embryo Technology for Cattle. W. Pond (ed). Marcel Dekker, Inc. New York, NY. (In press). Gómez, M.C., C. E. Pope, A. G. Leslie, A. Lyons, R.F. Harris, A. L. King, A. Cole, R. A. Godke and B. L. Dresser. 2004. Birth of African Wildcat cloned kittens born from domestic cats. Cloning & Stem Cells 6:247-258. Luster, S.M., M. Murakami, S.P. Leibo, R.S. Denniston Y. Echelard, and R.A. Godke. 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Roser. 2004 Androgen and estrogen regulation of the boar epididymis: concentrations and receptor localization. Biol. Reprod. (Special Issue): 133. Ducummon, C.C. and T. Berger. 2004 The monomeric G-protein RhoA is widely expressed in mammalian sperm, but does not play a role in actin cytoskeletal reorganization in porcine sperm. Biol. Reprod. (Special Issue): 185 COLORADO Barcelo-Fimbres, M. and G.E. Seidel, Jr. 2004. Optimizing sperm concentration to maximize monospermy and minimize polyspermy with bovine in vitro fertilization. J. Anim. Sci. 82 (Suppl. 1):371. Carnevale, E.M., W.D. Eldridge-Panuska, V. Caracciolo di Brienza, G.E. Seidel, Jr. and E.L. Squires. 2004. Embryo development rates after vitrification and transfer of equine embryos. Proc. 6th Int. Symp. Equine Embryo Transfer, Rio de Janeiro, p. 20. Coutinho da Silva, M.A., G.E. Seidel, Jr., E.L. Squires and E.M. Carnevale. 2004. Casein, native phosphocaseinate and caseinoglycopeptide enhance binding of equine sperm to bovine zonae pellucidae. Proc. 6th Int. Symp. Equine Embryo Transfer, Rio de Janeiro, p. 24. Coutinho da Silva, M.A., G.E. Seidel, Jr., E.L. Squires, Y.H. Choi and E.M. Carnevale. 2004. Binding of stallion sperm to equine and bovine zonae pellucidae: Effect of milk proteins and glucose. Reprod. Fert. Dev. 16:252 (abstr. #263). Coutinho da Silva, M.A., J.E. Stokes, G.E. Seidel, Jr., E.L. Squires and E.M. Carnevale. 2004. Fertilization of bovine oocytes using sperm selected by adhesion to oviduct epithelial cells in vitro. Proc. 15th Int. Congress Animal Reproduction, Brazil, Abstracts Vol. 2, p. 414. De La Torre Sanchez, J.F., M. Lane, J. Gibbons, D.K. Gardner and G.E. Seidel, Jr. 2004. Glucose metabolism of in vitro and in vivo produced bovine embryos. Reprod. Fert. Dev. 16:196 (abstr. #149). Horvath, G. and G.E. Seidel, Jr. 2004. Macromolecules for vitrifying bovine oocytes. Reprod. Fert. Dev. 16:171 (abstr. #99). Souza, A.H., R. Sartori, J.N. Guenther, D.Z. Caraviello, G.E. Seidel, Jr., J.L. Schenk and M.C. Wiltbank. 2004. Fertilization rate and embryo quality in superovulated Holstein heifers artificially inseminated with X-sorted or unsorted sperm. Proc. 15th Int. Congress Animal Reproduction, Brazil, Abstracts Vol. 2, p. 358. Stokes, J.E., E.M. Carnevale, E.L. Squires and G.E. Seidel, Jr. 2004. Preparation of bovine and equine sperm by Percoll or swim up for injection into bovine oocytes. Proc. 15th Int. Congress Animal Reproduction, Brazil, Abstracts Vol. 2, p. 471. Walker, D.F., L.F. Campos-Chillon and G.E. Seidel, Jr. 2004. Vitrification of in vitro produced bovine blastocysts by addition of cryoprotectant in one step. Reprod. Fert. Dev. 16:184 (abstr. #123). ILLINOIS Lopes, P.H.C., Malusky, S.A., Lima, A.S., Beebe, D.J., Wheeler, M.B. (2004) Mouse embryonic development from one-cell stage to blastocyst utilizing KSOM supplemented with amino acids in a microchannel device. Reproduction, Fertility and Development 16:205, abstr. 166. Marshall, K.M., Shanks, R.D., Hurley, W.L., Donovan, S.M., Wheeler, M.B. (2004) Effect of increased suckling stimulation on piglet weight gain and daily milk yield of sows transgenic for bovine a-lactalbumin. Reproduction, Fertility and Development 16:291, abstr. 342. Clark, S.G., Walters, E.M., Beebe, D. J., Wheeler, M.B. (2004) In vitro fertilization in microfluidic channels enhances monospermic penetration of swine oocytes. Reproduction, Fertility and Development 16:251, abstr. 262. Malusky, S.A. Wheeler, M.B. Isolation of Adult Adipose Derived Stem Cells from Transgenic Swine. International Society for Stem Cell Research (ISSCR), 2nd Annual Meeting, Abstract 248 page 124. INDIANA Brad, A.M., Spies, T., Olivier, F., Lane, M., Gardner, D.K., Bartels, P., Krisher, R.L. 2004. 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Evidence of a direct effect of P4 on IVF-derived Bovine 8-cell embryos. Reprod. Fertil. Develop. 17: (In press). Giraldo, A.M., J. W. Lynn, E.C. Pope, R. A. Godke and K. R. Bondioli. Lifespan and chromosomal stability of bovine and porcine fetal fibroblast cells cultured in vitro. Reprod. Fertil. Develop. 17: (In press). Gómez, M.C., C.E. Pope, A.M. Giraldo, L. Lyons, R.F. Harris, A. King, A. Cole, R.A. Godke and B.L. Dresser. 2004. Birth of African Wildcat cloned kittens. Reprod. Fertil. Develop.16:141. Klumpp, A. M., R.S. Denniston D. Paccamonti, S.P. Leibo, and R.A. Godke. 2004. Embryonic development after holding bovine oocytes in undiluted follicular fluid for a 6-hour prematuration period. Reprod. Fertil. Develop.16:278. Landry, A.M., H. DiMaggio, W. Burnside, L. Sarradet, J. Saenz, D.J. Landry, L.R. Gentry, K. Bondioli and R.A. Godke. The effect of hCG on circulating progesterone in goats at the end of the breeding season. Southern Section, Amer. Soc. Anim. Sci. (In press). Landry,A.M., M. Murakami, R.S. Denniston, J.L. Williams, Y. Echelard, and R.A. Godke Development of bovine aggregate embryos constructed from nuclear transfer embryos and electrofused IVF-derived embryos. Reprod. Fertil. Develop.17: (In press). Moisan, A.E., E.L.Chamberlain, S.P. Leibo, B.L. Dresser, K. Bondioli and R.A. Godke. Blastocyst formation from vitrified bovine oocytes, zygotes and two-cell embryos. Reprod. Fertil. Develop. 17: (In press). Moisan, A.E., J.W. Lynn, S.P. Leibo, R.S. Denniston and R.A. Godke. 2004. Volumetric changes of bovine oocytes resulting from dehydration in disaccharides solution. Reprod. Fertil. Develop.16:177. Nel, L., R.A. Godke, B.L. Dresser and P. Damiani. 2004. Isolation, culture and cryopreserva- tion of somatic cells obtained from fresh and cooled semen, and fresh milk of gulf coast native sheep. Reprod. Fertil. Develop.16:152. Nel, L., P. Damiani, B.L. Dresser and R.A. Godke. 2004. Freezing animal skin biopsies under field conditions. Proc. AZA Conference. (Abstr). Nel-Themaat, L., M.C. Gomez, A. Cole, G. Wirtu, K.R. Bondioli, B.L. Dresser R.A. Godke and C.E. Pope. Somatic cell isolation from semen by percoll gradients. Reprod. Fertil. Develop. 17: (In press). Roberts, J.L., R.P. DelVecchio, G.T. Gentry, Jr., D. Sanders, P.E. Humes and R.A. Godke. Effect of melengestrol acetate (MGA) and monensin supplementation on puberty and pregnancy rates in crossbred beef heifers. Southern Section, Amer Soc Anim Sci. (In press). Sansinena, M., J. Lynn, R. Denniston and R. Godke. Ooplasmic transfer after interspecies nuclear transfer: Presence of foreign mitochondria, pattern of migration, and effect on embryo development. Reprod. Fertil. Develop. 