Annual/Termination Reports:

[04/23/2010] [04/14/2011] [03/22/2012] [04/24/2013]

Report Information

Participants

Milan Shipka (AK);
Rick Rorie (AR);
Charles Rosenkrans (AR);
Gerrit Bouma (CO);
George Seidel (CO);
Quinton Winger (CO);
Rebecca Krisher (IL);
Ken Bondioli (LA);
Erdogan Memili (MS);
Brett White (NB);
Charlotte Farin (NC);
Mark Mirando (USDA);
Tom Bunch (UT);
Chris Davies (UT);
Clay Isom (UT);
Qinggang Meng (UT);

Brief Summary of Minutes

Minutes of the W-2171 Multi-State Project Meeting
February 19-20, 2010
Animal Reproductive Biology Laboratory
Colorado State University
Fort Collins, CO

Attendees (Station):

Milan Shipka (AK)
Rick Rorie (AR)
Charles Rosenkrans (AR)
Gerrit Bouma (CO)
George Seidel (CO)
Quinton Winger (CO)
Rebecca Krisher (IL)
Ken Bondioli (LA)
Erdogan Memili (MS)
Brett White (NB)
Charlotte Farin (NC)
Mark Mirando (USDA)
Tom Bunch (UT)
Chris Davies (UT)
Clay Isom (UT)
Qinggang Meng (UT)

Meeting was called to order by project Chair, Char Farin, at 8:30 a.m. Attendees made brief introductions, followed by introduction of our seminar speakers by George Seidel.

Gerrit Bouma and Quinton Winger presented Pluripotency in Trophoblast Stem Cells and Germ Stem Cells. Following the informative presentations, attendees asked questions and discussed the biology and potential future for stem cells.

Mark Mirando presented AFRI: Update about the USDA Reorganization and Comments on Preparing Competitive Proposals for AFRI Submission. Mark distributed a National Institute of Food and Agriculture Factsheet that described the mission, leadership, and structure of the newly formed organization. In addition, he summarized the 2009 funding for AFRI, and explained the anticipated changes for 2010 NIFA funding for research, education, and extension. Since the RFA had not been released, Mark did not have the liberty to address specifics for programs; however, he was very helpful in explaining the obvious and subtle changes to funding guidelines and answered as many questions as possible.

After a lunch break, the committee started station reports. The format for station reports this year was a 15 minute presentation followed by 5 minutes for discussion of potential collaboration. Most of the stations prepared an electronic presentation summarizing their work. Station reports concluded at 5:15 p.m. and meeting was suspended for the evening.

Committee meeting was reconvened Saturday at 8:17 a.m. Administrative representative, Milan Shipka, addressed our group and indicated the importance of developing genuine collaboration and continued productivity. The committee then discussed each of the two project objectives.

Objective 1:

Oocyte quality was discussed and the following questions/observations were made:

Why are in vitro matured oocytes not the same as in vivo matured oocytes?

We were reminded that blastocysts are not the endpoint, rather live births are the endpoint!

gene expression could/should be evaluated, possibly via miRNA and gene chips

oocyte quality questions: small vs. large follicles; prepubertal vs. postpubertal vs. lactating females

secreted items: miRNA, MHC proteins, cytokines...

How does aneuploidy impact our results? Post-natal death appears to be increased for bulls resulting from x-sorted sperm.

How do mosaic cells impact development?

Tool development was discussed. Possibilities included: specific chromosome paint(s), deep sequencing PCR with 2-3 markers per chromosome, expressed tag sequencing...

...its not just an egg problem, we need to continue evaluating sperm...

One approach for Objective 1 could be the evaluation of aneuploidy on in vivo and in vitro losses of oocytes and sperm. Assays would include: metabolic load, blastocyst percentage and quality, survival after cryopreservation, and offspring. Does cryopreservation induce aneuploidy? What about culture conditions? George Seidel agreed to lead the group in developing a funding proposal related to this topic. Char Farin and Rebecca Krisher agreed to manage the writing process.

Objective 2:

Reprogramming was the primary topic for discussion and the following questions/observations were made:

How successful are induced pluripotent stem cells? Relative to cloning?

How do we improve genetic reprogramming?

How does the oocyte conduct genetic reprogramming? Oocyte does considerable erasing of alterations (i.e. demethylation&), but not all modifications need to be erased. Which need to be modified?

transcriptional profiling could lead to answers

for consideration ...lack of cloning success is probably not a genetic defect, rather an epigenetic defect...

Business meeting was officially started at 10:20 a.m. Committee unanimously decided to hold the 2011 meeting in conjunction with IETS meeting in Orlando FL. Committee Chair for 2011, Charles Rosenkrans, agreed to check with IETS to determine the date and site preparation. First priority would be Friday January 7th from 8:00 to 5:00 without a guest seminar. Erdogan Memili was unanimously selected as the incoming project secretary.

Committee members thanked George Seidel for hosting our committee and for providing snacks and drinks for breaks, and for arranging tours of horse and laboratory facilities. In addition, committee thanked University of Arkansas Animal Science department for providing the bound annual committee report.

Utah committee members offered to host the committee for the 2013 meeting, and that invitation was unanimously accepted.

Project Chair, Char Farin, agreed to write thank you letters to our guest speakers (Drs. Bouma and Winger).

Meeting was adjourned at 10:45 a.m.

Respectfully submitted,

Charles Rosenkrans, Jr.
2009-2010 Secretary

Accomplishments

Objective 1: Understand the biology and underlying mechanisms of gamete development, fertilization, and embryogenesis<br /> <br /> Results indicate the length of the MGA-SelectSynch estrous synchronization protocol can be reduced and CIDR progesterone inserts can be used instead of MGA without adversely affecting estrous response in treated cows. <br /> <br /> Organic mineral supplementation improves some semen quality characteristics in bulls during hot weather but additional studies are needed to determine whether this improvement translates into improved pregnancy rates.<br /> <br /> Bovine heat shock protein 70 promoter and coding sequence are polymorphic. Those polymorphisms are associated with calving rates of beef cows.<br /> <br /> Decreased fertilizability was detected in oocytes retrieved from gilts exposed to elevated ambient temperature for four days, corresponding to a typical weaning to estrus interval.<br /> <br /> We were unable to improve in vitro embryo production using cAMP regulators under the conditions tested; more research is required. We did find a number of differences in microRNA content at different stages of oocyte maturation/zygote production. These provide a starting point for manipulating these molecules during in vitro embryo production. We found that a GnRH-induced LH surge does not result in GVBD of oocytes in vivo under luteal concentrations of progesterone.<br /> <br /> A low Na+ low Ca++ base medium did not improve bovine vitrification success. We developed a simplified method of cooling bovine embryos for vitrification. <br /> <br /> Studied expression profiles of in vitro produced and in vivo produced bovine fetuses at Days 90 and 180 of gestation in two important organs: liver and placenta.<br /> <br /> The accomplishment of this study was the development of homogeneous embryonic stem cell derived clonal germ cell lines that could be continually propagated and differentiated into haploid cells. This in vitro germ cell culture system will allow for in depth study of the mechanisms orchestrating germ cell development and problems in livestock germ cell differentiation that hinder production.<br /> <br /> Leptin and glucose may interact to regulate oocyte nuclear maturation in obese and/or diabetic individuals.<br /> <br /> The glutaredoxin pathway is involved in oocyte developmental competence in pigs.<br /> <br /> Microfluidic devices provide the opportunity to culture oocytes and embryos individually, with development equal to that of standard culture drops.<br /> <br /> 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. <br /> <br /> <br /> It has been established that Angus semen processed and frozen as early as 1960 and stored in LN is still viable and produces pregnancies at rates similar to semen frozen as recently as 2003.<br /> <br /> It was demonstrated that animal temperament measured by chute scores and chute exit velocity does not affect subsequent AI pregnancy rates in beef cattle.<br /> <br /> It was demonstrated that the addition of GnRH to a 14 day estrous synchronization protocol in White-tailed deer did not result in higher pregnancy rates. Transrectal ultrasonography in White-tailed dear as early as 73 days post-insemination allows for the differentiation of pregnancies derived from AI and intact bucks.<br /> <br /> It was demonstrated that laser assisted hatching in frozen-thawed bovine embryos does not increase the number of embryos hatching in vitro or establishing pregnancy upon transfer to recipients.<br /> <br /> Pluripotent-responsive, bovine NANOG promoter-GFP and tetracyline-responsive bovine OCT4 and SOX2 constructs have been produced to be used as tools for deciphering induction of pluripotency and differentiation in bovine embryonic derived cell lines. These have been transfected into the CT-1 line to determine how they regulate other key genes in this TE bovine cell line. Knock-down of endogenous gene expression using siRNA techniques has also been performed. Real-time PCR results are being collected and analyzed.<br /> <br /> Transcripts from an OCT4 pseudogene located on bovine chromosome 7 were detected in addition to transcript from the OCT4 gene. While full length Oct4 protein was detected by Western blot, the pseudogene mRNA does not appear to translated at a detectable level. A contiguous sequence has been reconstructed by RT-PCR which indicates that the cDNA is composed of a degenerate processed pseudogene of bOCT4 exons 1-4. <br /> <br /> Samples were collected from in vitro and in vivo produced bovine blastocysts, primary cell cultures established from ICM outgrowths, and from an established trophectoderm cell line (CT-1). The samples were processed and cDNA sequenced using the Illumina system. Data will be analyzed this year. <br /> <br /> Identified microRNA transcripts present in bull spermatozoa.<br /> <br /> Determined single nucleotide polymorphisms (SNPs) associated with bull fertility.<br /> <br /> Identified spermatozoa proteins associated with bull fertility. <br /> <br /> Contributed to better understanding of bovine genome in the Bovine Genome Sequencing and Annotation Consortium.<br /> <br /> Elucidated epigenetic regulators of early embryonic development, i.e., chromatin remodelers and DNA methyltransferases.<br /> <br /> Evaluated in vitro culture systems for porcine oocyte maturation, fertilization and embryo culture.<br /> <br /> Established an in vitro follicle culture system to identify the effect of excess insulin and IGF exposure on oocyte gene expression.<br /> <br /> Chronic in vitro exposure of follicles to insulin causes changes in 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.<br /> <br /> 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.<br /> <br /> 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 blocks embryonic development and acts via alteration of the cell cycle rather than apoptotic pathways. In addition, we have correlative data to suggest that GnRH binding to its receptor in early embryos activates the protein kinase C signaling pathway.<br /> <br /> Via biotin studies, 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.<br /> <br /> <br /> Transgenic pigs were produced expressing a spermatogonial marker gene.<br /> <br /> mRNA and proteins were successfully extracted from both freshly ejaculated and frozen samples of stallion spermatozoa. <br /> <br /> A significant correlation between immunostaining for the fertility-related protein, SP22, was found with total motility and progressive motility for semen collected during the breeding season.<br /> <br /> Identified potential candidate mRNAs associated with FSH-induced initiation of GVBD in murine and bovine cumulus-oocyte complexes matured in vitro.<br /> <br /> Used short interfering RNA approaches to assess the functional roles of Nr4A1 and Egr1 mRNAs during FSH-induced oocyte maturation in cattle.<br /> <br /> Determined that the administration of FSH using 3 subcutaneous injections in combination with a progesterone-releasing device was an effective method for superovulation of Holstein cows.<br /> <br /> Identified that bovine AIRN RNA is expressed in cattle.<br /> <br /> Found expression of bovine AIRN RNA in fetal tissue at the post-implantation and peri-implantation stages but not in pre-implantation stages of development.<br /> <br /> The modification of histones is an epigenetic mechanism used to regulate gene expression. Abnormal modifications of histones have been proposed to contribute to the reduced development to term and low success rate of embryos following somatic cell nuclear transfer (scNT). The inability to reprogram epigenetic markers such as histone modifications found in the fibroblast donor cell line, especially in histones associated with establishing totipotency, could add to the reprogramming deficiencies associated with scNT. Two histone modifications associated with transcriptional activation; H3K4m3 and H4K16ac, and one histone modification associated with repressing transcription; H3K9m2, were considered with their association to three genes known to contribute to maintaining totipotency: NANOG, POU5F1, and SOX2. The uChIP protocol was performed by using antibodies specific for each histone modification, follwed by real time PCR (qPCR) analysis in order to compare bovine cumulus cells, scNT blastocysts, and in vitro fertilized (IVF) blastocysts. The gene POU5F1 was not highly associated with any of the histone modifications regardless of the treatment group. The Sox2 gene showed high levels of association with all three histone modifications in both IVF and scNT blastocysts, but very minimal association with the modifications in cumulus cells. The gene Nanog was highly associated with all three histone modifications in the scNT embryos, while only highly associated with the modifications H3K4m3 and H3K9m2 in IVF embryos. <br /> <br /> Transcription factors play a critical role in gene regulation and the characterization of these DNA binding proteins is crucial for elucidating some of the interacting proteins that assist in regulating transcription. The interaction of proteins with DNA corresponding to different portions of the promoter regions of the bovine Oct4 and Sox2 genes was evidenced by a mobility shift in the band migration compared to the control DNA. The shifted bands were excised from the 6% polyacrylamide gel and were subsequently submitted to mass spectrometry analysis for identification of candidate proteins. Putative proteins identified are involved in diverse cellular transcriptional processes including: transcriptional activation; chromatin modification, decondensation, and distribution; nuclear architecture including core histone 2a; constitutive and alternative splicing; DNA repair; regulation of RNA polymerase II; and DNA stabilization.<br /> <br /> Intracellular calcium (Ca2+i) release, a hallmark of oocyte activation, is the end result of a complex series of signal transduction pathways which have yet to be completely characterized in the bovine model. It is well known that src family kinases (SFK) activate Phospholipase C (PLC) which converts phosphatidyl inositol (4,5)-bisphosphate into diacylglycerol and 1,4,5-inositol trisphosphate (IP3) which is directly involved in releasing Ca2+i from the endoplasmic reticulum. A two prong approach was undertaken to identify the mechanisms involved in the signal transduction pathway resulting in bovine activation. The first approach utilized immunoblotting to identify the presence of endogenous PLC isoforms in total bovine oocyte lysate using primary antibodies directed against four distinct isozymes of PLC. Immunoblotting was performed according to standard laboratory protocols and confirmed the presence of the PLC isoforms ´1, ´3, ´4, ³1, ²3 and ²4. The second approach involved microinjecting primary antibodies of ten different PLC isoforms into in vitro matured bovine oocytes at a 1:100 dilution. Following microinjection, oocytes were fertilized and cultured in vitro according to standard laboratory procedures (Reed et al., 1996). Activation and development were assessed by recording cleavage at 48 hours post fertilization. Microinjection of several PLC-specific antibodies resulted in no effect on cleavage rates while others significantly blocked development. Determining the presence and involvement of these intracellular signaling molecules in bovine oocyte fertilization and activation will result in better activation protocols for nuclear transfer (NT). <br /> <br /> Deficiencies in the success rate of somatic cell nuclear transfer are widely held to be epigenetic in nature, and arise from the limited ability of a differentiated donor cell to erase epigenetic signatures required for nuclear reprogramming. Following bovine somatic cell nuclear transfer DNA methylation signatures of two genes necessary for pluripotency and self-renewal, namely Nanog and POU5F1 (Oct-4) more closely resemble that of somatic cells rather than in vitro fertilized embryos. A retained methylation signature following scNT likely leads to interaction with methyl binding domain proteins capable of binding to methylated promoter regions and acting to silence gene expression. Using a modified version of Electrophoretic Mobility Shift Assay (EMSA) targeted to the genes Nanog and POU4F1 (Oct-4) we identified DNA binding proteins that interact specifically with the methylated promoter regions. We observed protein binding specific to a methylated DNA template that differs from the binding proteins specific to the non-methylated DNA template. These findings indicate that the gene promoter region is acted upon by different DNA binding proteins depending on its methylation status, and that the retention of a hyper-methylation signature could lead to the premature down-regulation of the genes Nanog and POU5F1.<br /> <br /> The practical application of somatic cell nuclear transfer (SCNT) in animal agriculture and biomedical research is limited at the present time to technical inefficiencies that contribute to high rates of embryo and fetal loss. One of those inefficiencies that has yet to be optimized in the animal system is the enucleation procedure. In this study we evaluated various enucleation methods that might lead to increased efficiency of chromosome removal in the bovine oocyte. The results of the study are summarized as following. 1. Three-tenth M of sucrose and 0.6ug/ml demecolcine will induce high cytoplasmic protrusions containing chromosomes (75% and 90%, respectively). 2. Most cytoplasmic protrusions induced by sucrose treatment will last longer than 30 min; whereas, demecolcine induced protrusions will disappear within 30 min, particularly when oocytes are exposed to solutions containing 7.5ug/ml cytochalasin B (CB). 3. Enucleation of 100% can be achieved upon removing cytoplasmic protrusions with a micropipette in 0.3M sucrose and 7.5ug/ml CB. The survival rate of oocytes using this procedure, however, is negatively impacted (reduced to 75%), and we therefore consider this as a limiting factor in its application . 4. One hundred percent enucleation is achieved by removing induced cytoplasmic protrusions in the presence of demecolcine. Oocyte survival rate is high when enucleation is performed in CB-free solution with or without demecolcine. 5. The cleavage and blastocyst rates of cloned embryos from sucrose or demecolcine-treated oocytes are similar. 6. Oocyte microtubules are depolymerized and mostly depleted in demecolcine or demecolcine+CB treated oocytes, but remain nearly intact in sucrose-treated oocytes. The results of this study indicate that the enucleation of bovine oocytes in sucrose+CB or demecolcine+CB solutions are inefficient. CB does not appear to be required to enucleate bovine oocytes, and demecolcine-assisted enucleation is just as efficient when not exposing oocytes to CB. <br /> <br /> Functional characterization of importin ±8 specifically expressed in bovine oocytes and early embryos.<br /> <br /> Our previous results support an important functional role for importin ±8 in early embryogenesis. We have initiated new experiments to test the ability of importin ±8, relative to other importin ±s, to bind and facilitate nuclear transport of known oocyte-specific transcription factors important for oocyte and early embryonic development, and the functional requirement of importin ±8 for meiotic maturation, fertilization and specific developmental events characteristic of the maternal-to-embryonic transition. Through data mining, we have identified the cDNA sequences for all bovine importin ± genes. The predicted importin ±1, ±3, ±4, ±5, ±6 and ±7 proteins are 529 aa, 521 aa, 521 aa, 538 aa, 536 aa and 536 aa, respectively. All proteins are predicted to contain an IBB domain and 8 ARM repeats. The importin ±8 protein shares 53%, 45%, 45%, 41%, 40% and 40% sequence identity with importin ±1, ±3, ±4, ±5, ±6 and ±7, respectively. Phylogenetic analysis revealed that importin ±8 belongs to the first subfamily of the importin ± family. Expression of mRNA for importin ±1, ±3, ±4, ±5, ±6 and ±7 in bovine tissues was determined by RT-PCR. All 6 genes are ubiquitously expressed. We have cloned the coding regions of these genes as well as the importin ±8 gene in frame of the glutathione S-transferase (GST) gene in pGEX-4T-1 vector and successfully expressed the GST-importin fusion proteins. We have also obtained the bovine cDNA sequences for a number of oocyte-specific genes including Oct4 and Nobox. We are currently evaluating the interactions of all importin ± proteins with Oct4 and Nobox proteins by GST pull-down assay. Furthermore, we have validated a model system where, following siRNA injection, spontaneous germinal vesicle breakdown can be inhibited for 48 h of culture in vitro in the presence of S-roscovitine. This model system will be used for studies of effects of importin ±8 reduction during meiotic maturation, fertilization and initial cleavage divisions.<br /> <br /> <br /> <br /> Objective 2: Refine methods for production of genetically modified animals to improve livestock production efficiency<br /> <br /> We have identified all members of the importin ± gene family in cattle, prepared plasmid constructs, and validated methodologies for future experiments to elucidate the functional roles of importin ±8 during early events of embryonic development.<br /> <br /> Work is currently in progress to determine the time-course protocols to assess the efficiency of non-homologous and homologous recombination in primary cells and in conjunction with cell cycle manipulations. Data suggest the time course of the effect is extended in primary cells and cell viability is decreased, especially following treatment with Ku 70 siRNA, which can be partially rescued by siRNA depletion of P53<br /> <br /> Investigated the feasibility of using frozen and immature oocytes in the generation of embryonic stem cells; studied genetic imprinting in naturally reproduced and cloned calves.<br /> <br /> For the first time in a domestic livestock species, porcine induced pluripotent stem cells were demonstrated to give rise to chimeric offspring demonstrating a true pluripotent state. These cells can undergo complex genetic manipulations to produce transgeneic animals that significantly improve livestock production.<br /> <br /> The present results suggest that there is no horizontal Tg transmission between T and C pigs due to rearing or mating. This work provides a critical step toward providing rigorous scientific data for risk assessment of transgenic livestock.<br /> <br /> ADSC and BMSC can be successfully differentiated into adipogenic and osteogenic lineages and maintain viability in an alginate hydrogel over time. Our conclusion, is that alginate is a viable scaffold material for the differentiation of mesenchymal stem cells for tissue engineering applications.<br /> <br /> The combination of microarray data and pairwise analysis uncovered novel and high reliable ICG for qPCR normalization in adult porcine stem cells induced into adipogenic and osteogenic lineages.<br /> <br /> Results suggest both ADSC and BMSC can differentiate towards the adipogenic lineage but with quantitatively different gene expression patterns.<br /> <br /> 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. 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.<br /> <br /> We have identified genes whose products might be playing a role in epigenetic control of molecular reprogramming of gene expression in cloned bovine embryos.<br /> <br /> Constructed lentiviral vectors containing a constitutively active promoter fused to selected siRNA designed to reduce GnRHR II mRNA levels in porcine cells and tissues.<br /> <br /> We have 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.<br /> <br /> Transgene copy number following transfection with alternative methods has been determined. It was determined that electroporation results in an average of 2-3 copies of the transgene while liposome-mediated transfection yields predominantly single copy insertions.<br /> <br /> Isolation and culture procedures for adipose derived stem cells from cattle and pigs have been established. These stem cell populations have been characterized for cell cycle characteristics, chromosomal stability in culture and epigenetic modifications. Bovine and eland adipose derived stem cells have been utilized as donor cells in somatic cell nuclear transfer procedures.<br /> <br /> Expression of pluripotency genes has been studied in bovine fetal fibroblasts and adipose derived stem cells. <br /> <br />

Publications

Refereed articles<br /> <br /> Caldwell, J. D., K. P. Coffey, W. K. Coblentz, J. A. Jennings, D. S. Hubbell, III, D. L. Kreider, M. L. Looper, and C. F. Rosenkrans, Jr. 2009. Performance by fall-calving cows grazing tall fescue pastures with different proportions stockpiled. Forage and Grazinglands doi:10.1094/FG-2009-0312-01-RS.<br /> <br /> Looper, M. L., G. E. Aiken, and C. F. Rosenkrans, Jr. 2009. Management strategies for cattle grazing toxic tall fescue during summer months. Forage and Grazinglands doi:10.1094/FG-2009-1102-03-RV.<br /> <br /> Looper, M. L., T. S. Edrington, T. R. Callaway, and C. F. Rosenkrans, Jr. 2009. Fate of Escherichia coli O157:H7 and Salmonella from contaminated manure slurry applied to soil surrounding tall fescue. Lett. Appl. Micro. 48:513-516.<br /> <br /> Looper, M. L., T. S. Edrington, and C.F. Rosenkrans, Jr. 2009. Influence of body condition and forage type on prevalence of Escherichia coli O157:H7 and Salmonella in grazing beef cows. Lett. Appl. Micro. 49:361-365.<br /> <br /> Looper ML, Rorie RW, Person CN, Lester TD, Hallford DM, Aiken GE, Roberts CA, Rottinghaus GE, Rosenkrans CF Jr. 2009. Influence of toxic endophyte-infected fescue on sperm characteristics and endocrine factors of yearling Brahman-influenced bulls. J Anim Sci. 87:1184-1191. <br /> <br /> Wang, H., M.L. Looper, Z.B. Johnson, R.W. Rorie and C.F. Rosenkrans. 2009. Involvement of signaling pathways in bovine sperm motility and effect of ergot alkaloids. In Vitro Cell Dev Biol Anim. 45:483-489.<br /> <br /> Looper, M. L., S. G. Black, S. T. Reiter, R. Okimoto, Z. B. Johnson, M. A. Brown, and C. F. Rosenkrans, Jr. 2010. Identification of polymorphisms in the enhancer region of the bovine prolactin gene and association with profitability traits of beef cattle. Prof. Anim. Sci. (in press).<br /> <br /> Rosenkrans, C., Jr., A. Banks, S. Reiter, M. Looper. 2010. Calving rates of Brahman-influenced cows are associated with heat shock protein 70 genetic polymorphisms. Anim. Repro. Sci. (in press).<br /> <br /> <br /> Berger, T., B. Nitta, Y. Ducolomb, and M. Betancourt. 2009. Interaction of potential porcine sperm ligands with the oocyte plasma membrane. Reprod Domest Anim. [Epub ahead of print]<br /> <br /> Berger, T., and B. M. Roberts. 2009. Reduced immunolabelling of a porcine oocyte membrane protein reflects reduced fertilizability of porcine oocytes following elevated ambient temperature. Reprod Domest Anim 44: 260-265.<br /> <br /> Bertolini, L.R., M. Bertolini, E.A. Maga, E.A., K.R. Madden and J.D. Murray. 2009. Increased gene targeting in Ku70 and Xrcc4 transiently deficient human somatic cells. Mol. Biotech. 41:106-114.<br /> <br /> Corbin, C. J. et al. 2009. Porcine hypothalamic aromatase cytochrome p450: Isoform characterization, sex-dependent activity, regional expression, and regulation by enzyme inhibition in neonatal boars. Biol Reprod 81: 388-395.<br /> <br /> Ahola, J.K., G.E. Seidel, Jr. and J.C. Whittier. 2009. Use of gonadotropin-releasing hormone at fixed-time artificial insemination at eighty or ninety-seven hours post prostaglandin F2± in beef cows administered the long-term melengestrol acetate Select Synch. Prof Anim Sci 25:256-261.<br /> <br /> Barcelo-Fimbres, M., Z. Brink and G.E. Seidel, Jr. 2009. Effects of phenazine ethosulphate during culture of bovine embryos on pregnancy rate, prenatal and postnatal development after embryo transfer. Theriogenology 71:355-368. <br /> <br /> Barfield, J.P., P.M. McCue, E.L. Squires and G.E. Seidel, Jr. 2009. Effect of dehydration prior to cryopreservation of large equine embryos. Cryobiology 59:36-41. <br /> <br /> Campos-Chillon, L.F., T.K. Suh, M. Barcelo-Fimbres, G.E. Seidel, Jr. and E.M. Carnevale. 2009. Vitrification of early-stage bovine and equine embryos. Theriogenology 70:349-354.<br /> <br /> Hyland, A., G.E. Seidel, Jr., R.M. Enns, R.K. Peel and J.C. Whittier. 2009. Intervals of five or seven days between controlled internal drug-release insertion, gonadotropin-releasing hormone, and prostaglandin F2± injections: Effects on pregnancy rate and follicular size. Prof. Anim. Sci. 25:150-154.<br /> <br /> Mahmoud, K.G., T.H. Scholkamy, Y.F. Ahmed, G.E. Seidel, Jr. and M.F. Nawito. 2009. Effect of different combinations of cryoprotectants on in vitro maturation of immature buffalo (Bubalus bubalis) oocytes vitrified by straw and open-pulled straw methods. Reprod. Dom. Anim. PMID 19090828.<br /> <br /> Purcell, S.H., J.D. Cantlon, C.D. Wright, L.E. Henkes, G.E. Seidel, Jr. and R.V. Anthony. 2009. The involvement of proline-rich 15 in early conceptus development in sheep. Biol. Reprod. 81:1112-1121.<br /> <br /> Roberts, R.M., G.W. Smith, F.W. Bazer, J. Cibelli, G.E. Seidel, Jr., D.E. Bauman, L.P. Reynolds and J.J. Ireland. 2009. Farm animal research in crisis. Science 324:468-469.<br /> <br /> Seidel, G.E., Jr. 2009. ASAS Centennial Paper: Future research in physiology and endocrinology. J. Anim. Sci. 87:384-389.<br /> <br /> Mansouri-Attia N, Sandra O, Aubert J, Degrelle S, Everts RE, Giraud-Delville C, Heyman Y, Galio L, Hue I, Yang X, Tian XC, Lewin HA, Renard JP. 2009. Endometrium as an early sensor of in vitro embryo manipulation technologies. Proc Natl Acad Sci U S A. 2009 Apr 7;106(14):5687-92. Epub 2009 Mar 18.<br /> <br /> Mansouri-Attia N, Aubert J, Reinaud P, Giraud-Delville C, Taghouti G, Galio L, Everts RE, Degrelle S, Richard C, Hue I, Yang X, Tian XC, Lewin HA, Renard JP, Sandra O. 2009. Gene expression profiles of bovine caruncular and intercaruncular endometrium at implantation. Physiol Genomics. 2009 Sep 9;39(1):14-27. Epub 2009 Jul 21.<br /> <br /> Curchoe CL, Zhang S, Yang L, Page R, Tian XC. 2009. Hypomethylation trends in the intergenic region of the imprinted IGF2 and H19 genes in cloned cattle. Anim Reprod Sci. 2009 Dec;116(3-4):213-25. Epub 2009 Feb 11.<br /> <br /> Suteevun-Phermthai T, Curchoe CL, Evans AC, Boland E, Rizos D, Fair T, Duffy P, Sung LY, Du F, Chaubal S, Xu J, Wechayant T, Yang X, Lonergan P, Parnpai R, Tian XC. Allelic switching of the imprinted IGF2R gene in cloned bovine fetuses and calves. Anim Reprod Sci. 2009 Nov;116(1-2):19-27. Epub 2009 Jan 20.<br /> <br /> Smith SL, Everts RE, Sung LY, Du F, Page RL, Henderson B, Rodriguez-Zas SL, Nedambale TL, Renard JP, Lewin HA, Yang X, Tian XC. 2009. Gene expression profiling of single bovine embryos uncovers significant effects of in vitro maturation, fertilization and culture. Mol Reprod Dev. 2009 Jan;76(1):38-47.<br /> <br /> Yang X, Guo XM, Wang CY, and Tian XC. 2009. Protocols for Large-Scale Derivation of Cardiomyocytes from Embryonic Stem Cells Chapter 22. In: Essentials of Stem cells. Elsevier, Lanza R (ed). (in press).<br /> <br /> Sung LY, Amano T, Smith SL, Tian XC and Yang X. 2009. Somatic Cell Nuclear Transfer and Derivation of Embryonic Stem Cells. Chapter 1. In: Methods in Stem Cell Medicine and Bioengineering (in press).<br /> <br /> Tian XC, Park J, Bruno R, French R, Jiang L, Prather RS. 2009. Altered gene expression in cloned piglets. Reprod Fertil Dev. 2009;21(1):60-6.<br /> Marjani SL, Le Bourhis D, Vignon X, Heyman Y, Everts RE, Rodriguez-Zas SL, Lewin HA, Renard JP, Yang X, Tian XC. 2009. Embryonic gene expression profiling using microarray analysis. Reprod Fertil Dev. 2009;21(1):22-30.<br /> <br /> Chang CC, Sung LY, Amano T, Tian XC, Yang X, Nagy ZP. Nuclear transfer and oocyte cryopreservation. Reprod Fertil Dev. 2009;21(1):37-44.<br /> <br /> West, F. D. et al. 2008. Enrichment and Differentiation of Human Germ-Like Cells Mediated by Feeder Cells and Basic Fibroblast Growth Factor Signaling. Stem Cells. 11: 2768-76.<br /> <br /> West, F. D. et al. 2010. Kit ligand and bone morphogenetic protein signaling enhances human embryonic stem cell to germ-like cell differentiation. Hum Reprod 25: 168-178.<br /> <br /> West, F. D. et al. Porcine Induced Pluripotent Stem Cells Produce Chimeric Offspring. Stem Cells and Dev (In Review; 12/01/09).<br /> <br /> West, F. D. et al. Isolation and Expansion of Meiotic Competent Clonal Germ Cell Lines Derived from Male Human Embryonic Stem Cells. Stem Cells and Dev (In Preparation; 01/06/09).<br /> <br /> Mônaco, E., Lima, A.S., Bionaz, M, Maki, A., Wilson, S.M., Hurley, W.L. Wheeler, M.B. 2009. Morphological and Transcriptomic Comparison of Adipose and Bone Marrow Derived Porcine Stem Cells. Open Tiss. Eng. Regen. Med. J. 2:20-33.<br /> <br /> Bleck, G.T., Wheeler, M.B., Hansen, L.B., Chester-Jones, H.,, Miller, D.J. 2009. Lactose Synthase Components in Milk: Concentrations of a-Lactalbumin and b1,4-Galactosyltransferase in Milk of Cows from Several Breeds at Various Stages of Lactation. Reprod. Dom. Anim 44:241247.<br /> <br /> Yuan, Y., Krisher, R.L. (2009) Effect of ammonium during in vitro maturation on porcine oocyte nuclear maturation and subsequent embryonic development. Animal Reproduction Science 117:302-307. doi:10.1016/j.anireprosci.2009.05.012. <br /> <br /> Paczkowski, M., Krisher R.L. (2010) Aberrant Protein Expression is Associated with Decreased Developmental Potential in Porcine Oocytes. Molecular Reproduction and Development 77:51-58. Published Online: 2 Sep 2009; DOI 10.1002/mrd.21102. <br /> <br /> Krisher, R.L., Wheeler, M.B. (2010). Towards use of microfluidics for individual embryo culture. Reproduction, Fertility and Development 22:32-39.<br /> <br /> Giraldo, A.M., M.N. Purpera, T.D. Vaught, D.L. Ayares, J W. Lynn, R. A. Godke and K R. Bondioli. 2009. Inhibition of DNA methyltransferase 1 expression in bovine fibroblast cells used for nuclear transfer. Reprod. Fertil. Develop. 21:785-795. <br /> <br /> Purpera, N.M., A.M. Giraldo, C. B. Ballard, D. Hylan, R. A. Godke and K. R. Bondioli. 2009. Effects of culture medium and protein supplementation on mRNA expression of in vitro produced bovine embryos. Molec. Reprod. Dev. 76(8):783-793.<br /> <br /> Hylan, D., A. M. Giraldo, J. A. Carter, G.T. Gentry Jr., K. R. Bondioli and R.A. Godke. 2009. Sex ratio of bovine embryos and calves originating from the left and right ovaries. Biol Reprod. 81:933-938. <br /> <br /> Owiny, O.D., D.M. Barry, M. Agaba and R.A. Godke. 2009. In vitro production of cattle x buffalo hybrid embryos using cattle oocytes and African buffalo (Syncerus caffer caffer) epididymal sperm. Theriogenology 71:884-894.<br /> <br /> Alapathi, R., M. Stout, J. Saenz, G. T. Gentry Jr., R.A. Godke and R.V. Devireddy. 2009. Ejaculated and epidydimal bovine sperm exhibit equivalent freezing response. Cryobiology 59: 164-170. <br /> <br /> Wirtu, G., C.E. Pope, D.L Paccamonti, R.A. Godke, B.L Dresser. 2009. Ultrasound-guided retrieval and in vitro maturation of eland (Taurotragus oryx) and bongo (Tragelaphus eurycerus isaaci) antelope oocytes. Anim. Reprod. Sci. 111:160-172.<br /> <br /> Hylan, D., A. M. Giraldo, K. R. Bondioli, R. A. Godke. 2009. Distribution of sexes within the left and right uterine horns of beef cattle. Theriogenology (In press)<br /> <br /> Godke, R.A., G.T. Gentry Jr. and K. R. Bondioli. New biotechnologies for the farm animal industry. So. Assn. Agri. Scientists. (Refereed) (In press)<br /> <br /> Pant, D., and C. L. Keefer. 2009. Expression of pluripotency-related genes during bovine inner cell mass explant culture. Cloning Stem Cells 11: 355-365.<br /> <br /> Rodriguez-Osorio, N., H. Wang, J. Rupinski, S. M. Bridges and E. Memili. Comparative functional genomics of mammalian DNA methylation. In press. Reproductive Biomedicine online. <br /> <br /> Bovine Genome Sequencing and Annotation Consortium including Memili, E., and others. 2009. The genome sequence of Taurine Cattle: A window to ruminant biology and evolution. Science 324(5926):522-528. <br /> <br /> Rodriguez-Osorio, N., Z. Wang, P. Kasinathan, G. P. Page, J. M. Robl and E. Memili. 2009. Transcriptional reprogramming of gene expression in bovine somatic cell chromatin transfer embryos. BMC Genomics 10:190. <br /> <br /> Uzun, A., N. Rodriguez-Osorio, H. Wang, A. Kaya, J.J. Parrish, V. Ilyin and E. Memili. 2009. Comparative Functional Genomics of Chromatin Remodeling Proteins HMGN3a and SMARCAL1 During Early Mammalian Embryogenesis. BMC Genomics 10:183. <br /> <br /> Feugang, J. M., A. Kaya, G.P. Page, L. Chen, T. Mehta, K. Hirani, L. Nazareth, R. A. Gibbs and E. Memili. 2009. Two stage genome wide association study identifies Integrin beta five as having a potential role in bull fertility. BMC Genomics 10: 176. <br /> <br /> Wang, H., J. M. Feugang, N. Rodriguez-Osorio, S. Jung, K. Garrison, C. Wolgemuth, L. Greer, E. Memili. 2009. Effects of culture media and inhibitors on biology of porcine embryonic development in vitro. Livestock Science 121: 102-107.<br /> <br /> Feugang, J. M, O. D. Camargo-Rodriguez and E. Memili 2009. Culture Systems for Bovine Embryos. Livestock Science 121:141-149.<br /> <br /> Cherrington, B.D., T.A. Farmerie, B.E. Bass, R.A. Cederberg, C.M. Clay, and B.R. White. 2009. Emergence of a new regulatory element in the proximal promoter of the gonadotropin releasing hormone receptor in Chinese Meishan swine. Biol. Reprod. (Provisionally Accepted).<br /> <br /> Montagner, M.M, A.R. Cropp, J.J. Swanson, R.A. Cederberg, P.B.D. Goncalves, and B.R. White. 2009. Role of gonadotropin-releasing hormone on mouse preimplantation embryonic development. Mol. Reprod. Dev. (Submitted).<br /> <br /> Montagner, M.M, P.B.D. Goncalves, G.A. Mills, R.K. Christenson, and B.R. White. 2009. Divergent survival of Meishan and white crossbred porcine embryos following vitrification using a microdroplet method. Reprod. Fert. Dev. (Submitted).<br /> <br /> Silva, C., J.R. Wood, L. Salvador, Z. Zhang, I. Kostetskii, C.J. Williams, and J.F. Strauss III 2009. Expression profile of male germ cell-associated genes in mouse embryonic stem cell cultures treated with all-trans retinoic acid and testosterone. Mol. Reprod. Dev. 76:11-21.<br /> <br /> Zhang, Z., R. Jaimez, X. Shen, D. Gude, H. Tang, J.R. Wood, E. Goldberg, and J.F. Strauss III. 2009. Autoregulation of SPAG16 expression: A single gene encoding an axoneme structural protein and a transcription factor that activates the axoneme protein promoter. Nucleic Acids Res. (Submitted).<br /> <br /> Farin CE, Farmer WT, Farin PW. 2010. Pregnancy recognition and abnormal offspring syndrome. Reprod Fertil Dev 22:75-87.<br /> Wrench N, Pinto CRF, Klinefelter GR, Dix DJ, Flowers WL, Farin CE. (2010). Effect of season on fresh and cryopreserved stallion semen. Anim Reprod Sci (accepted).<br /> <br /> Farmer WT, Farin PW, Piedrahita JA, Bischoff SR, Farin CE. (2010). Detection of antisense of insulin-like growth factor-2 receptor RNA non-coding (AIRN) in cattle. Reprod Fertil Dev (in revision).<br /> <br /> Farin CE, Alexander JE, Farin PW. (2010). Expression of messenger RNAs for insulin-like growth factors and their receptors in bovine fetuses at early gestation from embryos produced in vivo or in vitro. Theriogenology (in revision).<br /> <br /> Li, G.P., K.L. White , K.I. Aston , T.D. Bunch, B.A. Hicks, Y. Liu , and B.R. Sessions. 2009. Colcemid-treatment of heifer oocytes enhances nuclear transfer embryonic development, establishment of pregnancy and development to term. Mol. Reprod. Dev. 76:620-628.<br /> <br /> Aston, K.I., G.P. Li, B.A. Hicks, B.R. Sessions, A.P. Davis, Q.A. Winger, L.F. Rickords, and K.L. White. 2009. Global gene expression analysis of bovine somatic cell nuclear transfer blastocysts and cotyledons. Mol Reprod Dev. 76:471-82. <br /> <br /> Tejomurtula, J., Lee, K.B., Tripurani, S.K., Smith, G.W. and Yao, J. 2009. Role of Importin ±8, a new member of the importin ± family of nuclear transport proteins, in early embryonic development in cattle. Biology of Reproduction. 81: 333342. <br /> <br /> <br /> Books, non-refereed book chapters, proceedings, instructional media, theses/dissertations<br /> <br /> Delgado Callisaya, P.A. 2009. Factors influencing survival of bovine spermatozoa during storage and related to the ratio of X- to Y-chromosome bearing spermatozoa. M.S. Thesis, University of Arkansas, Fayetteville<br /> <br /> Seidel, G.E., Jr. 2009. Sperm sexing technology  the transition to commercial application. Theriogenology 71:1-3.<br /> <br /> Seidel, G.E., Jr. 2009. Artificial insemination of cattle with sexed semen. Proc. Meeting on Milk and Meat Production in Warm Climates, pp. 43-51.<br /> <br /> Wall, R.J., G. Labile, E. Maga, G.E. Seidel, Jr. and B. Whitelaw. 2009. Animal productivity and genetic diversity: Cloned and transgenic animals. In: Animal Agricultures Future through Biotechnology, Part 8, Council for Agricultural Service and Technology, Ames, IA, 16 pp.<br /> <br /> West, F. D. Differentation of Male Human Embryonic Stem Cells into Germ-Like Cells: A Developmental Model and an Assisted Reproductive Technology.<br /> <br /> Roberts, J. 2009. Title: Effect Priming with Melegestrol Acrtate (MGA) on Subsquent Fertility in Beef Heifers. MS Thesis. Louisiana State University. Baton Rouge, LA. Spring (May) 2009.<br /> <br /> Pennington, P 2009. Title: Characterization of the Common Eland (Taurotragus oryx) Estrous Cycle. MS Thesis Louisiana State University. Baton Rouge, LA. Summer 2009<br /> <br /> Willaims, K. 2009. Title: Characterization of Adult Porcine Adipose Stem Cells PhD Dissertation. Louisiana State University. Baton Rouge, LA. Summer 2009. (K. Bondioli and R. Godke Joint Advisors).<br /> <br /> Picou, A. 2009. Title: Isolation and Characterization of Bovine Adipose Derived Somatic Stem Cells for the use in Nuclear Transfer. MS Thesis. Louisiana State University, Baton Rouge, LA. Summer 2009.<br /> <br /> Wilson, J. 2009. Title: Determining Gene Copy Number in Transfected Caprine Fibroblasts. MS Thesis. Louisiana State University, Baton Rouge, LA. Summer 2009.<br /> <br /> Denniston, R. S., S. Michelet, K.R. Bondioli and R. A. Godke. Principles of Embryo Cryopreservation. In: Cryopreservation in Aquatic Species. Tiersch, T. R. and P. M. Mazik, Editors. World Aquaculture Society, Baton Rouge, Louisiana. (In press).<br /> <br /> Chandler J.E. and R. A. Godke. Cryopreservation of Cattle Sperm. In: Cryopreservation in Aquatic Species. Tiersch, T. R. and P. M. Mazik, Editors. World Aquaculture Society, Baton Rouge, Louisiana. (In press).<br /> <br /> Brauer, V.M. 2009. Transcriptional regulation of the porcine type II GnRH receptor gene. MS Thesis. University of Nebraska, Lincoln, NE.<br /> <br /> <br /> Abstracts<br /> <br /> Barcelo-Fimbres, M. and G.E. Seidel, Jr. 2009. Effects of lipolytic agents forskolin, epinephrine and caffeine on embryonic development and lipid content of bovine embryos produced in vitro. Reprod. Fertil. Develop. 21:154-155.<br /> <br /> Preis, K.A., G.E. Seidel, Jr. and D.K. Gardner. 2009. Effect of in vitro maturation medium on subsequent quantity and quality of developing embryos. Proc. American Society of Reproduction Medicine. In Press.<br /> <br /> Barfield, J.L., R. Sanchez, E.L. Squires and G.E. Seidel, Jr. 2009. Vitrification and conventional cryopreservation of equine embryos. Reprod. Fertil. Develop. 21:130.<br /> <br /> Amorim, L.E., C.A.A. Torres, E.A.M. Amorim, J.D. Guimaraes, J.F. Fonseca, L.G.B. Sigueira and G.E. Seidel, Jr. 2009. Effect of dietary urea on embryonic viability and development of Toggenburg goats. Reprod. Fertil. Develop. 21:157.<br /> <br /> Jiang L, SL Marjani, M Bertolini, H A Lewin, GB Anderson, X Yang, and XC Tian. 2010. Indistinguishable Transcriptional Profiles Between In Vivo- and In Vitro-Produced Bovine Fetuses (presented at the 36th annual meeting of IETS, Cordoba, Argentina, Jan 9-12, 2010)<br /> <br /> Chang CC, Lin CJ, Friedman J, Kort HI, Tian XC, Nagy ZP. The difference of spindle recovery during oocyte cryopreservation: vitrification vs. slow freezing. Fertil Steril 2009;92(3S):S86. Oral presentation (Atlanta, Georgia, USA; American Society for Reproductive Medicine in 2009 annual meeting.<br /> <br /> Yang, X, Amano T, Ma Y, He Z, Amano M, Lin C-J, Treaster S, Dym M, Tian XC. 2009. Genetically corrected ES cells derived from cloned embryos of infertile mice  a potential application of therapeutic cloning. Presented at the 6th Annual meeting of the Asian Reproductive Biotechnology Society, Siem Reap, Cambodia, Nov 16-20, 2009. p. 3.<br /> <br /> Franklin West, Kurinji Pandiyan, Kelly Robbins and Steve Stice. Differentiation of Primordial Germ Like Cells From Human Embryonic Stem Cells. Society for the Study of Reproduction; 2009 Sept: Pittsburgh, (PA). <br /> <br /> F.D. West, M.S. Natrajan, M.I. Roche-Rios, M.A. Bedell and S.L. Stice. Bone Morphogenetic Protein and KIT Ligand Signaling Enhance Human Embryonic Stem Cell Differentiation into Early Sperm Cells. Georgia Life Sciences Summit; 2008 Sept: Atlanta, (GA). <br /> <br /> Franklin West, Kurinji Pandiyan, Kelly Robbins and Steve Stice. Enrichment and Differentiation of Germ-Like Cells from Human Embryonic Stem Cells. Georgia Stem Cell Initiative; 2008 May: Athens, (GA). Recipient of 3rd place in poster competition. <br /> <br /> Franklin West, Kurinji Pandiyan, Kelly Robbins and Steve Stice. Enrichment of Germ-Like Cells in Human Embryonic Stem Cells Cultures. GTECH Industry Partners Symposium; 2007 Oct: Atlanta, (GA).<br /> <br /> F.D. West, K. Pandiyan, K. Robbins and S. L. Stice. Human Embryonic Stem Cells Differentiate into Sperm and Eggs Precursor Cells. Georgia Life Sciences Summit; 2007 Oct: Atlanta, (GA). <br /> <br /> Franklin West, Kurinji Pandiyan, Kelly Robbins and Steve Stice. Enrichment of Germ-Like Cells in Human Embryonic Stem Cell Cultures. Society for the Study of Reproduction 40th Annual Meeting; 2007 Jul 21-25: San Antonio, (TX).<br /> <br /> West F., Pandiyan K., Robbins K. R., and Stice S. L. 2006. Differentiation of Primordial Germ Like Cells from Human Embryonic Stem Cells. The University of Georgia Biomedical and Health Science Institute 2006 Annual Retreat; 2006 Sept 22; Athens (GA).<br /> <br /> Bionaz, M., Monaco, E., Lima, A.S., Lane, S.J., Kim, D., Hurley, W.L., Wheeler, M.B. 2009. Internal control genes for quantitative PCR of porcine mesenchymal stem cells during adipogenic and osteogenic differentiation in vitro. Reproduction, Fertility and Development 21:188-189.<br /> <br /> Kim, D., Maki, A.J., Kong, H-J., Monaco, E., Bionaz, M., Hurley, W.L., Wheeler, M.B. 2009. Multilineage potential of porcine bone marrow and adipose-derived mesenchymal stem cells in 3-D alginate hydrogels. Reproduction, Fertility and Development 21:237.<br /> <br /> Monaco, E., Lima, A.S., Wilson, S.M, Lane, S.J., Bionaz, M., Hurley, W.L., Wheeler, M.B. 2009. Adipogenic differentiation in vitro of porcine adult mesenchymal stem cells. Reproduction, Fertility and Development 21:238.<br /> <br /> Wheeler, M.B., Hurley, W.L., Lane, S.J., Mosley, J., Bressner, G.E., Monaco, E., Wilson, S.M. 2009. Risk analysis of alpha-lactalbumin transgene transfer to non-transgenic control animals during rearing and breeding. Reproduction, Fertility and Development 21:252-253.<br /> <br /> M. Paczkowski, J. Fleming-Waddell, C.A. Bidwell, R.L. Krisher. (2009) Maternal Age Alters Fetal and Placental Development and Expression of Methylated Genes. Reprod Fertil Dev. 21(1):194 (abstr. 191).<br /> <br /> Silva, E., Krisher, R. (2009) Leptin and Glucose Influence Porcine Nuclear Maturation. Reprod Fertil Dev. 21(1):226 (abstr. 256).<br /> <br /> Yuan, Y., R.L. Krisher. 2009. Glutaredoxin pathway genes are differentially expressed in mature porcine oocytes with varying developmental potentials. Biology of Reproduction 81 (Suppl. 1): 370.<br /> <br /> Ohlweiler, L.U., Mezzalira, J.C., Monaco, E., Mezzalira, A., Bertolini, M., Wilson, S.M., Ringwelski, J., Krisher, R.L., Rund, L., Wheeler, M.B. (2010) Pregnancy outcome after oviductal transfer of zona free 1 cell stage porcine embryos produced by hand made cloning. Reproduction Fertility and Development 22(1):194 (abst. 72).<br /> <br /> Mezzalira, J.C., Ohlweiler, L.U., Massie, A., Monaco, E., Silva, E.P., Yuan, Y., Mezzalira, A., Bertolini, M, Krisher, R.L., Wheeler, M.B. (2010) Effects of cell type, pre-activation protocol and culture conditions on development of porcine hand made cloned embryos. Reproduction Fertility and Development 22(1):193 (abst. 69).<br /> <br /> Gentry Jr., G.T., J.A. Pitchford, M. Chiasson, L.R. Gentry, K.R. Bondioli, D.L. Thompson and R.A. Godke. 2009. Effect of circulating leptin levels at synchronization on subsequent fixed-timed artificial insemination pregnancy rates in crossbred beef heifers. Reprod. Fertil. Develop. 21:104.<br /> <br /> Guerrero, C.A., G.T. Gentry, J. Saenz, K.R. Bondioli, and R.A. Godke. 2009. Birth of calves after artificial insemination with cryopreserved bovine caudal epididymal spermatozoa harvested from a postmortem bull. Reprod. Fertil. Develop. 21:105.<br /> <br /> Saenz. J.R., C. Dumas, B.L. Dresser, M.C. Gómez, R.A. Godke and C.E. Pope. 2009. Survival and in vitro functionality of cat epididymal spermatozoa following cryopreservation in extenders with or without egg yolk. Reprod. Fertil. Develop. 21:138.<br /> <br /> Kish, S.L., J.A. Wilson, A.A. Picou, J.W. Lynn, R.A. Godke and K.R. Bondioli. 2009. Streptolysn O and permeabilized and mitotic extract treated fetal goat fibroblast for use in nuclear transfer. Reprod. Fertil. Develop. 21:119<br /> <br /> Picou, A.A., R.A. Maclean, B.L. Dresser, R.G. Godke and K.R. Bondioli. 2009. Isolation and characterization of bovine adipose-derived somatic stem cells. Reprod. Fertil. Develop. 21:239<br /> <br /> Pennington, P.M., .L.R. Gentry, C.E. Pope, R.A. Maclean. D.L. Paccamonti, B.L. Dresser, K.R. Bondioli, R.A. Godke and G. Wirtu. 2009. Characterization of the common eland (Taurotragus oryx) estrous cycle. Reprod. Fertil. Develop. 21:181<br /> <br /> Stout, M.A,, J.R. Saenz, G.T. Gentry, S.P. Leibo, K.R. Bondioli and R.A. Godke. Comparison of post-thaw values of epididymal sperm from White-tail deer that was frozen using glycerol or DMSO as the cryoprotectant. Reprod. Fertil. Develop. 21:183. <br /> <br /> Williams, K.J., R.A. Godke and K. Bondioli. 2009. Characterization of porcine adipose tissue-derived adult stem cell surface proteins. Reprod. Fertil. Develop. 21:241<br /> <br /> Wirtu, G., P.M. Pennington, C.E. Pope, R.A. MacLean, J. Mercado, J. Galiguis, D.L. Paccamonti, R.A. Godke and B.L. Dresser. 2009. Behavioral aspects of estrus in the common eland antelope, Taurotragus ory). Reprod. Fertil. Develop. Reprod. Fertil. Develop. 21:183<br /> <br /> Carwell, D, J.A. Pitchford, G. Gentry Jr., H. Blackburn, K.R. Bondioli and R.A. Godke. Beef cattle pregnancy rates following insemination with aged frozen Angus semen. Reprod. Fertil. Develop. 22: (In press).<br /> <br /> Saenz, J.R., C. Dumasb, B. L. Dresser, M.C. Gómez R.A. Godke, C.E. Pope. Effect of egg yolk concentration in test buffered extender on survival and in vitro functionality of domestic cat epididymal spermatozoa following cryopreservation. Reprod. Fertil. Develop. 22: (In press).<br /> <br /> Pennington, P.M.,C. E. Pope, R. A. MacLean, B. L. Dresser, K. R. Bondioli, R. A. Godke, and G. Wirtu. Cortisol levels in the common eland (Taurotragus oryx) during intensive and nonintensive handling periods. Reprod. Fertil. Develop. 22: (In press).<br /> <br /> Chiasson, M.K., J.A. Carter, K.R. Bondioli, R.A. Godke and G.T. Gentry. Laser-assisted zona pellucida hatching in frozen-thawed bovine embryos. Reprod. Fertil. Develop. 22: (In press).<br /> <br /> Lambe J., W. Forbes, B.M. Olcott, D E. Sanders, R A. Godke and G.T. Gentry. Effect of GnRH on fixed-tmed artificial insemination pregnancy rates of White-tail deer. Reprod. Fertil. Develop. 22: (In press).<br /> <br /> Schiffmacher, A., and C.L. Keefer. 2009. Deciphering induction of pluripotency in a bovine model. Biology Reproduction 81(S1): abstr. 656.<br /> <br /> Robertson, L. R., J. M. Feugang, N. Rodriguez-Osorio, A. Kaya and E. Memili 2009. MicroRNAs of Bull Spermatozoa. Annual Conference of the International Embryo Transfer Society (IETS) in San Diego, CA, January 2009. Journal of Reproduction, Fertility and Development. <br /> <br /> Brauer, V.M., J.R. Wiarda, R.A. Cederberg, and B.R. White. 2009. Transcriptional regulation of the porcine gonadotropin releasing hormone II receptor gene. Biol. Reprod. 81(Supplement 1):352.<br /> <br /> Cederberg, R.A., V.M. Brauer, J.G. Kerl, J.R. Wiarda, and B.R. White. 2009. Characterization of the porcine Type II GnRH receptor gene. Biol. Reprod. 81(Supplement 1):371.<br /> <br /> Lee, C., R.A. Cederberg, and B.R. White. 2009. Glucocorticoid responsiveness of the porcine GnRH receptor (GnRHR) gene is conferred by an element(s) located between -290/-270 bp of proximal promoter. Biol. Reprod. 81(Supplement 1):161.<br /> <br /> Hicks JE, Farin, CE. 2009. Candidate mRNAs regulating meiotic resumption in bovine cumulus-oocyte complexes. Reprod Fertil Dev. 21:221 (abst #247).<br /> Farin PW, Dowdall KM, Hicks JE, Farin CE, Whisnant CS. 2009. Subcutaneous administration of follicle stimulating hormone for superovulation of Holstein cows. Reprod Fertil Dev. 21:243-244 (abst #293).<br /> <br /> <br /> Miscellaneous publications (semi-technical/lay publications)<br /> <br /> Ata, M.A., K.P. Coffey, J.D. Caldwell, A. N. Young, D. Philipp, E. Kegley, G.F. Erf, D.S. Hubbell, III, and C.F. Rosenkrans, Jr. 2009. Immune function responses of spring-born calves weaned from wild-type or novel-endophyte infected tall fescue. AR Agr. Exp. Sta. Research Series 574:60-62.<br /> <br /> Banks, A., M. Looper, S. Reiter, L. Starkey, and C. Rosenkrans, Jr. 2009. Associations between heat shock protein 70 genetic polymorphisms and calving rates of Brahman-influenced cows. AR Agr. Exp. Sta. Research Series 574:10-13.<br /> <br /> Caldwell, J., K. Coffey, D. Kreider, D. Philipp, D. Hubbell, III, J. Tucker, A. Young, M. Looper, M. Popp, M. Savin, J. Jennings, and C. Rosenkrans, Jr . 2009. Post-weaning performance by spring and fall-born calves weaned from full access, limited access, or no access to wild-type endophyte-infected tall fescue pastures  year 1. AR Agr. Exp. Sta. Research Series 574:49-51.<br /> <br /> Caldwell, J., K. Coffey, D. Kreider, D. Philipp, D. Hubbell, III, J. Tucker, A. Young, M. Looper, M. Popp, M. Savin, J. Jennings, and C. Rosenkrans, Jr. 2009 Performance by spring and fall-calving cows grazing with full access, limited access,or no access to wild-type endophtye-infected fescue  2 year summary. AR Agr. Exp. Sta. Research Series 574:52-54.<br /> <br /> P. A. Delgado, P.A., T.D. Lester and R.W. Rorie. 209. Effect of a Low-Sodium, Choline-Based Diluent on Viability of Bovine Sperm Stored at Refrigerator Temperatures. Ark Agri. Exp. Sta., Res. Series 574:77- 79.<br /> <br /> Larson, M., M. Sales, S. Reiter, H. Brown, Jr., M. Brown, M. Looper, and C. Rosenkrans, Jr. 2009. Effects of forage type and CYP3A28 genotype on beef cow milk traits. AR Agr. Exp. Sta. Research Series 574:18-21.<br /> <br /> Looper, M. L., S. T. Reiter, D. M. Hallford, and C. F. Rosenkrans, Jr. 2009. Influence of body condition and forage type on endocrine factors and calving rate of postpartum beef cows. AR Agr. Exp. Sta. Research Series 574:89-91.<br /> <br /> Moubarak, A. S., S. Nabhan, Z. B. Johnson, M. L. Looper, and C. F. Rosenkrans, Jr. 2009. Metabolism of ergot alkaloids by steer liver cytochrome P450 3A. AR Agr. Exp. Sta. Research Series 574:29-31.<br /> <br /> Murphy, K., M. Sales, S. Reiter, H. Brown, Jr., M. Brown, M. Looper, and C. Rosenkrans, Jr. 2009. Polymorphisms in the regulatory region of bovine cytochrome P450. AR Agr. Exp. Sta. Research Series 574:14-17.<br /> <br /> Starnes, A. R., A. H. Brown, Jr., Z. B. Johnson, J. G. Powell, J. L. Reynolds, S. T. Reiter, M. L. Looper, and C. F. Rosenkrans, Jr. 2009. Relationships between prolactin promoter polymorphisms and Angus calf temperament scores and fecal egg counts. AR Agr. Exp. Sta. Research Series 574:22-24.<br /> Chiasson, M. K., J. Carter, K. R. Bondioli, R.A. Godke and G.T. Gentry. 2009. The use of laser-assisted embryo hatching prior to the transfer of frozen-thawed in vivo-produced beef cattle embryos. LSU AgCenter Beef Cattle/Forage Res. Rep. (In press)<br /> <br /> Gentry, Jr., G.T., J.A. Roberts and R.A. Godke. 2009. Influence of age, body weight and body condition on plasma leptin concentrations in beef cattle. LSU AgCenter Beef Cattle/Forage Res. Rep. (In press)<br /> <br /> Regular speaker at the Farm & Family program for Mississippi Public Radio/National Public Radio. Topics included: Undergraduate Research in Bioinformatics, Graduate Research in Reproductive Biology<br /> <br />

Impact Statements

1. Reducing the length of estrous synchronization protocols could increase their appeal and use by livestock producers. Improving fertility during hot weather breeding would improve the profitability of cow-calf operations. Genetic markers with proven association to cattle fertility could be used to increase the profitability of cow-calf enterprises.
2. Our research with microRNAs may lead to greatly improved understanding of the processes of oocyte maturation, fertilization, and embryonic development. This could lead to improved methods of in vitro embryo production. The profound inhibitory effect of progesterone on GVBD in vivo is of great importance for future experiments and for numerous biotechnological interventions. Our refinements in vitrification procedures for bovine embryos are being used as a foundation to make this procedure more efficacious.
3. We compared bovine fetuses produced by the in vitro fertilization technology to those of conventional breeding. We found few differences in the global expression profiles between the two types of fetuses at Days 90 and 180 in two important organs, liver and placenta. These data are important references to farmers who desire embryos of selected sex produced in the laboratory, and will enhance the general acceptance of embryos produced in vitro. The transfer of bovine embryos of selected sex has the potential to improve farmers profit up to 50%.
4. Development of embryonic stem cell derived germ cell lines provides an in vitro system to study germ cell development.
5. The identification of the important role of the glutaredoxin pathway in oocytes as a critical component of oocyte development competence provides researchers with the opportunity to manipulate culture conditions to alter oocyte competence during in vitro maturation.
6. The demonstration that semen stored in LN for greater than 40 years remained viable and was able to produce pregnancies provides justification for storage of genetic material in the form of cryopreserved gametes to guard against unforeseen lose of genetic variability in livestock populations. The lack of correlation between temperament measures and pregnancy rates from subsequent AI suggests that additional methods of measuring stress in livestock are needed. Farming of deer in the US is a potential niche enterprise for agricultural producers. The studies conducted on fixed time AI in White-tailed deer provide incremental knowledge to increase the efficiency of such operations. Some commercial services have suggested that laser assisted hatching can be used to increase pregnancy rates following transfer of frozen-thawed bovine embryos. Our studies do not validate these claims.
7. 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).
8. Identified microRNAs and proteins transcripts in bull spermatozoa that can be used to better understand biology of spermatozoa and improve bull fertility. In addition, we identified comparative functional genomics of mammalian DNA Methyl Transferases (DNMTs) which can lead to better understanding of epigenetic control of mammalian reproduction and development.
9. 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. 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. Manipulation of the interaction between GnRH and its receptor in preimplantation embryos could culminate in novel contraceptive procedures. Determination of biotin effects on oocyte development could lead to establishment of appropriate levels of biotin supplementation for women.
10. Development of methods to identify and isolate gonocytes and spermatogonia will aid in improving understanding of the regulation of these stem cell types. Such information will support development of novel methods for creating transgenic animals. Identification of simple and effective methods for superovulation of cows will benefit embryo transfer practitioners and producers. Identification of fertility-related characteristics of stallion spermatozoa will aid in indentifying semen ejaculates that may be better suited for successful cryopreservation. Understanding the regulation of fetal development will lead to methods for identifying normal and abnormal growth patterns of fetuses resulting from the transfer of embryos produced through assisted reproductive technologies as well as from pregnancies in which maternal nutrition is manipulated. Understanding the regulation of fetal growth and development will lead to improvements in systems used for producing embryos in vitro.
11. The information presented in this report will have the most application to the basic science of early embryo development. In study 1 we have shown that aberrant reprogramming of histone modifications in bovine scNT does occur and can contribute to the low success rate of scNT embryos. In study 2 we showed that by using the electrophorectic mobility shift assay in conjunction with mass spectrometry analysis we can identify the functionally relevant DNA binding proteins which provides additional insight into the transcription factors involved in the gene regulation of the bovine Oct4 and Sox2 genes. In study 3 we showed that by activating NT embryos in a more biological manner we can ultimately improve the efficiency of the NT process as well as identifying messages associated with activation.
12. We observed that the gene promoter region is acted upon by different DNA binding proteins depending on its methylation status, and that the retention of a hyper-methylation signature could lead to the premature down-regulation of the genes Nanog and POU5F1. In study 5 we showed that the enucleation of bovine oocytes in sucrose+CB or demecolcine+CB solutions are inefficient. CB does not appear to be required to enucleate bovine oocytes, and that demecolcine-assisted enucleation is just as efficient when not exposing oocytes to CB. In summary, data obtained from the 5 studies will eventually contribute to new and improved methods that will enhance the rate of success using NT technologies and lead to a better understanding of the various biochemical pathways in early embryo development.
13. Understanding the functions of this novel oocyte-specific importin ± 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.
14. We have demonstrated an increase in the production of homologous recombinants relative to non-homologous recombinants in the test cell line, which suggests this method may ultimately be of practical value in increasing the ability to genetically engineer embryonic stem cells, primordial germ cells, or somatic cells suitable for nuclear transfer-based cloning, thus enhancing the availability of transgenic livestock for use in agriculture.
15. We aimed to improve the technology for genetically modifying livestock by employing frozen oocytes for the purpose of generating embryonic stem cells. Embryonic stem cells are important in genetic modification for disease resistance, special production trait generation and pharmaceutical protein production. The demonstration that frozen and immature oocytes can be used efficiently for stem cell generation will greatly reduce the cost of genetic modifications. Additionally, we studied the imprinting status of IGF2R in cloned animals. Understanding gene expression abnormalities in cloned animals will help improve the efficiency of the technology and safety of cloned offspring.
16. Porcine induced pluripotent stem cells will increase the ability to perform complex genetic manipulations in creating transgenic livestock.
17. The production of a-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.
18. 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.
19. Somatic cell nuclear transfer represent a powerful tool for the production of genetically modified livestock animals. The study of transfection procedures and isolation and culture of alternative donor cells especially somatic stem cells will likely increase the efficiency of this technology. The characterization of somatic stem cells isolated from fat of pigs and cattle will provide valuable information concerning the utilization of these cells for nuclear transfer procedures. Induced pluripotency is an important new technology for creating pluripotent cell lines. These cells have important implications for technologies such as tissue regeneration and nuclear transfer in both veterinary and human medicine. Little is known about this process in livestock species and the expression of critical pluriopotency- associated genes is an important first step in the development of this technology.
20. Identified transcriptome profiles of bovine embryos generated by IVF and somatic cell nuclear transfer. These functional genomics markers can be used to improve the cloning efficiency.

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Report Information

Participants

Milan Shipka (AK)
Charles Rosenkrans (AR)
George Seidel (CO)
Peter Farin (NC)
Carol Keefer (MD)
Ken Bondioli (LA)
Erdogan Memili (MS)
Brett White (NE)
Kent Bondioli (LA)
Matt Wheeler (IL)
Mark Mirando (USDA)
Pablo Ross (CA)
Kurt Youngs (IA)
Jianbo Yao (WV)

Brief Summary of Minutes

Accomplishments

Publications

Impact Statements

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Report Information

Participants

Shipka,Milan (mpshipka@alaska.edu) Administrative Advisor, University of Alaska;
Rorie, Rick (rrorie@uark.edu) University of Arkansas;
Berger, Patricia (tberger@ucdavis.edu) University of California, Davis;
Ross, Pablo (pross@ucdavis.edu) University of California, Davis;
Seidel, George (George.Seidel@ColoState.EDU) Colorado State University;
Youngs, Curt (cryoungs@iastate.edu) Iowa State University;
Bondioli, Kenneth (kbondioli@agcenter.lsu.edu) Louisiana State University;
Keefer, Carol (ckeefer@umd.edu) University of Maryland;
Memili, Erdogan (em149@ads.msstate.edu) Mississippi State University;
White, Brett (bwhite2@unl.edu) University of Nebraska
Isom, S Clay (clay.isom@usu.edu) Utah State University

Brief Summary of Minutes

Minutes of the W-2171 Multi-State Project Meeting
January 6, 2012
Renaissance Glendale Hotel and Spa
Phoenix, Arizona

Attendees (Station):
Milan Shipka (AK)
Rick Rorie (AR)
Patricia Berger (CA)
Pablo Ross (CA)
George Seidel (CO)
Curt Youngs (IA)
Ken Bondioli (LA)
Carol Keefer (MD)
Erdogan Memili (MS)
Brett White (NE)
Clay Isom (UT)
Mark Mirando USDA by conference call

The meeting was called to order by the project Chair Erdogan Memili at 1:00 PM.
Following correction of several name spellings the minutes of the previous meeting were approved.

Attending stations presented brief summaries of the reports included in the distributed Technical Report.

Dr. Mark Mirando joined the meeting through teleconference and reviewed the handouts concerning the National Institute of Food and Agriculture (NIFA) that had been distributed to the members.
The first Director of NIFA resigned in May 2011 and Dr. Chavonda Jacobs-Young is serving as Acting Director. Interviews for a replacement have been conducted and results sent to the White House.

Current priorities for funding will remain the same or 2012.
It was summarized that NIFIA faired fairly good in the 2012 appropriated budget. Funding for the AFRI competitive programs remained essentially the same compared to last year.
2012 RFAs for Global Food Security, Food Safety, Sustainable Bioenergy and the Fellowships Grant Programs have been released. The RFA for the Foundation Program is expected to be released in March 2012. Letters of intent would be due approximately 6 weeks after the release and applications would be due approximately 3 months after the release.

The informal group of Rosenkrans, Memili, Ross and Bondioli reported on their efforts to create a collaboration from within the group to apply for an anticipated large integrated grant for reproduction. This group met after the previous meeting and on several occasions throughout the year by telephone or email. An outline of potential topics for such an application was generated and distributed to the project members. During these discussions it was anticipated that the 2012 Food Security RFA would include an opportunity to apply for a planning grant to assemble a team and develop a proposal. When the 2012 RFA was released there was not a provision for a planning grant but called for full proposals addressing Translational Genomics for Improved Fertility in Animals. The group decided that there was not sufficient time to assemble a competitive team and proposal for this opportunity.

Additional opportunities for collaborations within the project membership were discussed and encouraged. One opportunity that was discussed includes collaboration between groups with experience and availability of deep sequencing technology and groups with physiological models where this technology would be appropriate. Pablo Ross (CA) and Erdogan Memili (MS) reported that they had established such collaboration.
Trish Berger (CA) suggested the possibility of a collaboration to establish a proposal addressing the Climate Change Priority. Such a proposal could address heat stress and mitigating this effect of climate change through genetics and Biotechnology.

The meeting next year cannot be associated with the annual IETS meeting because this meeting is outside of the US. It was agreed that the meeting would be held at Utah State University with a target date of late February. It was tentatively planned that the meeting would be 1.5- 2 days over Friday-Saturday and include a keynote speaker and short presentations from the attending stations.

Pablo Ross was unanimously selected as the incoming project secretary.

The meeting was adjourned at 5:00 PM.

Respectfully submitted.

Ken Bondioli
2011-2012 Secretary

Accomplishments

ACCOMPLISHMENTS.<br /> Objective 1:<br /> Understand the biology and underlying mechanisms of gamete development, fertilization, and embryogenesis.<br /> Osteopontin is a fertility-associated protein found in higher concentrations in the seminal plasma of bulls that produce higher conception rates. Bulls were evaluated for polymorphisms within the osteopontin gene promoter as potential markers for fertility.<br /> A modified long-term progestin-Select Synch estrous synchronization protocol was developed and evaluated for use in for beef cattle.<br /> Semen collected during hot weather has poor viability after cryopreservation. A study evaluated the effects of organic and inorganic trace mineral supplementation on semen collected and cryopreserved during hot weather.<br /> <br /> Data suggest porcine oocyte fertilizability is very sensitive to short-term preovulatory heat stress. Together with previous observations on detrimental effects of slightly elevated temperature on in vitro maturation, these data suggest elevated ovarian temperature contributes to the reduced oocyte quality. RNASeq analysis in single blastocyst was successfully achieved, allowing for global gene expression profiling, SNP discovery, allelic specific expression analysis, and of characterization of unannotated bovine genes.<br /> <br /> MicroRNAs clearly are present in bovine oocytes and preimplantation embryos, and furthermore likely have important regulatory functions in these cells. We also demonstrated that the antioxidant cysteamine could counteract the negative effects of culturing embryos in 5% CO2 in air relative to using lower oxygen concentrations. We showed that vitrification of in vivo-produced bovine embryos could be accomplished satisfactorily in 0.25-ml plastic straws suitable for direct transfer, although results were not satisfactory for in vitro-produced embryos.<br /> <br /> We collected liver and placental tissue samples from in vitro produced and in vivo control bovine fetuses at days 90 and 180 of gestation. We used a bovine 13K oligonucleotide microarray to investigate the transcriptional profiles in both tissues of IVP fetuses, and compared them with those of their age-matched in vivo counterparts.<br /> <br /> We cultured mouse oocytes under microgravity condition simulated by NASAs rotary cell culture system, examined the maturation rate and observed the spindle morphology (organization of cytoskeleton) during the mouse oocytes meiotic maturation.<br /> <br /> The presence of osteopontin (SPP1) mRNA in oocytes and cumulus cells and the larger mRNA abundance before maturation suggests a role of this protein prior maturation of oocytes.<br /> <br /> Microfluidic devices provide the opportunity to culture oocytes and embryos individually, with development equal to that of standard culture drops. These results demonstrate that this microfluidic system provides an efficient way to successfully mature oocytes individually.<br /> The post-thaw motility of stallion spermatozoa was higher in 5% egg yolk than 20% egg yolk, but jack spermatozoa exhibited higher post-thaw motility when cryopreserved in high than low egg yolk. For both species, post-thaw motility was higher for spermatozoa cryopreserved in 60 mM ²-CD than 0 mM. Spermatozoa cryopreserved in low egg yolk exhibited better CASA parameters than those cryopreserved in high egg yolk for both species, and the use of 60 mM ²-CD likewise resulted in poorer post-thaw CASA parameters in both species. All post-thaw treatments designed to induce acrosome reaction caused a decrease in viability compared with the control. Post-thaw viability of stallion spermatozoa remained relatively constant over time whereas viability of jack spermatozoa decreased by nearly 30% from 0 to 90 min. Post-thaw treatment of stallion sperm cells with b-CD increased sperm acrosome reaction from 5% (90 min) to 22% (90 min). The percentage of acrosome-reacted control jack sperm cells went from 11% (0 min) to 31% (90 min), whereas those treated with b-CD went from 12% (0 min) to (85% (90 min). <br /> <br /> It was demonstrated that bovine epididymal sperm had lower levels of cryopreservation induced acrosome reaction but similar levels of cryopreservation induced capacitation compared to ejaculated sperm and that epididymal sperm were less dependent upon the capacitation agent, heparin to support in vitro fertilization. <br /> <br /> It was demonstrated that cat epididymal sperm could be frozen in a completely defined extender and that addition of cryoprotectant after gradual cooling was beneficial to post-thaw survival. <br /> <br /> Increasing fertility in dairy cattle is an important goal. Male infertility represents a part of the overall infertility in dairy cattle, and can be partitioned into compensatory and non-compensatory components where compensatory refers to infertility which can be overcome by increasing sperm number and non-compensatory infertility represents the remainder, presumably due to molecular and genomic defects. Through estimation of single nucleotide polymorphism (SNP) association with non-compensatory bull fertility, identifying regions of the genome influential to this trait is possible. Use of this information in selection can allow for an increase in cattle fertility resulting in economic benefits. In this study, high density SNP genotypes and non-compensatory fertility data from 795 Holstein sires were used to examine SNP association with fertility. A Bayes B analysis was performed to develop information for genomic selection and to identify genomic regions associated with non-compensatory fertility. A cross-validation approach was used to assess effectiveness of the models within the original set of 795 bulls. Correlations of predicted and observed fertility values were approximately 0.145 in cross-validation. <br /> <br /> Fertility is one of the most economically important traits controlling animal reproduction. Despite significant economic impact, there are no reliable markers to predict semen quality. The objective of this study was to identify spermatozoal proteins associated with bull fertility, ability of the sperm to fertilize the oocyte and support embryonic development. To accomplish our objectives, we isolated total spermatozoal proteins from four Angus bulls with different fertility records. Next, differentially expressed proteins were determined using two dimensional in gel electrophoresis (2D-DIGE) followed by sequencing of the most differentially expressed proteins. Immunoblotting experiments were conducted to confirm expression of key proteins detected by 2D-DIGE. Our results from 2D-DIGE experiments showed around 2000 detectable spermatozoal proteins. Of these comprehensive lists of proteins, we identified 80 of the proteins with highest differentially expression in spermatozoa from high and low fertility bulls. Diverse sets of differentially proteins known to play roles in sperm motility, metabolism and cell morphology were identified. These proteins included Outer dense fiber of sperm tails 2 (ODF2) and Manganous superoxide dismutase (MnSOD), Heat Shock Protein, Tubulins, Alpha enolase, and Acrosomal vesicle protein-1. Expression profiles of ODF2 and MnSOD were confirmed using immunoblotting. The findings are significant because they help us understand fundamental biology of the male gamete and early development. In addition, the identified proteins can be used as molecular markers to predict bull fertility, an economically important trait.<br /> <br /> Transgenic pigs were produced expressing a spermatogonial marker gene.<br /> <br /> The full-length bAIRN ncRNA has been characterized as a transcript whose length is approximately 65kb that is transcribed from a promoter located approximately 440bp upstream of DMR2 on the IGF2R antisense strand. <br /> Analysis of serum progesterone levels at the time of embryo transfer in relation to conceptus development at Day 17 of gestation was performed and was found to vary based on embryo production source (in vivo vs. in vitro).<br /> <br /> The results of immunohistochemistry indicate a co-localization of FAK protein clusters that involves specific oocyte membrane integrin proteins during fertilization. These findings illustrate the formation of focal adhesions via colocalization of signalling molecules with integrin ² subunits in bovine oocytes and further support the hypothesis that induction of signalling pathways is initiated with sperm-oocyte binding in the bovine.<br /> <br /> These data illustrate the formation of focal adhesions indicated by colocalization of ±V and FAK at the site of spermatozoa binding to the vitelline membrane. These results provide evidence that spermatozoa interact with integrins, known outside-in signaling complexes, at the level of the vitelline membrane in bovine oocytes. These events could serve as the initiation of signaling cascades associated with bovine oocyte activation and require further investigation.<br /> <br /> A defined culture medium, USU6 was compared to three other successful undefined embryo culture media/conditions: mSOFaaci, CR1aa, and CR1aa with bovine cumulus cell co-culture. There was no significant difference among cleavage rates for USU6 (88.5%), CR1aa with co-culture (85.1%), CR1aa (82.2%), and mSOFaaci (76.4%). The day 7 blastocyst rates for CR1aa with co-culture (35.7%) was not different than CR1aa alone (31.1%) but was significantly higher than USU6 (27.3%) and mSOFaaci (7.5%). The day 9 hatched blastocyst rates were significantly higher for USU6 (20.0%) and CR1aa with co-culture (17.4%) compared to CR1aa alone (2.6%) and mSOFaaci (0.0%). These data indicate a defined media, USU6 is equivalent or better for bovine IVF embryo development in vitro to the hatched blastocyst stage compared to other common undefined embryo culture media/conditions.<br /> <br /> The results of the study are summarized as following: 1. Histone H3K9me3 and H3K9/18ace levels were very prominent in GVs. 2. H3K9/18ace levels in fibroblast nuclei in GV oocyte cytoplasts were enhanced 2 h post fusion, and then declined by 4-6 h post fusion. 3. H3K9me3 level of fibroblast nuclei in GV oocyte cytoplasts was enhanced 4 h post fusion, and then declined or restored to original level 6 h post fusion. 4. H3K9me3 level of fibroblast nuclei in non-enucleated GV oocyte cytoplasm was similar to that of the changes in enucleated GV oocytes. 5. GV oocyte extract-treatment reduced the H3K9me3 level of fibroblast nuclei, especially when treated cells were cultured overnight. <br /> <br /> We have performed co-immunoprecipitation experiments to evaluate the interaction of importin ±8 with oocyte-specific nuclear factors (such as Oct-4 and Nobox). Hela cells were co-transfected with constructs expressing Nobox-flag and importin ±8-myc. Whole cell lysates were immunoprecipitated with anti-flag antibody and analyzed by western blotting using anti-myc antibody. Our data indicate that importin ±8 interacts with Nobox, an oocyte-specific transcription factor important for folliculogenesis and early embryonic development.<br /> <br /> <br /> Objective 2:<br /> Refine methods for production of genetically modified animals to improve livestock production efficiency.<br /> cAMP levels during sheep oocyte in vitro maturation were characterized and reported.<br /> The levels of cAMP in sheep oocytes were successfully elevated by treatment with forskolin and IBMX, resulting in delayed oocyte maturation without compromising the rate of oocytes reaching the MII stage; however, no difference in parthenogenetic blastocyst production was observed between treated and control oocytes.<br /> <br /> We investigated the possibility of replacing reprogramming viral factors with small molecules for the purpose of induce pluripotency or trans-differentiation. To this end, we evaluated the effects of carcinogens at nongenotoxic levels on somatic cells by identifying 16 candidate chemicals through biology-oriented in silico high-throughput screening, and established a reprogramming protocol of 16-day treatment.<br /> <br /> We evaluated the reprogramming potency of purified mouse Klf4 protein linked with the cell-penetrating peptide of HIV transactivator of transcription (TAT) or Drosophila Penetratin protein at the N- or C-terminus in conjunction with the other 3 pluripotent factors (Oct3, Sox2 and C-myc) carried by retrovirus.<br /> <br /> We have secured a $1.5 Million grant from the Bill and Melinda Gates Foundation to develop naturally resistance chickens.<br /> <br /> Overall data uncovered a high similarity at the transcriptional level between ADSC and BMSC both prior differentiation and during differentiation. Those data support ADSC being particularly similar to BMSC.<br /> <br /> Results indicate that CD34+ cells were similar to CD34- in the in vitro osteogenic differentiation but have lower in vivo healing capacity. The uADSC have a greater healing capacity than sorted cells. Overall our data indicate that the sorting of ADSC cells is not of clinical relevance.<br /> <br /> Circular defects in the posterior region of the pig mandible with diameters equal or greater than 25 mm will result in limited healing without additional medical intervention and can be termed critical-sized defects. This porcine model will allow for the rapid development and testing of new approaches for the repair of damaged bone, which is especially prevalent in the craniofacial area.<br /> <br /> The narrower microwell system appeared to have promoted better in vitro development than the conventional and the DMPS-based microwell systems, with no impact on subsequent in vivo development. However, the IVC in the WOW-PDMS system supported reasonable rates of development, in accordance with the current literature.<br /> <br /> It was demonstrated that treatment of bovine adipose stem cells with the epigenetic modifying agents zebularine and valproic acid for 5 days resulted in increased expression of the pluripotency associated transcription factors Oct 4 and Nanog but the transcription factor Sox 2 was not affected. <br /> <br /> It was demonstrated that exposure of bovine fetal fibroblasts to cellular extracts from bovine adipose stem cells resulted in increased expression of the pluripotency associated transcription factor Sox 2. Exposure of fetal fibroblasts to extracts for human embryonic stem cells had no affect on any of the pluripotency associated transcription factors.<br />

Publications

Impact Statements

1. Haplotypes based on polymorphisms within the promoter region of the bovine OPN may prove useful in selection of bulls with improved fertility. A long-term progestin-select synch estrous synchronization protocol was developed that allows for use of different progestin sources and reduces the overall treatment time by 10 days without loss of effectiveness.
2. Progress to date suggests short-term cooling of sows during the weaning to fertilization interval may be an economic means to increase pork productivity per unit input.
3. RNASeq analysis of single embryos enables the characterization of genetic mechanisms of preimplantation embryo development which will lead to a better understanding of embryogenesis and gamete development. MicroRNA studies are in the basic research category, but show great promise for eventual practical application in addition to utility for probing fundamental mechanisms regulating oocyte maturation and early development of embryos.
4. The ability to use a simple chemical cysteamine, as a substitute for expensive and cumbersome systems to lower oxygen concentrations from those found in air could have utility in numerous situations for keeping embryos viable. Coupling vitrification of embryos with direct embryo transfer is a most useful option for the embryo transfer industry.
5. We demonstrated the expression profiles of liver and placenta from early and mid-gestation do not differ significant between in vitro and in vivo produced bovine fetuses. These data would serve to enhance the application of the IVF technology both in humans and in agriculture.
6. The identification of the important role of the osteopontin in oocytes as a critical component of oocyte development competence provides researchers with the opportunity to manipulate culture conditions to alter oocyte competence during in vitro maturation.
7. Our results impact the equine industry because they indicate response of stallion sperm to cryopreservation differs from that for jacks. Protocols developed for the stallion are not appropriate for use with jacks.
8. The study of differences between epididymal and ejaculated sperm response to capacitation and cryopreservation will aid in the development of in vitro fertilization methods for epididymal sperm for bovine and other species.
9. The ability to freeze felid sperm in a defined extender without potentially contaminated animal proteins will aid in preservation of genetic resources for these species.
10. Identified SNP markers shed light into fundamental biology and molecular genetic mechanisms regulating male fertility. In addition, these markers can be used to predict bull fertility that will translate into significant cost savings both for producers and for consumers. Similarly, identified sperm proteins help us better understand sperm physiology, fertilization and early embryonic development. Furthermore, these markers can be used to determine fertility of bulls.
11. Development of methods to identify and isolate gonocytes and spermatogonia will aid in improving understanding of the regulation of these stem cell types. Such information will support development of novel methods for creating transgenic animals.
12. Understanding the regulation of conceptus and fetal development will lead to methods for identifying normal and abnormal growth patterns of fetuses resulting from the transfer of embryos produced through assisted reproductive technologies as well as from pregnancies in which maternal nutrition is manipulated. Understanding the regulation of conceptus and fetal growth will lead to improvements in systems used for producing embryos in vitro.
13. Importin ±8 is required for oocyte meiotic maturation and early embryogenesis, and interacts with known oocyte-specific nuclear factors. Understanding the functions of this oocyte-specific importin ± 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.
14. Improvements in sheep in vitro oocyte maturation conditions will result in improved developmental capacity of in vitro produced embryos, which is an essential part of animal transgenesis.
15. We developed a strategy to screen small molecule inventory and to reprogram somatic cells for pluripotency induction and trans-differentiation induction. This method is of importance in the generation of pluripotent stem cells in both humans and domestic animals without the changes in the genomic DNA. Genetic modification of animals can be generated without the concern of foreign gene integration. The method will have a major impact in cellular reprogramming.
16. The development of germline competent porcine iPSCs that do not produce tumors in chimeric animals presents an attractive and powerful translational model to study the efficacy and safety of stem cell therapies and perhaps to efficiently produce complex transgenic animals. In addition the chimera competent quail iPSCs and in vitro differentiated cells offer insight into the conserved nature of reprogramming and genetic tools that were only previously available in mammals. This iPSC technology will be used in the BMGF grant to develop disease resistant livestock.
17. 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 as well as the development of large livestock for biomedical applications.
18. The demonstration of factors and/or conditions resulting in upregulation of pluripotency associated transcription factors in bovine somatic cells will likely aid in the development of bovine induced pluripotent stem cells. These cells will be useful as donor cells in nuclear transfer for the production and propagation of genetically modified livestock.

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Report Information

Participants

Rosenkrans, Charles - U of Arkansas
Berger, Trish - U of California Davis
Winger, Quinton - Colorado State U
Tian, Cindy - U of Connecticut -
Stice, Steven U of Georgia
Bondioli, Kenneth - Louisiana State U -
Rivera, Rocio U of Missouri
Schmidt, Ed - Montana State U
White, Bret - U of Nebraska Lincoln -
Bunch, Thomas - Utah State U
Ison, Clay - Utah State U
Polejaeva, Irena - Utah State U
Wang, Zhongde - Utah State U
White, Kenneth - Utah State U
Zobell, Dale - Utah State U
Shipka, Milan - U of Alaska Fairbanks

Brief Summary of Minutes

Meeting convened by Dr. Ken Bondioli (LSU) at 1 PM on February 14, 2013.
Dr. Mark Mirando was unable to join by telephone due to illness. Initial discussion included issues about loss of intellectual capital, e.g. limited appointment of new faculty with retirements and funding. This was followed by station reports (Arkansas-Rosenkrans, California-Berger, Colorado- Winger, Connecticut-Tian, Georgia-Stice, Louisiana-Bondioli, Missouri-Rivera, Montana-Schmidt, and Nebraska-White). Utah (Bunch, Isom, Polejaeva, Wang, and Aston) deferred most of their discussion to the following day. Lively discussion of research followed presentations with collaborations potentially developing from these exchanges and continued into dinner and lunch.
The meeting was reconvened at 8:07 on Friday, February 15. Following the presentations from Utah, and comments from Dr. Milan Shipka, liaison to USDA, on impact statements, the group as a whole discussed the wording of our impact statements. Following extensive discussion of these drafts, a copy was to be submitted to all members by email after the meeting. The group then proceeded to the topic of the rewrite, due January 15, 2014. Since this deadline for submission occurs during the IETS meeting in 2014, the decision was made to have the W2171 meeting on Friday, January 10, before the IETS meeting. The rewrite will need to be complete before our next meeting. Pairing of a more experienced and a relatively new member to the group was made for each objective. For Objective 1 related to efficieny of gamete and embryo development, Charles Rosenkrans and Clay Isom agreed to be the writing team. For objective 2 related to efficiency of transgenic animal production, Matt Wheeler (not present) and Rocio Rivera were appointed as the writing team. Clay Isom was nominated, seconded, and voted in as Secretary of the group. The group requested that Ken Bondioli and Milan Shipka on behalf of the group write a letter thanking Clay Isom for the much appreciated organization for this meeting. The meeting was adjourned and members shared lunch at the union and several participated in afternoon tours of Utah State facilities generously provided by the Utah members.

Accomplishments

PROGRESS OF WORK AND PRINCIPAL ACCOMPLISHMENTS:<br /> OBJECTIVE 1<br /> Understand the biology and underlying mechanisms of gamete<br /> development, fertilization, and embryogenesis<br /> OBJECTIVE 2<br /> Refine methods for production of genetically modified animals to improve<br /> livestock production efficiency<br /> ACCOMPLISHMENTS:<br /> 1) The success of an artificial insemination program utilizing sorted semen might be<br /> improved by delaying insemination beyond the typical 10 to 14 hours after onset<br /> of estrus that is currently used for conventional, unsorted semen.<br /> 2)Managing beef cows in such a manner that they are not consuming toxic forage<br /> during the breeding season and have adequate body condition during pregnancy<br /> will result in heifers that have greater fecundity.<br /> 3) Genotyping bulls based on mutations associated with the heat shock protein 70<br /> gene may lead to dairy cows with improved reproductive rates.<br /> 4) Preliminary sow breeding trials build upon previous basic research and<br /> suggest very short term cooling in the weaning to breeding interval may alleviate<br /> summer subfertility in swine.<br /> 5) RNA-Seq analysis of single bovine oocytes enabled quantification of<br /> polyadenylated transcript abundance and subsequent UTR analysis providing<br /> insight into the mechanisms facilitating posttranscriptional regulation during<br /> oocyte maturation and pre-implantation embryogenesis. RNA-seq analysis of<br /> single equine ICM and TE provides a comprehensive characterization of genes<br /> expressed in these compartments of the blastocyst.<br /> 6) Characterization information on goat MSC, and show that<br /> significant differences can exist between MSC isolated from different tissues and<br /> from within the same tissue.<br /> 7) Determined the expression patterns of Lin28 in the sheep placenta. <br /> 8) Timing of expression suggest unique roles of Lin28A and Lin28B.<br /> Identified cell-secreted vesicles in ovarian follicular fluid and demonstrated their<br /> uptake by granulosa cells.<br /> 9) Identified ACVR1 protein in exosomes isolated from follicular fluid of equine<br /> ovaries.<br /> 10) Identified gross morphological, histological and molecular abnormalities in<br /> placentomes from prenatal androgenization in ewes.<br /> 11) Identified gene expression alterations in placentomes, of prenatal<br /> androgenization of ewes, in genes that regulate epigenetic DNA modifications<br /> like histone demethylase KDM4D that could contribute to environmental<br /> programming of the placenta.<br /> 12) Detection of Lin28A and Lin28B in sheep trophectoderm cells suggests a<br /> pluripotent stem cell population exists in sheep placenta.<br /> 13) Identified four reprogramming factors will not<br /> reprogram somatic cells without LIF. LIF in fact actively reprogram the<br /> epigenetic modifications of the somatic cells in addition to maintaining the<br /> pluripotency of already reprogrammed cells.<br /> 14) With the understanding on the role of LIF in nuclear reprogramming, we now<br /> have more understanding of the reprogramming mechanism. The knowledge<br /> obtained from our study will bring us closer to producing safer and more<br /> completely reprogrammed cells using chemical approaches.<br /> 15) Although avian ESC and primordial germ cell lines have been established they<br /> have not been used in gene targeting studies, mostly likely because they have<br /> not been clonally isolated nor are they highly proliferative in extended cultures.<br /> 16) Overall data indicated that the transcriptomes of the two MSC are similar<br /> under both conditions. In addition, despite the limited differentially expressed<br /> genes between the two MSC, the enrichment of several functions/pathways<br /> might indicate differences in therapeutic application.<br /> 17) It was demonstrated that cryopreserved ejaculated and epididymal bovine<br /> sperm could be used within the same in vitro fertilization protocol. The inclusion<br /> of the capacitating agent heparin increased blastocyst production for both<br /> epididymal and ejaculated sperm.<br /> 18) Studies demonstrated that unlike for red deer, increased bicarbonate and Ca2+<br /> are not necessary to induce capacitation of white-tailed deer epididymal<br /> spermatozoa. Although sheep serum did not affect the number of live acrosomeintact<br /> spermatozoa, its presence did alter the lipid architecture of the sperm<br /> plasma membrane.<br /> 19)Initial parameters for the utilization of synthetic mRNA for induction of bovine<br /> pluripotent stem cells were determined and it was demonstrated that transfection<br /> of bovine fetal fibroblasts with synthetic mRNA encoding human Oct 4 resulted in<br /> the stimulation of endogenous bovine Oct 4.<br /> 20) A pluripotency reporter gene consisting of the bovine NANOG promoter fused<br /> with a nuclear localized green fluorescent protein (GFP) gene was demonstrated<br /> to faithfully reflect regulatory mechanisms controlling bovine NANOG expression<br /> in embryos and following iPSC reprogramming.<br /> 21) The abundance of<br /> miRNAs in the spermatozoa and the differential expression in sperm from high<br /> vs. low fertility bulls suggests that the miRNAs possibly play important functions<br /> in the regulating mechanisms of bovine spermatozoa Identification of specific<br /> microRNAs expressed in spermatozoa of bulls with different fertility phenotypes<br /> will help better understand mammalian gametogenesis and early development.<br /> 22) It was demonstrated that misregulation of the KvDMR1 imprinted<br /> cluster occurs in LOS.<br /> 23) It was demonstrated that culturing bovine embryos on a collagen<br /> matrix improves development to the blastocyst stage.<br /> 24) Carried out experiments to determine the effect of adipocytokines on the<br /> expression maternal effect genes associated with cumulus cell maturation in<br /> cumulus-oocyte complexes isolated from obese (Lethal Yellow, high fat fed<br /> C57BL/6) and normal-weight (C57BL/6) female mice after ovulation.<br /> 25) Provided important evidence that fluctuations in endocrine hormones and proinflammatory<br /> cytokines associated with an obese phenotype alters either the<br /> transcriptional activity of target genes or post-transcriptional stability of target<br /> mRNAs in the female reproductive tract which likely contributes to poor oocyte<br /> quality and reduced fertility.<br /> 26) Studies performed in a bovine model of fertility provide evidence that the ability of<br /> the follicle to convert androgens to estrogen uniquely regulates gene expression<br /> in granulosa cells and the oocyte. Likewise, the consequence of androgen<br /> excess is a direct or indirect negative effect on oocyte mRNA abundance, which<br /> likely impacts the developmental competence of the oocyte.<br /> 27) Constructed lentiviral vectors containing a constitutively active promoter fused to<br /> selected siRNA designed to reduce GnRHR II mRNA levels in porcine cells and<br /> tissues.<br /> 28) Established a genetically altered line of swine with reduced levels of GnRH II<br /> receptors using a non-replicative lentiviral delivery system.<br /> 29) Performed immunohistochemistry and immunofluorescence procedures using an<br /> antibody specific for the GnRH-II receptor on fixed testicular tissue.<br /> 30) Examined serum LH and testosterone concentrations in boars treated with either<br /> a GnRH-I or GnRH-II agonist.<br /> 31) Transgenic pigs were produced expressing a spermatogonial marker gene.<br /> 32) Differences in mRNA expression were identified between in vivo produced<br /> conceptuses recovered on Day 15 of gestation that were classified based on<br /> length.<br /> 33) The full-length bAIRN ncRNA has been characterized as a transcript whose<br /> length is approximately 65kb that is transcribed from a promoter located<br /> approximately 440bp upstream of DMR2 on the IGF2R antisense strand.<br /> 34) In vitro exposure of bovine embryos to TNFa resulted in an inhibition of<br /> development to the blastocyst stage.<br /> 35) Analysis of serum progesterone levels at the time of embryo transfer in<br /> relation to conceptus development at Day 17 of gestation was performed and<br /> was found to vary based on embryo production source (in vivo vs. in vitro).<br /> 36) GFP marker in aggregated embryos could track the<br /> development of cloned blastomeres.<br /> 37) The PLC isotypes ´3,<br /> ´4, and ³2 decreased Ca2+<br /> ioscillations and cleavage rates, indicating they are<br /> involved in both IP3R and RyR activation. PLC ´4 and ·2 did not impact Ca2+<br /> i but<br /> did significantly decrease cleavage rates.

Publications

1) Rosenkrans, C. F., Jr., A. R. Mays, G. E. Aiken, and M. L. Looper. 2012.<br /> Prolactin genomics and biology in herbivores. In: C. A. Young, G. E. Aiken, R.<br /> L. McCulley, J. R. Strickland, and C. L. Schardl, editors, Epichloae, Endophytes<br /> of Cool Season Grasses: Implications, Utilization and Biology. The Samuel<br /> Roberts Noble Foundation, Ardmore Oklahoma, USA, p 28-33.<br /> http://www.noble.org/plant-symbionts/isfeg7<br /> 2) Looper, M. L., G. E. Aiken, and C. F. Rosenkrans, Jr. 2012. New perspectives<br /> in fescue toxicosis and ryegrass staggers. In: C. A. Young, G. E. Aiken, R. L.<br /> McCulley, J. R. Strickland, and C. L. Schardl, editors, Epichloae, Endophytes of<br /> Cool Season Grasses: Implications, Utilization and Biology. The Samuel<br /> Roberts Noble Foundation, Ardmore Oklahoma, USA, p 1-4.<br /> http://www.noble.org/plant-symbionts/isfeg7<br /> 3) Moubarak, A. S., Z. B. Johnson, and C. F. Rosenkrans, Jr. 2012. Liver<br /> cytochrome P450 system as affected by endophyte infected tall fescue seed<br /> extracts and ergot alkaloids. Agricultural Sciences 3:1-4.<br /> 4) Giraldo, A. M., S. Ball, and K. R. Bondioli. 2012. Production of transgenic and<br /> knockout pigs by somatic cell nuclear transfer. