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W1171: Germ Cell and Embryo Development and Manipulation for the Improvement of Livestock

Annual/Termination Reports (SAES-422): [02/18/2005] [05/29/2006] [07/05/2007] [05/01/2008] [04/05/2009]

Date of Annual Report: 02/18/2005

Report Information:
  • Annual Meeting Dates: 02/04/05 to 02/05/05
  • Period the Report Covers: 02/2004 to 02/2005

    • Participants:
    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.

    Accomplishments:
    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.

    Impact Statements:
    Last Modified: unknown

    Date of Annual Report: 05/29/2006

    Report Information:
  • Annual Meeting Dates: 01/07/06 to 01/07/06
  • Period the Report Covers: 01/2005 to 12/2005

    • Participants:
    Brief Summary of Minutes of Annual Meeting:
    1. The meeting was called to order by Rebecca Krisher.

    2. Comments from W-1171 administrators

    Dr. C.Y. Hu (W-1171 administrative advisor): Even though there was no numerical decrease in the funding amount for the National Research Initiative (NRI) last year, the real money (purchasing power) decreased. During the last 10 years the real money (purchasing power) has decreased by as much as 25%. The current push is to have more formula funds converted to NRI. It is proposed that formula funds be cut by 50% in the first year with the remaining 50% being cut in the second year. If enacted, this may enlarge the current USDA budget. However, directors of experiment stations nation-wide want formula funds increased by 100%. Theoretically, 25% of all formula funds should be spent on national projects, and accountability now is being examined more closely. This particular multi-state project (W1171) is highly productive in contrast to some projects that have had no progresses in the past 10- 20 years.

    Now and in the future, it is highly important to stress the impact statements in the annual progress reports. It is advisable to include the dollar amount saved by applying the research results, and use past tense. For multi-state projects, including W-1171, we need to have a collaborative impact. We need to strengthen our efforts in conducting collaborative projects. We are strong in our individual projects, but for a multi-state project collaborative impact/efforts are even more important.

    The W-1171 project was renewed in 2004, and it is scheduled to terminate on September 30, 2009. If a renewal is necessary and justified, the process must begin in mid-year 2007. Different stations may have to compete for renewal.

    The CREATE 21 idea was also briefly introduced. CREATE 21 is the acronym for Creating Research, Extension and Teaching Excellence in the 21st century. Potentially, a new institution may be created for studies of food, animal and natural resources, to be housed in the USDA. The new Director will be named by the president. It may be supported by competitive, intramural research and pork barrel funds.

    Dr. Mark Mirando (CSREES representative): An update on CSREES personnel and competitive programs was provided. It was specifically mentioned that 20% of all funds are saved for integrated projects. Awards are being made at higher budget amounts (up to $450,000). Proposals are welcomed from extension personnel. The integrative project proposals cant have objectives just for research, teaching, extension or education alone, but should combine objectives and goals for at least two of the four major areas. Last year, the NRI reproduction program funded one integrated proposal.

    Attention was drawn to proposals for conferences. To receive funding, conferences must be national in scope and have large attendance (100 people or more). The budgets for large conferences may be up to $10,000, whereas budgets for smaller conferences typically are funded at a level of $3,000 - 6,000. The funding rate for conference awards is about 90%.

    A question was raised by Jerry Yang regarding the status of a white paper on the use agricultural animals as models for biomedical research. Dr. Mirando commented that $1 billion may end up in the National Science Foundation. The funds may be in the form of pork barrel monies and NRI competitive programs. This is up to the decision by Congress who often listens to the National Academy of Sciences. The National Academy of Sciences has high regards for NRI and will favor competitive programs, but other constituents may speak differently to Congress.

    3. The minutes from the 2005 W-1171 Technical Committee Meeting were approved unanimously (moved by Tom Bunch and seconded by Cindy Tian).

    4. New Members Update: Cindy Tian will contact Jianbo Yao (jianbo.yao@mail.wvu.edu), West Virginia University and John Gibbons (jgibbns@clemson.edu), Clemson University to invite them to join the project.

    5. Election of a New Secretary for 2006: Curt Youngs was elected as secretary for the upcoming year, and Cindy Tian will assume the duties of chairperson and preside over the 2007 meeting.

    6. Date and Location of 2007 meeting: The 2007 W-1171 annual meeting will be held in Kyoto, Japan, the evening before the IETS meeting. Cindy Tian will contact Jennifer Gavel to arrange for a meeting room and time.

    7. Station reports: Due to time constraints, individual station reports by PIs were skipped so that more time could be allocated to discussion of collaborative projects among different stations. (It was felt that PIs were capable of reading the annual reports that were compiled prior to the meeting.) The attendees were divided into 4 different focus groups for discussion (stem cells, oocytes, embryo production, placental function).

    8. Discussion of Joint Research Projects: Reports from each group were given on their plans for collaboration. Stem cell group: collaboration on adipose stem cell expression profiling; Oocyte group: a research proposal will be finalized and submitted to members of the group via e-mail after the meeting ; Embryo production group: continue with experiments devised at the 2005 meeting. Placental function group: collaborative experiments will be finalized after the meeting.

    9. The meeting was adjourned.

    Accomplishments:
    The Arkansas Station demonstrated the use of sexed semen to produce embryos in vitro and evaluated survival after transfer to recipients. Fully mature oocytes may preferentially incorporate Y-bearing spermatozoa, but this does not exclude normal development after fertilization with X-bearing spermatozoa. Male embryos may develop at a higher rate after fertilization than females.

    The Colorado station confirmed that in vitro-produced bovine embryos cultured with phenazine ethosulfate in the CDM-1/CDM-2 system are much more normal with respect to lipid content than embryos produced without this compound. Cerulenin was not effective in decreasing lipid content of embryos. Fructose resulted in higher percentages of blastocysts per oocyte than glucose. A mini Percoll system was developed for isolating normal stallion sperm from small aliquots of frozen semen. Bovine embryos were vitrified efficaciously with PVA and Ficoll 70 as the only macromolecules in vitrifying media.

    The California station obtained results to further our understanding of the effects of environmental temperature stress during oocyte maturation and of porcine epididymal development, which affects sperm maturation. Although epididymal development may be affected by altering steroid levels, and both alpha and beta forms of the estrogen receptor and the androgen receptor are present during development, altering estrogen levels did not appear to affect receptor levels. Abnormal development of placentomes was observed already at Day 30 of gestation, which likely account for placental dysfunction in somatic cell nuclear transfer conceptuses.

    The Connecticut station compared the expression profiles of embryos from nuclear transfer, IVF and superovulation by DNA microarray; produced thousands of sexed bovine IVF embryos and conducted a large ET trial in China and obtained high pregnancy rates.

    The Illinois station developed a method to decrease polyspermy in swine during in vitro fertilization using a microchannel culture system, which provides a culture environment that more closely mimics the in vivo environment. Mammary-specific transgenic over-expression of IGF-I significantly increased milk IGF-I and IGFBP content, and provides a method to manipulate lactation persistency in swine. Adult stem cells were isolated from adipose tissue in swine and successfully differentiated them into fat-producing mature adipocytes after transplantation in vivo.

    The Indiana station further elucidated the mechanisms of meiosis and cytoplasmic maturation in porcine oocytes. Glucose metabolism is critical for both the initiation of nuclear maturation, and is also reflective of developmental potential. Five proteins that are differentially present in oocytes of low and high developmental potential were isolated and identified, possibly reflecting mechanisms involved in the acquisition of developmental competence. Efforts to identify differentially expressed genes in oocytes of low and high quality were initiated. This research will allow scientists to develop markers of oocyte quality that can be used to identify oocytes before embryo transfer that will be more likely to result in healthy offspring.

    The Iowa station demonstrated that a 250 mg dose of rbST was inadequate to enhance superovulatory response in Jersey cows.

    The Louisiana station continued effort to save germplasm from deceased bulls. Epididymal sperm harvested from testes of deceased males were found to be viable following both immediately after collection as well as following freezing and thawing. In collaboration with Illinois established that adding a small amount of progesterone to the in vitro culture medium can increase the number and the quality of cattle embryos produced from standard in vitro fertilization. Documented that long-term, continuous culture of cattle fibroblasts enhances the chances of aneuploidy (abnormal number of chromosomes).

    The Maryland station determined the mRNA and protein expression patterns of the transcription factor NANOG during preimplantation development. NANOG mRNA and protein expression was initiated at 8-cell stage. The NANOG mRNA expression is downregulated in trophectoderm of goat blastocysts, and protein appears to be sequestered and degraded in a process involving nucleolar localization. Goat and bovine NANOG mRNA was detected, and the full open reading frame (ORF) was sequenced. The sequence (AY786437) was updated from the initial partial ORF submitted to GenBank. A fusion construct consisting of the bovine NANOG promoter and GFP (green fluorescent protein) gene has been demonstrated to be functional. The expression patterns of key components in pathways known to be involved in embryonic stem cell maintenance were identified in bovine blastocysts and ICM outgrowths. This knowledge will aid in the derivation and characterization of ruminant embryonic stem cell lines.

    The Oregon station demonstrated that plasminogen activator inhibitor-1 is a potent reversible inhibitor of bovine endodermal cell migration on vitronectin in vitro. It is likely bovine endodermal cells use the urokinase-type plasminogen activator receptor to bind vitronectin during migration from the inner cell mass. The timely appearance of plasminogen activator inhibitor-1, of either endodermal or trophectodermal origin, may provide the signal for completion of the extra-embryonic endoderm. The North Carolina station identified gene expression differences between porcine placentae differing in efficiency, identified differences between placentas from in vivo- and in vitro-produced bovine embryos in vascular morphometry as well as gene expression, and confirmed conservation of genomic imprinting in swine placenta for over 10 known imprinted genes. A somatic cell nuclear transfer method was developed for swine that can generate both transgenic and non-transgenic offspring at high efficiencies. mRNAs associated with the resumption of meiosis in mammalian oocytes (cattle and mice) were identified. Bovine oocytes were prevented from undergoing meiotic maturation by blocking expression of a novel mRNA associated with meiosis. Demonstrated that COC held in meiotic arrest can be used in subsequent in vitro fertilization procedures without detrimental effects on oocyte developmental capacity.

    The Utah station demonstrated that a linear arginine-glycine-aspartic acid (RGD) peptide can block fertilization and that the amino acids surrounding the RGD sequence have an impact on the biological activity of the RGD receptor. Identified 2 proteins containing a RGD tripeptide sequence whose expressions differs between oocyte adsorbed and nonadsorbed proteins. Generated a list of sperm ligands that apparently interact with oocyte receptors, as well as a list of oocyte receptor proteins that apparently interact with sperm ligands. Learned that the tyrosine kinase inhibitor genistein inhibited oocyte activation, while tyrosine kinase activator sodium orthovanadate induced oocyte activation. The specific Src family kinase inhibitor PP1 inhibited oocyte activation, whereas an antibody directed against focal adhesion kinase, a mediator of integrin associated pathways, blocked oocyte activation. Wortmannin, a potent inhibitor of PI3-kinase had no effect on oocyte activation. These data indicate the involvement of tyrosine kinases, specifically one or more Src family kinases and focal adhesion kinase. Learned that eExposure of bovine oocytes to nicotine, at levels of 4, 6 or 10 mM, during oocyte maturation increased the incidence of aneuploidy, and exposure of oocytes to 10 mM nicotine inhibited the percentage of oocytes that extruded the first polar body. Immunoflurencence staining revealed irregular anaphase I and telophase I spindles after oocyte exposure to nicotine at levels of 1.2 mM or higher. Demonstrated that oocytes derived from cows have a greater capacity to reprogram donor cell DNA following nuclear transfer as compared with heifer oocytes (based on in vitro development to the 2-cell stage and to the compacted morula/blastocyst stages). Nuclear transfer embryos derived from cow oocytes resulted in significantly higher rates of pregnancy establishment than embryos derived from heifer oocytes as well as higher pregnancy retention to 90 and 180 days of gestation and to term. Following delivery more nuclear transfer calves derived from cow oocytes tended to be healthy and normal than those derived from heifer oocytes. Observed that exposure of donor cell nuclei to oocyte cytoplasm for more than 2.5 hours prior to activation resulted in abnormal chromatin morphology, and reduced development to compacted morula/blastocyst stages in vitro; however following transfer of embryos to recipients there was no difference in pregnancy rates throughout gestation. Found that removal of cumulus cells and centrifugation of maturing bovine oocytes affected the pattern of spindle microtubule distribution and division of chromosomes.

    The USDA Station (ARS Biotechnology and Germplasm Lab ) adapted the small amplified RNA-SAGE technique, which enables reliable analysis of as little as 50 ng total RNA, for use in porcine conceptus transcriptome studies. Utilization of SAR-SAGE enables a 33-fold reduction in the number of embryos needed to establish gene expression profiles in day 6 in vivo- and in vitro-derived embryos. Characterized mesoderm-related changes (via brachyury expression) in the embryonic disc of elongating (ovoid, tubular, and filamentous stages) porcine embryos and demonstrated that the progression of trophectoderm elongation and embryonic disc differentiation in the pig conceptus are independent of each other as is the case for other ungulates. To facilitate rapid and more extensive cellular analyses of boar sperm before and after storage and cryopreservation, we developed different techniques using dual staining flow cytometric to measure 1) formation of reactive oxygen species using two different fluorescent probes, 2) mitochondrial inner membrane charge potential, and 3) the physiological effect of the respiration inhibitor, menadione. Stable and long-lived bovine trophectoderm cell lines from NT, IVP, parthenogenetic, and in vivo embryos were characterized by 2-D gel electrophoresis and MALDI-TOF mass spectrophotometric analysis. New stable endoderm and trophoblast cell lines from in vivo-derived porcine embryos were established. The endoderm cell lines have been characterized thoroughly.

    The Virginia station learned that the post-thaw morphological evaluation of sperm samples revealed a decrease in the percentages of normal spermatozoa in the 3 wk-PI samples in comparison with the Pre-insult samples for Bulls I and Bull III. The percentage of vacuolated spermatozoa increased significantly for Bull II. There was no apparent change in abnormal sperm populations for Bull IV. The cleavage and blastocyst formation rates and embryo development scores were affected by the interaction of bull by sample collection time. For Bulls I and III (severe responders) the scrotal insulation effects persisted from the time of cleavage through blastocyst formation. In the second study zygotes were cultured and subpopulations were removed from culture at Day 4 and 8 and subjected to either the TUNEL or caspase assay. The apoptotic index and caspase intensity were recorded and no differences in the apoptotic index were found in embryos generated from the semen samples for Bull I. The apoptotic index for Bull III for the 3 wk-PI embryos (34.9%) was significantly lower than the activity for the Control (43.2%) and 2 wk-PI embryos (41.4 %). On Day 8 caspase intensity increased significantly for both Bull I (217 ± 147) and Bull III (229 ± 98) for the 3 wk-PI embryo groups compared to the equivalent embryo groups for Bull II (98 ± 115) and Bull IV (90 ± 111).

    The Wisconsin station showed that in vitro produced cattle embryos transferred to surrogate dams can be recovered by usual flushing techniques up to day 14. Recovered embryos can be examined or otherwise evaluated and then retransferred for term gestation to new surrogate dams. Further we have shown that embryos resulting from flow cytometry sorted semen were compromised in developmental potential.

    Impact Statements:
    1. Production of offspring of pre-determined sex would be of tremendous economic benefit to livestock producers. Current studies using sexed semen demonstrated proof of concept. Widespread application of this technology could improve the efficiency of in vitro production of preimplantation embryos of a pre-determined gender and potentially improve embryo survival after transfer to recipients.
    2. In vitro production of preimplantation embryos with normal lipid content should improve cryopreservation success and should result in more normal offspring, making these procedures feasible for routine on-farm applications. Vitrification systems for bovine embryos contaiiing no products of animal origin should facilitate use of this simple method of cryopreservation for many purposes, including exporting embryos.
    3. Somatic-cell nuclear transfer procedures frequently produce conceptuses with compromised developmental capacity during and after gestation, creating public, scientific, and producer concern over practical use of the tool in animal agriculture. Documentation of the timing of alterations in placentome development will enable scientists to elucidate the underlying mechanisms that influence abnormal development of nuclear transfer offspring.
    4. Studying the expression profiles of nuclear transfer embryos is the first step in identifying the cause of the low efficiency of the cloning technology and will provide insights into how the process can be improved through regulation of embryonic gene expression. Regulating the epigenetic status of donor cells will significantly improve nuclear transfer efficiency and improve the health of animals produced via nuclear transfer.
    5. Products dervied from animals produced via somatic cell nuclear transfer were characterized. The analyses of these food products are of vital importance for regulatory agencies to decide on the safety of products obtained from animals produced via nuclear transfer for human consumption.
    6. Identification of regulatory mechanisms controlling trophectoderm 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. nuclear transfer and transgenics).
    7. Implementation of SAR-SAGE reduces the number of embryos and the methodology provides a more efficient mechanism for the genomic evaluation of embryos very early in development
    8. The differences in developmental efficiency between nuclear transfer embryos derived from cow and heifer cytoplasts demonstrate that subtle differences in oocyte biology can have significant effects on subsequent development of nuclear transfer embryos. Therefore, cow oocytes have a developmental advantage over heifer oocytes in nuclear transfer.
    9. Previously, embryos transferred on day 7 could not be experimentally manipulated or, since they reside deep in the uterus, be evaluated except by indirect means such as ultrasound. This technique allows another week of evaluation before any particular embryo is allotted to valuable gestational space.
    10. Being able to produce pregnancies from epididymal sperm from testes would allow the livestock producer to save germplasm from injured or deceased breeding males. This information would be of key importance to cattle seedstock producers.
    Last Modified: unknown

    Date of Annual Report: 07/05/2007

    Report Information:
  • Annual Meeting Dates: 01/06/07 to 01/07/07
  • Period the Report Covers: 10/2005 to 09/2006

    • Participants:
    Brief Summary of Minutes of Annual Meeting:
    The meeting was called to order at 5:00 p.m. by Secretary Curt Youngs (Chairperson Cindy Tian was attending the IETS pre-conference symposium and did not join the meeting until after it had started). Members were welcomed, and Curt expressed regrets to the group from Tom Bunch (Utah) and C.-Y. Hu (Hawaii) who were unable to attend this years annual meeting. George Seidel moved and Matt Wheeler seconded a motion to approve the minutes of last years annual meeting. The motion carried unanimously.

    Curt Youngs asked one of our new members, Jianbo Yao, to introduce himself to the group. Jianbo earned his PhD at McGill University in Canada and did post-doctoral training in human genetic diseases in Montreal. While at Michigan State University he worked with Paul Coussens and George Smith to develop a cDNA library from bovine oocytes. At West Virginia University, he collaborates with Paul Lewis and Keith Inskeep.

    Bob Godke moved and Matt Wheeler seconded a motion to elect Brett White as secretary. Motion carried unanimously.

    Neither C.-Y. Hu (administrative advisor) nor Mark Mirando (CSREES) were present at the meeting, so the group did not receive comments from the administrative advisor or CSREES representative.

    The date and location of next years annual meeting was discussed. The W-1171 group had previously decided to hold their annual meeting in conjunction with the annual conference of the International Embryo Transfer Society (IETS). The 2008 IETS conference will be held January 6-8, 2008 in Denver, Colorado, USA. Members raised concerns about the usual timing of the W-1171 meeting. Typically the W-1171 meeting is held at the same time as the IETS pre-conference symposium, and this has been a recurring conflict for the W-1171 members who are speakers and/or attendees at the IETS pre-conference symposia. After lengthy discussion, a decision was made to explore the possibility of having the W-1171 meeting on the Monday evening and Tuesday evening of the annual IETS conference.

    The idea of sponsoring a symposium or mini-symposium at the annual IETS conference was discussed. Potential topics that were suggested for the symposium were embryo development & manipulation, the first cell cycle, hyperactivated sperm motility, maternal/embryonic transition, and cortical granules.

    Curt Youngs led a discussion regarding the upcoming midterm review and evaluation of the W-1171 multistate project by RCIC. There are four main areas that will be reviewed: progress report, linkages, funding, and information & technology transfer. The administrative advisor (C.-Y. Hu) is responsible for providing a brief evaluation of the activities and success of the W-1171 multistate project to RCIC using a special form (Appendix I of the MRP manual).

    The initial topic of discussion pertained to section 2 of this review (linkages). The following linkages were identified: Colorado & Illinois, Colorado & Connecticut, Colorado & North Carolina, Illinois & Wisconsin, Illinois & Nebraska, Illinois & Connecticut, California & Connecticut, Louisiana & Wisconsin, Louisiana & Iowa, Iowa & Beltsville, Nebraska & Indiana, North Carolina & Beltsville, North Carolina & Clay Center, Indiana & Illinois, Connecticut & Beltsville, Indiana & Beltsville. These linkages have involved collaborative research, graduate student training, publication of joint manuscripts, and submissions of grants.

    Rather than taking limited time to discuss each and every section of the upcoming midterm evaluation and review, it was decided that is would be more time efficient if four subcommittees were formed. Subcommittee one is headed by Bob Godke (assisted by Curt Youngs) and will focus on the progress report. Subcommittee two is headed by Carol Keefer (assisted by Matt Wheeler) and will focus on linkages. Subcommittee three is headed by Cindy Tian (assisted by Jerry Yang) and will focus on funding. Subcommittee four is headed by Rebecca Krisher (assisted by Curt Youngs) and will focus on information and technology transfer. Curt Youngs will send out an e-mail request to the W-1171 membership no later than January 31, 2007 requesting that each station prepare concise information to provide to each subcommittee chairperson. Responses to the subcommittee chairpersons will be due February 15, 2007. Subcommittee chairpersons will have one month to compile and prepare their sections of the midterm evaluation and review, and these need to be sent to Curt Youngs no later than March 15, 2007. Curt Youngs will compile the four sections into a single, cohesive report and send the report to Gary Anderson and George Seidel for editorial review. Because C.-Y. Hu needs to have the report submitted no later than May 15, 2007, George and Gary should submit the revised report to C.-Y. no later than April 20, 2007. George requested that Rebecca, Cindy, and Curt e-mail to him the compiled W-1171 station reports for the past three years.

    Discussion was held regarding the establishment of a W-1171 web site. This web site could serve as a public source of scientifically-based information about embryo and reproductive technologies such as transgenic animals, nuclear transfer, sperm sexing, etc. A suggestion was made that one portion of the web site should be password protected so that only members of W-1171 could have access. This web site could be a site for chat rooms, exchange of experimental methods, reaction to research proposals, etc. Richard Fayrer-Hosken indicated that he could take the lead of developing such a web site if the group would give him some guidance and direction on what they wanted.

    Station reports were given, although members were asked to be very brief and to highlight only extremely novel findings and/or discuss ideas for new collaborative research projects. The members were asked to read the complied station reports on their own time after adjournment of the meeting.

    The W-1171 members broke out into the three main joint research groups (oocytes, stem cells, and embryo production). Before discussions began, members were reminded that the success of the multistate project hinges on productive collaborations and that the viability of W-1171 is contingent upon joint research efforts (and not on individual station research efforts). The groups were asked to share research findings, evaluate new possibilities for collaborative research efforts based on those findings, and to chart a plan of action for the coming year.

    Bob Godke moved and Brett White seconded a motion to adjourn the meeting. The motion carried unanimously, and the meeting was adjourned at 10:20 p.m.

    Respectfully submitted,

    Curt Youngs 2006 W-1171 Acting President and Secretary

    Accomplishments:
    Work during the preceding year suggests aromatase inhibition during the postnatal interval may have statistically significant effects on accessory sex gland development but suggests such effects will not be biologically significant given the previously observed positive effects on Sertoli cell proliferation. Our data suggest specific oocyte plasma membrane proteins may be affected by heat-stress during oocyte maturation and used as biomarkers of heat-stress induced reduction in oocyte quality during development. Our data provide further evidence for involvement of two specific oocyte proteins in the fertilization process.

    Although there were no statistical differences in the sex ratio of single- or super-ovulating beef cattle, there is compelling evidence to continue this line of research. There may indeed be a bull effect that can be exploited in future experiments to enhance the efficacy of this sorting procedure, and there may also be other mechanisms (such as trypsin treatment) that may aid the binding of the HeiferPlus® product to the sperm head. There may also be other sorting methods (electromagnetic) that are favorable to flow cytometery to sway the sex ratio without the associated low pregnancy rates and high variability and costs.

    Understanding the interplay among hormones and the establishment of one or more dominant follicles will provide enabling knowledge about the fertilizability of oocytes from specific follicles within a follicular wave. Further, investigation of the follicular hierarchy is the focus of at least two submitted USDA-CSREES Standard Grant Proposals in which Clemson University will serve as a collaborator. The ability to supply a follicular wave with a pure, reliable, and consistent source of FSH will decrease the considerable variation associated with embryo transfer and will eliminate potential prion disease transmission. We showed that reducing oxygen concentrations to 5%, which approximate physiological levels in the follicle and oviduct, improved the quality of resulting mouse embryos. Similar studies with maturing bovine oocytes in our laboratory show no detrimental effect of lowering oxygen during oocyte maturation.

    We showed that culturing embryos in the presence of phenazine ethosulfate (PES) had no detrimental effects on pregnancy rates in cattle. Embryos cultured with PES survive cryopreservation better than controls.

    We achieved high calving rate of sexed IVP bovine embryos and improved the bovine IVP system by improving fertilization and vitrification conditions.

    To study oocyte developmental competence, we have determined that we can reliably isolate adequate amounts of mRNA from 150 oocytes to hybridize to a microarray. For validation of microarray results, we can test up to 9 genes by real time PCR from a pool of 25 oocytes.

    We have shown that large-scale transfer of IVP Holstein heifer embryos, produced with gender-selected semen, to beef recipients is a feasible production scheme. Producers can use IVP to produce high genetic merit claves from lower genetic merit recipients.

    Oocytes harvested from Brahman and Angus breed donor cows were individually evaluated for lipid content. Using buoyant density gradients and Nile Red staining, the Brahman oocytes were found to have significantly more intracellular lipids than the oocytes from the Angus cows.

    Sexed semen was used to successfully to produce in vitro fertilized cattle embryos. These gender-selected Holstein embryos were transferred nonsurgically to mature crossbred beef cows. All these gender-selected embryos produce female calves. Mature crossbred beef cow recipient females had no difficulty giving birth with the typically large Holstein heifer calves.

    Our lab has identified two novel Lef1 isoforms, Lef12,3,6 and Lef16 that are expressed in bovine preimplantation embryos. These transcription factors may play a critical role in trophectoderm and placental differentiation.

    Fusion constructs consisting of the bovine NANOG promoter and GFP gene and the bovine Nanog protein and GFP have been produced and demonstrated to be expressed following transfection. These constructs will aid in functional studies concerning the role of Nanog in maintenance of pluripotency in early ruminant embryos.

    We have determined transcriptomes, functional blue prints, of bovine oocytes and early embryos.

    We have identified many proteins in the bovine GV stage oocytes and its surrounding cumulus cells.

    With respect to oocyte maturation, we: " Determined gene transcription is not required for EGF-induced oocytes maturation. " Determined that FSH-induced maturation requiring gene transcription over-rides EGF pathway when both hormones are present in maturation medium. " Identified potential candidate mRNAs associated with FSH-induced initiation of GVBD in murine and bovine cumulus-oocyte complexes matured in vitro. " Used short interfering RNA approaches to assess the functional role of NR4a1 and other mRNAs of interest during FSH-induced oocyte maturation in cattle.

    With respect to embryo and fetal development, we: " Determined that expression of IGF1 and IGF2R mRNAs was significantly decreased in the livers of fetuses resulting from in vitro-produced embryos at day 70 of gestation. " Determined that expression of IGF2R was significantly decreased in skeletal muscle of bovine fetuses from in vitro-produced embryos at day 70 of gestation. " Developed a new classification system for reporting of normal and abnormal outcomes following the transfer of IVF or SCNT embryos. The wide-range of potential developmental abnormalities of fetuses, placentas and calves are included in the new term, Abnormal Offspring Syndrome (AOS). " Confirmed conservation of genomic imprinting in swine placenta for over 15 known imprinted genes.

    We gained new information regarding the developmental biology of the early bovine embryo and factors affecting the establishment of extraembryonic endoderm. Identifying critical factors involved in this process may provide insights into aberrant mechanisms that predispose the embryo to pregnancy loss due to abnormal development and/or implantation failure.

    Genomic analysis of in vivo and in vitro porcine blastocysts: We constructed and characterized Serial Analysis of Gene Expression (SAGE) libraries representing comprehensive gene expression profiles of in vivo- and in vitro-derived day 6 pig blastocysts.

    Poultry sperm studies: a. Established methodology to differentiate distinct testicular cell populations and progenitor spermatogonia within the turkey testis. b. Developed transplantation protocol for the successful transfer of donor germ cells and repopulation of germ-cell-free recipient seminiferous tubules. c. Characterization of the turkey and chicken sperm glycocalyx composition. d. Identification of glycocalyx modulations occuring during in vitro storage of poultry semen. e. Confirmation of the ability of turkey sperm to acquire exogenous phospholipids during in vitro storage.

    Integrins facilitate attachment of cells to the extra-cellular matrix, often binding the arginine-glycine-aspartic acid tri-peptide motif, thus facilitating cell migration, mediating cell-cell adhesion, linking the extracellular matrix with cytoskeletal elements, and acting as signaling molecules. Adhesion activates signaling mechanisms that regulate integrin function, cytoskeletal assembly, cell behavior, and protein synthesis. Immunofluorescence was used to determine the presence of integrin a and b subunits on the surface of bovine oocytes using a panel of monoclonal antibodies specific for aL, aM, aX, aV, a2, a4, a6, b1, b2, and b3 antigens, with multiple antibodies for each subunit. Confocal microscopy indicated the presence of aV, a6, a4, a2, ß1, and ß3 integrin subunits on the plasma membrane of bovine oocytes. The presence of these subunits was verified by RT-PCR analysis using primers designed based on known gene sequences of bovine integrin subunits, or by using sequence information using bovine expressed sequence tags compared with known human and murine integrin subunit gene sequence information. Previously unpublished sequence information for bovine a6 and b3 integrins was determined. The presence of these integrin subunits on the bovine oocyte vitelline membrane supports the hypothesis that sperm-oocyte interactions in the bovine are mediated by integrins.

    The ability of synthetic RGD-containing peptides to induce intracellular calcium transients similar to those observed at fertilization by spermatozoa in the bovine has been reported (Campbell et al., 2000; Sessions et al., 2006). These results also indicated the ability of synthetic RGD-containing peptides to induce activation and subsequent parthenogenetic development to the blastocyst stage, although, at numbers lower than observed with control in vitro fertilization (IVF). Evidence has been provided indicating the important effect of surrounding regions on the biological activity of the RGD sequence (Zhu and Evans, 2002; Sessions et al., 2006). The current experiments were designed to use natural RGD-containing sequences (disintegrins) to understand their effects. A total of three RGD-containing snake venom peptides (Kistrin (K), Elegantin (Ele), and Echistatin (Ech)) and one non-RGD-containing venom (Erabutoxin B (EB; control) were used at three concentrations (0.1, 1 and 10 mg /ml) to induce parthenogenetic development to the blastocyst stage and in conjunction (1.0, 5.0 and 10 µg/ml) with spermatozoa to evaluate competitive inhibition of fertilization and subsequent development. A (P< 0.01) higher number of bovine oocytes developed to the blastocyst stage after incubation with K, Ele and Ech at 1.0 µg/ml and was not different (P> 0.01) from IVF control. Fertilization was significantly reduced (P<.01) at all concentrations of K, Ele and Ech as compared to IVF control. No reduction (P>.05) was observed in EB (non-RGD) treated oocytes. These results support the involvement of a disintegrin-integrin interaction at fertilization in the bovine resulting in activation and subsequent development.

    Integrins have been shown to be involved in the process of fertilization and many integrin-ligand interactions are mediated through the recognition of an arginine-glycine-aspartic acid (RGD) sequence. Despite the fact the RGD domain is a principal player in determining the functional characteristics of an adhesive protein, increasing evidence has accumulated implicating the amino acids flanking the RGD sequence in determining the functional properties of the RGD-containing protein. A set of linear peptides in which the amino acid sequence in and around the RGD tri-peptide was modified was synthesized to better understand the specificity of the RGD-receptor interaction. Mature oocytes were fertilized in vitro in the presence of RGD-containing and RGD-modified peptides. Both the RGD-containing and RGD-modified peptides impaired the ability of sperm to fertilize bovine oocytes, illustrated by a reduction in cleavage. The linear modified RGD containing peptides were also examined for their ability to induce parthenogenetic development with the objective of providing a linear RGD peptide with greater biological activity than the one (GRGDSPK) used previously (Campbell et al. 2000). The data demonstrate the specificity of the receptor for the RGD sequence, further implicate the involvement of integrins in the process of bovine fertilization, and illustrate the importance of the amino acids surrounding the RGD sequence in determining the binding and functional properties of RGD-containing peptides. The data support the findings that a linear RGD peptide can block fertilization and that amino acids around the RGD sequence have an impact on the biological activity of the receptor.

    The effects of nicotine on nuclear maturation and meiotic spindle dynamics of bovine oocytes and subsequent embryonic development were investigated. Maturation rates (85%-94%) were similar to the control (86%) at concentrations from 0.01 to 1.0 mM, but decreased significantly (P<0.01 or 0.001) at 2.0 to 6.0 mM. Haploid complements of metaphase II oocytes in 0.01 to 1.0 mM nicotine (approximately 90%) were similar to the control, while significantly lower (ranged from 63% to 76%, P<0.05 or P<0.01) haploid oocytes were observed in the 2.0 to 6.0 mM nicotine groups. The majority of diploid (2n=60) oocytes without a PB1 occurred in the 3.0 to 6.0 mM nicotine treatments. Spindle microtubules changed from characteristically being asymmetrical in the controls to being equally distributed into two separate chromosome groups in the nicotine treatments. Nicotine inhibited the movement of anaphase or telophase chromosomes to the cortical area. The inhibited two chromosome groups became two spindles that either moved close in proximity or merged entirely together resulting in diploidy within the affected oocyte. Nicotine treatment significantly reduced the rate of cleavage and blastocyst development after parthenogenetic activation. Diploidy and cell number were dramatically reduced in the resultant blastocysts. We conclude that nicotine can alter the normal process of bovine oocyte meiosis by causing a diploid state in the oocyte that subsequently interferes with embryonic development.

    Immunofluorescent staining and cytogenetic preparation techniques were performed to examine bovine oocyte maturation, dynamic nuclear changes, and meiotic spindle patterns after oocytes were exposed to nicotine at various concentrations and at different times. Nicotine at concentrations of 0.01 and 0.1 mM resulted in higher (P<0.05) maturation rates (92.9% and 93.6%, respectively) than the controls (84.1%); concentrations of 0.5 to 2.0 mM had maturation rates similar to the control; concentrations of 3.0 to 6.0 mM significantly decreased (P<0.01 or 0.001) maturation rates. Haploid compositions of metaphase II oocytes in 0.01 to 1.0 mM nicotine (approximately 90%) were similar to the control, while significantly lower (ranged from 63% to 75%, P<0.05 or P<0.01) haploid oocytes were obtained in the 3.0 to 6.0 mM nicotine groups. Extremely swollen and twisted spindles formed after oocytes were exposed to concentrations e 2.0 mM. Spindle microtubules were equally distributed between the separated two groups of chromosomes in nicotine groups, while in the control, microtubules were asymmetrically distributed to the separated two groups of chromosomes, and the majority of the microtubules were oriented and distributed to the first polar bodies. Nicotine exposure at 3.0, 4.0, 5.0, and 6.0 mM for 24 hr resulted in 23.1, 32.5, 41.2, and 43.1% diploid oocytes, respectively. Diploidy resulted from the inhibition of anaphase or telophase chromatid movement. The inhibited two groups of chromatids became two spindles that either moved close in proximity or merged entirely together. Nicotine exposure affected oocyte maturation in a dose-dependent manner resulting in disfiguration of meiotic spindles and causing diploidy.

    Examination of histological sections gave evidence of the formation of eosinophilic cells, blood islets, and contractile elements with no evidence of a forming neural system. Blood islets and eosinophilic cells were associated with the contractile elements forming an apparent vascular tissue matrix. Contractile elements were positive for the formation presence of calcium activated ATP-ase activity, and were unresponsive to acid reversal of calcium activated myo-fibriliary ATP-ase activity at pH 4.3 and pH 4.5, indicating a similarity to cardiac muscle fibers.

    We have demonstrated level of nuclear reprogramming of gene expression using DNA microarrays in bovine SCNT derived blastocysts.

    The expression patterns of key transcription factors, Nanog, Oct4 and Sox2 in ICM explants were determined. This knowledge will aid in the derivation and characterization of ruminant embryonic stem cell lines.

    Our study showed that culturing bovine fibroblast cells in vitro enhanced various nuclear epigenetic modifications that are likely detrimental to nuclear reprogramming during the cloning procedure in animals.

    In a recent study we were able to isolate and culture stem cell like cells from porcine subcutaneous fat cells (adipocytes).

    The enhanced lactation potential of the transgenic gilts synergizes with suckling intensity to stimulate increased milk production during early lactation, as well as stimulating piglet growth. These animals provide a method to manipulate lactation persistency in swine.

    Cultured bone marrow derived porcine mesenchymal stem cells have been differentiated into osteoblastic cells when cultured on chitosan/biphasic calcium phosphate porous scaffolds for up to four weeks. This allows development of engineered bone replacement tissue for humans and animals.

    We improved NT efficiency by using PHA; studied the reprogrammability of cells at different differentiation stages; studied gene reprogramming in porcine NT by using X-linked genes as markers.

    We observed that loading extra cholesterol into membranes of oocytes improved their survival after vitrification. Similar studies by others with sperm of several species show that adding cholesterol improves survival after cryopreservation.

    An efficacious method of vitrification of bovine and equine pre-compaction embryos was developed. This likely would also work for sheep and goat embryos.

    We developed a SCNT method in swine that can generate both transgenic and non-transgenic clones at high efficiencies.

    We have increased usefulness of Affymetrix porcine arrays.

    We have determined which microarray platform is best suited for swine studies.

    Embryo-derived cell line models: We established stable and long-lived porcine endoderm cell lines from in vivo embryos and characterized cell lines transcriptomic and proteomic of candidate genes analyses as well as by morphology and enzymatic activity.

    Porcine embryo stem cells: To better understand the potential importance of several well-known human and/or stem cell and differentiation associated factors in the potential maintenance or spontaneous differentiation associated with in vitro culture, transcript expression analysis of those factors was examined in undifferentiated porcine epiblasts and the effect of short-term of culture was determined. Furthermore, we demonstrated that the porcine epiblast expression profile more closely resembles that of the human embryonic stem cell.

    Cloning is one of several new assisted reproductive techniques being developed for clinical use in the equine industry. Potential uses of equine cloning include: (1) the preservation of genetics from individual animals that would otherwise not be able to reproduce, such as geldings; (2) the preservation of genetic material of endangered and/or exotic species, such as the Mongolian wild horse (Przewalskis horse); and (3) because of the companion animal role that horses fill for some individuals, it is likely that some horse owners will have individual animals cloned for emotional fulfillment.Although equine cloning has been successful, like other species, it remains a very inefficient process (<3% success). In most species, the inefficiency of cloning results from a high incidence of embryonic, fetal and/or placental developmental abnormalities that contribute to extremely high rates of embryonic loss, abortion and stillbirths throughout gestation and compromised neonatal health after birth. The present review describes some of the ultrasonographic, endocrinological and histopathological characteristics of successful (produced viable offspring) and unsuccessful (resulted in pregnancy failure) cloned equine (mule and horse) pregnancies we have produced.A total of 21 cloned mule pregnancies were established using fetal fibroblast cells, whereas a total of seven cloned horse pregnancies were established using adult cumulus cells. Three of the cloned mule conceptuses were carried to term, resulting in the birth of three healthy clones.

    We have successfully developed a course in embryo transfer and related technologies that utilizes research advancements made by members of the W-1171 multistate research project.

    Impact Statements:
    1. Reducing oxygen tension during in vitro oocyte maturation will benefit all sorts of assisted reproductive biotechnologies, such as cloning, ICSI, and IVF by producing healthier embryos.
    2. In vitro embryo production systems using sex-selected semen will allow the genetic improvement livestock as well as more efficient production of heifers for milk production and steers for beef production.
    3. Being able to select the sex of the future calf at the time of breeding the cow will allow cattle producers to improve animal production efficiency. We have demonstrated that this technology is capable of producing gender-selected calves under field conditions.
    4. This first comprehensive proteome analysis of bovine oocytes and cumulus cells not only provides a foundation for signaling and cell physiology at the germinal vesicle stage of oocyte development, but are also are valuable for comparative studies of other stages of oocyte development at the molecular level.
    5. Using the new classification system of development of placentae, fetuses, and calves following the transfer of IVF or somatic cell nuclear transfer embryos will result in more precise reporting of these developmental outcomes.
    6. Genomic analysis of in vivo and in vitro porcine blastocysts has enabled the identification of several biological processes, most involving mitochondria, that are compromised with in vitro embryo culture.
    7. The identification of specific integrins and their role in sperm fusion will contribute to increased fertilization rates during in vitro embryo production.
    8. The pluripotency-related transcription factor NANOG can be used as a marker of pluripotency and will aid greatly in the derivation of embryonic stem cells in cattle, goats and other ruminant livestock.
    9. The production of alpha-lactalbumin and insulin-like growth factor transgenic swine allows for improvement of lactation in swine and has profound effects on increasing the efficiency of milk and meat production.
    10. Several students who have taken the embryo transfer course are now employed in the commercial embryo transfer industry, as well as in human infertility clinics.
    Last Modified: 06-Jul-2007

    Date of Annual Report: 05/01/2008

    Report Information:
  • Annual Meeting Dates: 01/05/08 to 01/06/08
  • Period the Report Covers: 10/2006 to 09/2007

    • Participants:
    Brief Summary of Minutes of Annual Meeting:
    The meeting held in Mineral Hall B was called to order at 5:00 p.m. by Chair Curt Youngs. Bob Godke moved and Matt Wheeler seconded a motion to approve the minutes of last years annual meeting. The motion carried unanimously.

    Mark Mirando, CSREES Representative, presented a short report. Mark discussed the USDA budget for the upcoming year. He indicated that there was an additional $20 million in Hatch funds for 2007 compared to 2006. This was due to the fact that all special grant funds were rolled into Hatch funds last year. In 2008, however, these special grant funds were removed from the budget. He also indicated that there was a $1.5 million increase in Smith Lever funds and funding for integrated projects within NRI increased from 22 to 26% this past year. Mark indicated that the USDA would be moving to electronic submission of research proposals for all programs and that fillable forms in portable document format (PDF) would potentially be available next year. In addition, he promoted two upcoming Grant Writing Workshops to be held in Salt Lake City, UT and Washington, D.C. Finally, Dr. Mirando indicated that more funds were budgeted for the NRI. Practically, this means that the Animal Reproduction program could fund one additional research proposal. A good discussion with the committee followed.

    The W-1171 Administrative Assistant, C. Y. Hu, provided his report to the committee. First, he apologized for being unable to attend the 2007 annual meeting in Kyoto, Japan. Next, he indicated that the Western states were really hurt by the reduction in special grants last year, estimating that Hawaii lost approximately $7 million. Funds went to CSREES but not necessarily to all the experiment stations, therefore only certain stations benefitted from these additional funds. In addition, C. Y. mentioned that Congress always passes appropriations in March after the end of the fiscal year on October 1. However, last year they just approved a continuing appropriation. He also suggested that it has been a tough 1½ to 2 years for experiment station directors. The Farm Bill has required lots of haggling by the directors, utilizing much of their time. Additionally, a great deal of debate ensued over agricultural subsidies. Currently, NIFA is in both the House and Senate versions of the Farm Bill. Further, he indicated that they lost IFAFS funding as well.

    C. Y. indicated that the budget picture for the upcoming year looks unfavorable. There has been $195 million allocated to the NRI program. Another $195 million is budgeted for Hatch funds. Of these Hatch funds, 25% has to be used for experiment stations in each state to support formula funds. As an additional note, C. Y. mentioned that in the future, many areas of USDA would be combined in an attempt to streamline and incorporate a hierarchy similar to NIH.

    In regard to the W-1171 committee, C. Y. pointed out that the committee had its mid-term review this past year. He reported that the review team was very happy with the performance of the committee. In addition, he indicated that a revised project for the W-1171 committee would be due by the 2009 annual meeting. He expects a reasonably good draft by the next annual meeting. He suggested that our accomplishments were relatively easy to document. However, he stressed the importance of documenting impacts of our research. It is important to focus on integrated, multistate linkages. Again, collaborations among institutions and impacts could not be stressed enough. Both C. Y. and Mark Mirando agreed that 3 to 5 good impact statements would be sufficient for the renewal proposal and that each impact statement should be no more than 2 sentences. C. Y. warned that the majority of impact statements simply reiterate the objectives or methods, suggesting that the committee avoid this pitfall. He also indicated that the impact statement should not include the impact will be&

    Suggested examples of terminology to include in impact statements were: 1) Increased conception rates, reproductive efficiency or birth rates. 2) Decreased production costs. 3) Improved animal welfare. 4) Increased global competitiveness.

    Both C. Y. and Mark Mirando indicated that impact statements should be general in nature. It is important to identify critical gaps in the knowledge base and suggest how these gaps will be filled.

    Brett White will become Chair of the committee for the 2008 calendar year. Because there were no nominations for secretary, both Peter and Char Farin volunteered to serve as Co-Secretaries for the 2008 calendar year. Peter and Char will assume duties of Committee Co-Chairs for the 2009 calendar year.

    The 2009 W-1171 annual meeting will be held in conjunction with the IETS meetings in San Diego, California (January 4-7). It was suggested that the committee try to meet around the end of the meeting but prior to the post-conference symposia. Brett White will contact Jennifer Gavel to arrange a room and time.

    To avoid time constraints, individual station reports were excluded in order to allocate additional time to discuss the objectives for the upcoming rewrite. At 5:55 pm, the committee began to discuss the objectives for the renewal proposal. Discussion of Objective 1 from our last proposal revolved around the idea of incorporating physiological genomics into a similar objective. It was noted that at least six members of the committee were performing this type of research. However, the committee agreed that this terminology might be too specific for an objective encompassing the research of so many scientists and thus chose to keep the language more general. Bob Godke moved and Char Farin seconded a motion that the first objective stand as is, Understand the biology and underlying mechanisms of gamete development, fertilization, and embryogenesis.

    Discussion of the second objective from our previous proposal ensued. Chairman Youngs suggested including genetically enhanced animals instead of genetically modified animals in this objective. Bob Godke moved to entitle the second objective, Refine methods for production of genetically enhanced animals to improve livestock production efficiency. The motion was seconded by Peter Farin and the committee passed the motion unanimously.

    Next, the committee focused on the rewrite. A recommendation was brought forward to divide the writing into specific sections. Erdogan Memili, Rebecca Krisher and Brett White volunteered to serve as overall coordinators for the sections of the rewrite. Two important components of the proposal identified were the critical reviews of the literature and the project methods. The committee agreed that members assisting with the writing concentrate on the impacts and linkages among research stations. The question was raised whether the committee needed to include year by year milestones. Our Administrative Assistant, C. Y. Hu, indicated that this format was not required. Finally, Chair Youngs asked that all committee members be responsible and timely in responding to information sent out via email regarding the renewal proposal.

    Charlie Rosenkrans moved and Rebecca Krisher seconded a motion to adjourn the business meeting. The motion carried unanimously, and the business meeting was adjourned at 6:30 p.m.

    The committee met at 8:30 a.m. on January 6, 2008 in the same room to discuss collaborative projects for the rewrite. Before discussions began, Chairman Youngs suggested that we have a good draft of the rewrite by September 1, 2008, taking advantage of time flexibility over the summer months. Secretary White would send out a reminder around May 1, 2008 encouraging everyone to complete their delegated parts of the proposal. Members were reminded that the success of the multistate project hinges on productive collaborations and that the viability of W-1171 is contingent upon joint research efforts (and not on individual station research efforts). The W-1171 members broke out into the two objectives that were agreed upon the previous day. The groups were asked to identify new possibilities for collaborative research efforts. Moderators of each group were to provide a summary of the ideas derived by each group to the Secretary, Brett White, so that he could compile the findings and send them out to the committee for further input. Following group discussions, the meeting was adjourned.

    Respectfully submitted,

    Brett White 2007 W-1171 Secretary

    Accomplishments:
    We examined gene expression in oocytes of good and poor quality, identifying alterations in the transcriptome of oocytes matured in vitro compared to those matured in vivo that correlate to developmental competence. These results indicated that gene expression is altered in oocytes matured in vitro compared to those in vivo. Based on the functional annotations of genes differentially expressed, dysregulation of gene expression resulting in altered DNA methylation and an up regulation in cell death pathways are potential developmental mechanisms influenced by in vitro culture conditions that correlate to reduced embryonic developmental potential. Of particular interest the functional category of gene expression. This category includes genes involved in the regulation of gene expression, including those genes controlling histone modifications and chromatin structure. Our microarray results demonstrate that genes in this category are specifically associated with differences in oocyte quality.

    Developmental potential of oocytes derived from sows and gilts was determined after in vitro maturation, fertilization and culture. This data confirms that gilt derived oocytes matured and cultured in vitro have a similar ability to undergo nuclear maturation compared to sow derived oocytes. Gilt oocytes, however, display a decreased ability to undergo embryonic developmental, suggesting that gilt oocytes have not fulfilled all the required mechanisms for cytoplasmic maturation and normal embryonic development.

    Member scientists used computer assisted sperm analysis (CASA) to evaluate the effects of techniques routinely used for selection of motile sperm prior to IVF on sperm quality parameters, and the effects of PHE on sperm parameters of semen varying in quality. Results suggest that the method used for selection of motile sperm can influence parameters related to motility and sperm head morphology. The beneficial effects of PHE on sperm over time, particularly those of poor quality, were identified.

    The effects of tobacco smoke compounds on sex ratio were investigated for potential application to sex selection in livestock. Results suggest that these tobacco compounds reduce the male to female sex ratio of bovine embryos, confirming previous reports in humans.

    Other researchers acquired data suggesting that oocyte receptors can be used as an estimate of oocyte quality and that the interval prior to fertilization is sensitive to heat stress in the pig.

    Investigators have developed and validated a simple, objective method of quantifying lipids in bovine embryos. They also found that injecting inositol triphosphate at the time of ICSI of bovine oocytes resulted in improved cleavage rates, as well as a non-significant increase in blastocyst production.

    Validated a method to study how bovine oocytes modify mRNA during maturation, mostly by selective degradation of specific mRNAs. The assay is sensitive enough such that one can inject labeled constructs and recover them in a modifed form some hours later in sufficient quantities to make meaningful measurements.

    Found that mRNA for the gene, periattachment factor, increased dramatically in elongating ovine embryos, from non-detectable on day 11 to a peak on day 16, and subsequent decline to very low levels by day 30. This timing is very similar to what is seen in bovine embryos. Since mRNA for this molecule rises sharply during elongation and falls to nearly undetectable concentrations when elongation ceases, it likely has a very important function that we are trying to understand. This molecule could be key to understanding the high incidence of embryonic death that normally occurs at this stage.

    Compared differences in gene expression of embryos fertilized and developed in vivo from in vivo matured oocytes to those that are fertilized in vitro but cultured in vitro from in vivo matured oocytes and those that were fertilized and cultured in vitro from in vitro matured oocytes.

    Developed miniaturization technologies toward microfluidics and small mechanical systems, microelectromechanical systems (MEMS), creating a means for dynamic culture on a volumetric scale, reportedly more consistent with the needs of the embryo.

    We have shown that either chemical (cycloheximide) or electrical methods are adequate and efficient for the activation of embryonic structures reconstructed by the hand-made cloning technique.

    Identified two novel Lef1 isoforms expressed in bovine preimplantation embryos. Studies using siRNA knockdown are ongoing to determine the role of this transcription factor in blastocyst formation.

    Determined in vitro culture conditions supporting better development of porcine oocytes in vitro.

    Provided functional genomics blue prints (proteomics) of bovine oocytes and spermatozoa.

    We have determined an important role for GnRH during embryogenesis, having a receptor mediated effect on 1-cell embryos that occurs within the first 36 hours of embryo development.

    Established biotin effects on oocyte maturation in mice, identified genes that are up- or down-regulated in oocytes from biotin-deficient compared to biotin-normal mice, and examined intracellular signaling cascades and molecular mechanisms underlying biotin effects on oogenesis and subsequent development.

    Found that production of bovine embryos in vitro using a serum based system was associated with increased body weight, normal liver and kidney morphology, and increased concentrations of blood urea nitrogen of fetuses during late gestation.

    Identified that bovine Air ncRNA is expressed in cattle and determined expression of bovine Air ncRNA in fetal tissue at the post-implantation and peri-implantation stages but not in preimplantations stages of development. Found that method of embryo production altered expression of bovine Air ncRNA in fetal tissue at day 70 of gestation. Determined the status of a range of imprinted genes in swine. Annotated and compared several platforms for porcine gene expression profiling studies.

    Identified potential candidate mRNAs associated with FSH-induced initiation of GVBD in murine and bovine cumulus-oocyte complexes matured in vitro. Used short interfering RNA approaches to assess the functional roles of NR4a1 and Egr1 mRNAs during FSH-induced oocyte maturation in cattle. Determined that a spermicidal compound can be encapsulated in cyclodextrin without loss of spermicidal activity. Found that ejaculated equine spermatozoa contain measurable amounts of various mRNAs for genes associated with testicular spermatogenesis. Determined that sperm protein (SP)-22 was specifically localized in equine spermatozoa and that SP-22 expression was altered with season of collection.

    Provided new information regarding the developmental biology of the early bovine embryo.

    Evaluated the efficacy of the commercially available semen sexing kit; HeiferPlus". Although more research is necessary (as is usual with binary data), perhaps a more compelling observation is the potential that incubation alone of bovine semen prior to AI may sway the sex ratio in favor of the female in superovulating (but not single ovulating) cows.

    The chemical composition of estrous synchronization implants used in this research must be manipulated to allow ease of insertion and removal, but the protein release profiles (in vitro and in vivo) are encouraging. Ultimately, the variety of the FSH loaded into the implant (recombinant or pituitary derived, glycosylated or not) may determine the type of material used to construct the implant. Further research is necessary to determine the precise FSH (and perhaps LH) release profile to reliable stimulate follicular waves to produce multiple dominant follicles, fertilizable oocytes, and developmentally competent embryos.

    We studied the role of disintegrin-integrin dynamics in sperm-oocyte interactions.

    Investigated differential gene expression of somatic cell nuclear transfer embryos and placental tissue as compared to controls.

    Examined the effects of nicotine on bovine meiotic maturation in vitro and subsequent embryonic development.

    Since the mRNA and protein of the novel importin ± are extremely abundant in oocytes and early cleavage stage embryos, but barely detectable in morula and blastocyst stage embryos, we believe that this novel importin ± is a maternal effect gene that is essential for early embryogenesis.

    Compared gene expression in cloned and control pigs and calves. Examined the methylation patterns of the intergenic region of H19-IGF2 in cloned calves.

    Found that enhanced lactation potential of transgenic gilts (bovine alpha-lactalbumin) synergizes with suckling intensity to stimulate increased milk production during early lactation, as well as stimulating piglet growth. These animals provide a method to manipulate lactation persistency in swine. Preliminary results indicate absence of the transgene in control animals after co-habitation and post-mating with transgenic animals. This work provides a critical first step toward providing rigorous scientific data for risk assessment of transgenic livestock.

    Determined that the use of multi-factorial directed differentiation using high-speed robotic systems will enable the examination of large matrices of culture and differentiation conditions for stem cells. Furthermore, this approach enables analysis of gene expression and other cell characteristics related to differentiation and cell function from cells cultured under essentially unlimited conditions. Using the automated microscale system in large factorial experiments allows analysis of the basic mechanisms underlying stem cell development in vitro, and ultimately in vivo.

    Determined that Noggin, a cytokine, helps maintain expression of the pluripotency transcription factor Nanog during ICM explant culture.

    Identified functional genomics (transcriptomics) of bovine cloned embryos from IVF and several rounds of chromatin transfers.

    Established divergent survival rates for cryopreserved embryos from Chinese Meishan and white crossbred lines of swine. This difference in survival rate occurs within the first 24 hours following thawing.

    Determined the most efficient method for SCNT cloning in pigs. Unlike cattle and sheep, SCNT in pigs does not generate large offspring but instead increases the incidence of IUGR.

    Tested different methods for enhancing homologous recombination in somatic cells.

    Identified single nucleotide polymorphisms in the promoter regions of the prolactin and HSP-70 genes of cattle that are related to cattle fecundity.

    Boar libido was not detectably reduced by lowering endogenous estrogens in an attempt to increase sperm production.

    Impact Statements:
    1. 1. Elucidation of specific molecular mechanisms critical to oocyte competence will allow development of effective strategies to alter precise pathways critical to developmental potential of oocytes.
    2. 2. Application of the results of these studies should lead to refinement and improvement of procedures for in vitro production of embryos, and possibly AI pregnancy rates.
    3. 3. Understanding gene expression differences among embryos produced by various assisted biotechnology techniques has the potential to improve culture media and embryo production efficiency through in vitro production systems.
    4. 4. Development of efficient IVP systems using microscale technologies will enable the automation of such techniques for production of large numbers of embryos in vitro, accelerating genetic improvement and selection of superior livestock.
    5. 5. Identification of specific mRNAs regulating the resumption of meiosis in mammalian oocytes may provide novel opportunities for the development of gene-targeted contraceptive methods useful for the manipulation of reproductive cycles in livestock.
    6. 6. Research in bovine embryo physiology may provide insights into aberrant mechanisms that predispose the embryo to pregnancy loss due to abnormal development and/or implantation failure.
    7. 7. Swaying the sex ratio of offspring will assist livestock producers in reaching their genetic, financial, and reproductive goals.
    8. 8. Identification of mRNAs and proteins in spermatozoa that are known to be associated with the progression spermatogenesis may provide potential markers for predicting fertility.
    9. 9. Isolation of cytokines that extend expression of the pluripotency-related transcription factor, NANOG, will aid in derivation of embryonic stem cells, providing an invaluable tool for improved production traits, disease resistance and production of biopharmaceuticals via genetic engineering.
    10. 10. Isolation and differentiation of mesenchymal lineage stem cells in vitro, as well as transplantation into live animals with corresponding proper differentiation, will allow stem cell therapy for production parameters such as lactation and muscle growth in livestock.
    Last Modified: 05-Jan-2009

    Date of Annual Report: 04/05/2009

    Report Information:
  • Annual Meeting Dates: 01/04/09 to 01/05/09
  • Period the Report Covers: 10/2007 to 09/2008

    • Participants:
    Brief Summary of Minutes of Annual Meeting:

    URL: Copy of minutes
    Accomplishments:
    Objective 1:

    A low sodium medium was found to be a viable alternative to synthetic caudal epididymal fluid medium for maintaining sperm viability during storage.

    Grazing bulls on endophyte infected fescue was shown to reduce sperm motility and morphology, especially during elevated ambient temperatures.

    Reduced oocyte fertilizability due to reduced sperm receptors, support the use of these receptors as biomarkers of oocyte quality, and support the hypothesis that a short term elevated ambient temperature during oocyte maturation reduces oocyte quality.

    We have been studying various ways of making in vitro-produced embryos more like in vivo embryos, particularly with respect to lipid content. Forskolin-treated embryos had much less lipid than controls. Caffeine and epinephrine were not effective at doses used.

    We attempted to apply IVP procedures in a practical way by ET to lactating dairy cows. Success rates were considerably lower than with AI using commercially available oocytes and semen. Sexed semen worked reasonably well with AI.

    A fairly efficacious method of freezing small equine embryos was developed that appeared superior to a standard vitrification procedure, which performed more poorly in this experiment than in previous experiments for unknown reasons.

    Investigated strategies to improve oocyte freezing survival.

    Utilizing a novel embryonic stem cell differentiation culture system, BMP and KIT ligand signaling were demonstrated to have a significant effect on the derivation of germ cells from ESCs. This provided further insight into important signaling events in germ cell development and highlights the potential use of this system to study developmental problems hindering livestock production.

    The development of miniaturization technologies toward microfluidics and small mechanical systems, microelectromechanical systems (MEMS), has created means for dynamic culture on a volumetric scale reportedly more consistent with the needs of the embryo.

    The presence of SPP1 mRNA in oocytes and cumulus cells and the larger mRNA abundance before maturation may suggest a role of this protein prior maturation of oocytes. Additional studies will be required to determine the specific role of SPP1 in oocyte maturation in the pig.

    Fusion using 2 pulses, 0.9 Kv/cm or 1.2 Kv/cm, for 30µs was more efficient for embryonic structure reconstruction in the hand-made cloning technique, when comparated to 0.6 Kv/cm for 30µs.

    Ammonium has deleterious effects on both nuclear and cytoplasmic oocyte maturation in vitro.

    Gene expression is altered in oocytes matured in vitro compared to those in vivo. Dysregulation of gene expression in the oocyte resulting in altered DNA methylation and an up regulation in cell death pathways are potential developmental mechanisms influenced by in vitro culture conditions that correlate to reduced embryonic developmental potential.

    Aberrant metabolism, incorrect mRNA post-transcriptional processing and increased apoptosis are some of the mechanisms contributing to reduced developmental potential in gilt oocytes.

    Leptin and glucose may interact to regulate oocyte nuclear maturation in obese and/or diabetic individuals.

    Ossabaw swine with MetS develop reproductive parameters reminiscent of PCOS, and thus may represent a complete animal model of the disease.

    Abnormal fetal and placental development and differential expression of imprinted genes in the ovary and brain of aged mice suggests that epigenetic regulation of oocyte and fetal development is impaired with advanced maternal age.

    Methods have been established which allow propagation of the TE model CT1-cells to be grown in a feeder-free system. This allows cleaner determination of gene expression without issues of feeder cell contamination.

    CT-1 cell line expresses TE lineage associated transcription factors: Cdx2, Ets2, Err², Id2, Sox15, Elf5, Hand1 and Gata6, along with the pluripotency-associated transcription factor, Oct4 (POU5F1).

    Provided functional genomics blue prints (transcripts) of bovine spermatozoa.

    Determined comparative functional genomics of chromatin remodeling proteins HMGN3A and SMARCAL1 during early mammalian embryogenesis.

    Established an in vitro follicle culture system to identify the effect of excess insulin and IGF exposure on oocyte gene expression.

    Chronic in vitro exposure of follicles to insulin changed the expression of NIMA-related kinase 2 (Nek2), Nek4 and TACC1. Similarly, insulin treatment of granulosal cell primary cultures induced alterations in Nek2, Nek4 and TCAA1 mRNA abundance.

    Determined the expression profile of Nek2, Nek4 and TCAA1 during the follicular and luteal phases of the estrous cycle in superovulated mice. Interestingly, Nek2 and Nek4 mRNA and protein expression seemed to be uncoupled with mRNA abundance peaking during the follicular phase and protein abundance peaking during the luteal phase.

    We have determined an important role for GnRH during embryogenesis, having a receptor-mediated effect on 1-cell embryos that occurs within the first 36 hours of embryo development.

    A specific antagonist of GnRH completely blocked embryonic development and acted via alteration of the cell cycle rather than apoptotic pathways.

    We have accomplished the following: (i) established biotin effects on oocyte maturation in mice; (ii) identified genes that are up- or down-regulated in oocytes from biotin-deficient compared to biotin-normal mice; and (iii) examined intracellular signaling cascades and molecular mechanisms underlying biotin effects on oogenesis and subsequent development.

    Identified that bovine Air ncRNA is expressed in cattle.

    Found expression of bovine Air ncRNA in fetal tissue at the post-implantation and peri-implantation stages but not in preimplantation stages of development.

    Found production of bovine embryos in vitro using a serum based system was associated with increased body weight, normal liver and kidney morphology, and increased concentrations of blood urea nitrogen of fetuses during late gestation. Examined the status of swine imprinted genes in a range of tissues.

    Updated an annotation that is freely available that increases the usefulness of the porcine Affymetrix microarrays.

    Identified potential candidate mRNAs associated with FSH-induced initiation of GVBD in murine and bovine cumulus-oocyte complexes matured in vitro. Used short interfering RNA approaches to assess the functional roles of Nr4A1 and Egr1 mRNAs during FSH-induced oocyte maturation in cattle. Identified conditions used for maturation of cumulus-oocyte complexes that influence the developmental competence of immature oocytes selected based on brilliant cresyl blue staining. Research in cell migration events occurring in the early embryo may provide valuable insights into mechanisms that predispose the embryo to pregnancy loss due to abnormal development and/or implantation failure.

    Increasing the proportion of female offspring (embryos or calves) by manipulating semen (via incubation, etc.) or the female (frequent ultrasound, or hormonally) will be beneficial to the beef or dairy producer without the added cost and potential negative effects (reduced pregnancy rates, bis-benzimidazole dye, etc.) of sexed semen protocols.

    Better understanding of the ovarian and hormonal dynamics in cattle that become pregnant (or not) following artificial insemination will provide a platform to manipulate Interferon tau profiles (via a trophoblastic vesicle) in order to enhance pregnancy rates following embryo transfer (especially with viability-compromised embryos).

    Recombinant FSH produced from non-mammalian expression systems will provide a safer, less expensive, and perhaps more effective method of producing embryos from hyperstimulated cattle. Currently the superovulation and embryo transfer industry is dependent upon FSH products purified from mammalian pituitaries and are of non-domestic origin.

    Oocytes microinjected with FAK siRNA had significantly lower cleavage rates compared to control oocytes.

    Oocytes pre-incubated with the function blocking antibodies for the ±V and ²1 subunits had significantly lower cleavage rates (p <.05) compared to all other treatment groups.

    We have identified and characterized a new member of the importin ± gene family and demonstrated its potential role in transporting oocyte-specific nuclear proteins and its requirement during early embryogenesis.

    Objective 2:

    The understanding of the mechanism of gene expression differences in cloned vs control animals has the potential to improve the survival of cloned animals and therefore increase cloning efficiency. Improving the efficiency of embryonic stem cell derivation has major implication in the generation of bovine embryonic stem cells which are still not available due to culture condition inadequacy.

    Studied genetic imprinting in naturally reproducted pigs and compared gene expression and DNA methylation in cloned and control pigs. Additionally we improved the efficiency of stem cell generation.

    The accomplishments of these studies are the development of a novel differentiation culture system that is capable of producing large numbers of germ-like cells that undergo advanced stages of development from embryonic stem cells. These cells represent an important germ cell source for the production of transgenic animals, leading to the improvement of livestock production.

    Preliminary results indicate absence of the transgene in control animals after co-habitation and post-mating with transgenic animals. This work provides a critical first step toward providing rigorous scientific data for risk assessment of transgenic livestock.

    Adipose-derived stem cells demonstrated a clear osteogenic differentiation and similar expression and pattern of the two osteogenic genes most abundant in MSCs (COL1A1 and SPARC). However, the higher abundance of SPP1 and BGLAP and the different behavior of SPP1 in MSCs suggest a different transcription profile between the two cell types.

    Using a 13K porcine DNA microarray, ADSCs and MSCs had a very similar transcriptome before differentiation. Adipogenic differentiation exhibited a larger number of genes affected by time compared to osteogenic differentiation. Microarray analysis uncovered several genes that were unique for each of the two types of differentiation. Interestingly, adipogenic differentiation was distinguished by genes involved in triacylglycerol transport and storage but not de novo synthesis.

    The use of multi-factorial directed differentiation using high-speed robotic systems will enable the examination of large matrices of culture and differentiation conditions for stem cells. Furthermore, this approach enables analysis of gene expression and other cell characteristics related to differentiation and cell function from cells cultured under essentially unlimited conditions. Using the automated microscale system in large factorial experiments allows analysis of the basic mechanisms underlying stem cell development in vitro, and ultimately in vivo.

    The two components of lactose synthase, ±-lactalbumin (±-LA) and ²1,4-galactosyltransferase (B4GALT), are each correlated to protein concentration and individually correlated to the concentration of other milk components and stage of lactation.

    Polymorphism in the CYP7 gene locus affected cerebrum weight and behavior and dietary cholesterol (C) tended to increase cerebrum C concentration in neonatal pigs. These findings in neonatal pigs have considerable potential importance in human infant nutrition and behavioral development.

    Our lab has shown that, while selective ablation of differentiating cells in ICM explant culture is not sufficient for short-term pluripotent cell survival, supplementation with Noggin prolongs proliferation of undifferentiated cells.

    The cytokine Noggin can upregulate Nanog expression sufficiently to permit further studies that will help identify more optimal conditions.

    Identified genes whose products might be playing a role in epigenetic control of molecular reprogramming of gene expression in cloned bovine embryos.

    Developed a successful procedure for cryopreservation of porcine embryos using a microdroplet procedure. In addition, we have established divergent survival rates for cryopreserved embryos from Chinese Meishan and white crossbred lines of swine. This difference in survival rate occurs within the first 24 hours following thawing.

    Generated Yucatan SCNT clones

    Isolated neural stem cells, amniotic fluid stem cells, bone marrow mesenchymal stem cells and have preliminary evidence of isolation of porcine induced pluripotent cell (iPS)

    Utilized AFS to generate transgenic swine by SCNT

    Methylation patterns of the scNT blastocysts show more similarity to the fibroblast donor cell line used for scNT as compared to the IVF blastocysts.

    Impact Statements:
    1. Being able to identify and/or predict a skewed sex ratio in individual ejaculates of semen may lead to a practical, cost effective method to control the sex of offspring in livestock species.
    2. Improvement in semen storage procedures could enhance the efficiency and use of artificial insemination, especially in species where the semen cannot be successfully cryopreserved.
    3. Identification of causes of infertility due to endophyte infected fescue could lead to better management practices and improved cow herd productivity and profitability.
    4. In vitro-produced embryos have excess lipid content, and developing a method to decrease lipids will aid in improving systems of producing embryos in vitro.
    5. The knowledge gained in oocyte preservation improvement will be important in preservations of elite livestock, endangered species as well as human eggs.
    6. The development of efficient IVP systems using microscale technologies will enable the automation of such techniques for production of large numbers of embryos in vitro, which will speed the genetic improvement and selection of superior livestock.
    7. Manipulation of specific molecular mechanisms critical to oocyte competence, including DNA methylation, cell death pathways, cellular metabolism and mRNA processing, will allow scientists and clinicians to positively alter the developmental potential of oocytes.
    8. Identification of regulatory mechanisms controlling ICM and trophectoderm lineage differentiation will provide a better understanding of fetal and 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).
    9. Identified spermatozoal transcripts in bull spermatozoa can be used to understand biology of spermatozoa and improve bull fertility. In addition, identification of comparative functional genomics of chromatin remodeling proteins, HMGN3A and SMARCAL1, can lead to better understanding of epigenetic control of mammalian embryonic development.
    10. Defining genes that are important for establishing an oocyte of high quality will identify new targets for reversing the deleterious effects of a persistent follicle and increase the fertility of dairy and beef cattle.
    11. A better understanding of the biological mechanisms underlying GnRH regulation of early embryonic development could lead to new methodologies that reduce early embryonic loss in livestock species and improve in vitro development rates of human embryos. Also, manipulation of the interaction between GnRH and its receptor in preimplantation embryos could culminate in novel contraceptive procedures.
    12. Determination of biotin effects on oocyte development could lead to establishment of appropriate levels of biotin supplementation for women.
    13. Understanding the regulation of fetal development will lead to methods for identifying normal and abnormal growth patterns resulting from ARTs, and improved systems for producing embryos in vitro.
    14. Understanding mechanisms regulating the resumption of meiosis in mammalian oocytes will lead to methods for manipulating the timing of meiotic maturation. Improving methods for manipulating the timing of meiotic maturation will improve the efficiency and effectiveness of in vitro embryo production.
    15. Identification of specific mRNAs regulating meiotic maturation will allow the development of systems to improve the developmental competence of in vitro matured mammalian oocytes by specifically arresting nuclear maturation while facilitating cytoplasmic maturation during continued culture. It will also provide novel opportunities for development of gene-targeted contraceptive methods useful for the manipulation of reproductive cycles in livestock and other mammalian species.
    16. Platform comparison and annotation of Affymetrix arrays will help other investigators make a better choice and get more information when planning gene expression profiling studies on swine.
    17. Improvements in livestock reproductive efficiency will provide consumers food and fiber products at reduced cost.
    18. Identification of the novel importin ± and understanding of its functions in controlling early events of embryonic development may ultimately lead to the development of new strategies to improve the efficiency of nuclear transfer and reproduction in cattle.
    19. The production of alpha-lactalbumin and IGF transgenic swine allow for improvement of lactation in swine production systems. These observations have profound effects on increasing the efficiency of milk and meat production. Further, the risk assessment on the safety of these animals will provide needed information to enable their entrance into the food supply.
    20. The ability to isolate and differentiate mesenchymal lineage stem cells in vitro and the transplant them back into live animals with corresponding proper differentiation will allow stem cell therapy for production parameters such as lactation and muscle growth in livestock.
    21. The identification of cytokines which extend expression of pluripotency-related transcription factor NANOG in bovine embryonic cells 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.
    22. Results from freezing studies with embryos from different lines of swine could provide improved methods for cryopreservation of swine embryos so that valuable germplasm, including genetically altered animals, can be maintained for future use.
    Last Modified: 06-Apr-2009
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