17: (In press) Wirtu, W., C. E. Pope, P. Damiani, B.L. Dresser, R.A. Godke and B.D. Bavister. 2004. Insulin improves frequency of cleavage and subsequent development of bovine embryos in vitro. Reprod. Fertil. Develop.16:203. Wirtu, G. C. E. Pope, A. Cole, R. A. Godke and B. L. Dresser. 2004. Behavioral training and handling of adult female eland antelope (Taurotragus oryx) in a hydraulic chute. Proc AZA Conference. (Abstr.) MARYLAND He, S., Pant, D., Bischoof, S., Melican, D., Gavin, W., Keefer, C.L. (2004). Expression of embryonic stem cell (ESC) markers in preimplantation goat embryos. Biol. Reprod. (S1):212, abst. 522. He, S., Pant, D., Bischoff, S., Gavin, W., Melican, D., Keefer, C.L., (2005). Expression of pluripotency-determining factors Oct-4 and Nanog in preimplantation goat embryos. Reprod. Fert. Dev.: in press. USDA Beltsville Blomberg, L.A., J.R. Dobrinsky, and K.A. Zuelke. 2004. Steroidogenic acute regulatory protein (STAR) gene expression coincides with increased estrogen synthesis in porcine embryos. Society for the Study of Reproduction, Vancouver, British Columbia, Canada, August 3. Donovan, D.M., R.J. Wall, D. Pritchard, and N.C. Talbot. 2004. Expression of a group B streptococcus bacteriophage lysine in mammalian cells. American Society for Microbiology annual meeting. Guthrie, H.D., R.J. Wall, V.G. Pursel, J.A. Foster-Frey, D.M. Donovan, G.R.Welch, and W.M. Garrett. 2004. Follicular expression of a human beta-cell leukemia/lymphoma-2 (BCL-2) transgene does not decrease atresia or increase ovulation rate in swine. Society for the Study of Reproduction annual meeting. Vancouver, British Columbia, Canada, August 2. Mitchell, A.D. and R.J. Wall. 2004. In vivo evaluation of changes in body composition of transgenic mice expressing the myostatin pro domain using dual energy X-ray absorptiometry. Society of Experimental Biology annual meeting. Powell, A.M., N.C. Talbot, K.D. Wells, D.E. Kerr, V.G. Pursel, and R.J. Wall. 2004. Effect of roscovitine on fibroblasts ability to form blastocysts and establish pregnancies after bovine nuclear transfer. Annual Meeting of the International Embryo Transfer Society, January 10-14; Reproduction, Fertility, and Development 16: 155. Pursel, V.G., A.D. Mitchell, and R.J. Wall. 2004. Progress on using transgenes to produce lean pork. In: Present and Future of Transgenic cloned pigs. 2nd International Symposium of Research for Transgenic Cloned Pigs. Daejeon, Korea. Rodriguez, K.F., L.A. Blomberg, K.A. Zuelke, J. Miles, J. Alexander, and C. Farin. 2004. Use of serial analysis of gene expression (SAGE) for identification of candidate genes associated with gonadotropin-induced oocyte maturation in the mouse. Society for the Study of Reproduction, Vancouver, British Columbia, Canada, August 2. Talbot, N.C., O.M. Ocon, A.M. Powell, and A.D. Ealy. 2004. Interferon-tau expression from the trophectoderm outgrowths of primary bovine blastocyst culture: comparison between IVP, NT, and parthenogenote embryos. Annual Meeting of the International Embryo Transfer Society, January 10-14; Reproduction, Fertility, and Development 16: 153. Talbot, N.C. and A.M. Powell. 2004. Effect of bovine pituitary extract on hatching and post-hatching development of IVP bovine embryos. Annual meeting of the International Embryo Transfer Society, January 10-14; Reproduction, Fertility, and Development 16: 202. Zhao, B., R.J. Wall, and J. Yang. 2004. Prevention of high-fat diet induced insulin resistance by transgenic interruption of myostatin function in mice. North American Association for the Study of Obesity annual meeting. UTAH Aston, K.I, G-P. Li, B. Hicks, B.R. Sessions, B..J. Pate, L.N. Meerdo, D.S. Hammon, T.D. Bunch and K.L. White. 2004. The developmental competence of bovine nuclear transfer (NT) embryos derived from cow verses heifer ooplasts. SSR. Bunch, T.D. and G-P. Li. 2004. Conditioned media effects chromosome composition of bovine cloned emblryos. Proc. Pac Div. AAAS. Pate, B.J., G-P. Li, K.I. Aston, T.D. Bunch, K.D. Campbell, D.S. Hammon, L.F. Rickords and K.L. White. 2004. Specific integrins subunits present on the vitelline membrance of bovine oocytes. SSR. WISCONSIN Fischer-Brown, A., R. Monson,D. Northey, T. Kuhlka and J. Rutledge. 2004. Pregnancy and parturition following transfer of bovine embryos cultured in two media under two oxygen concentrations. Reprod, Fert and Devel:16(1,2):197. Nguyen, B.X., N.T. Uoc, L.V. Ty, R.L. Monson, M.L. Leibfried-Rutledge, N.H.duc, L.C. Bui, Q.X. Huu, N.V. Hanh, N.T. Tanh, N.V. Linh and J.J. Rutledge. 2004. Production of tropical dairy calves by embryo transfer using local Laisind (Bos incicus) recipients and intercontinental fresh IVF shipped embryos. Reprod, Fert and Devel:16(1,2):249-250. Wilson, R.D., K.A. Weigel, P.M. Fricke, M.L. Leibfried-Rutledge, D.L. Matthews, V.R. Schutzkus, and J.J. Rutledge. 2004. System for in vitro production with sexed sperm in commercial dairy herds. J. Dairy Sci: Vol. 87 (suppl.1): 258. Wilson, R.D., K.A. Weigel, P.M. Fricke, M.L. Leibfried-Rutledge, D.L. Matthews, and J.J. Rutledge. 2004. In vitro production of Holstein embryos using Beltsville method sex-sorted sperm. J. Dairy Sci: Vol 87 (suppl. 1): 366. Wilson, R.D., K.A. Weigel, P.M. Fricke, M.L. Leibfried-Rutledge, d.L. Matthews, J.J. Rutledge, and V.R. Schutzkus. 2004. In vitro production of Holstein embryos using sex sorted semen. Reprod, Fert and Devel:16(1,2):263. d. Miscellaneous Publications (Semi-Technical/Lay Publications): CONNECTICUT We transferred two technologies to a start-up company spun off from the University of Connecticut: 1) NT with long-term cultured cells for gene knockout; and 2) embryo/oocyte droplet vitrification technology. We also filed disclosure on a novel cloning technology in the pig. LOUISIANA Roberts, J.L., R.P. DelVecchio, G.T. Gentry, Jr., D. Sanders, P.E. Humes and R.A. Godke. Effect of melengestrol acetate and monensin supplementation on pregnancy rates in crossbred beef heifers. Louisiana Beef Cattle Rep. (In press). USDA Beltsville Smith 3rd, J.E., W.D. Rees, M.P. Richards, and R.J. Wall. 2004. Codon-optimization of threonine biosynthetic genes for mammalian cells. Beltsville Area Research Center Poster Day. March 24. 2004. WISCONSIN Fischer-Brown A. and J. Rutledge. 2004. Practical and research applications of elongation stage bovine embryos. IETS newsletter 21(3):11-15. Rutledge, J.J.2004. Technology Innovations to Enhance Livestock Agribusiness. Babcock Dairy Update, Babcock Institute of International Dairy Research and Development, 204 Agriculture Hall, College of Agricultural and Life Sciences, University of Wisconsin, Madison, WI 53706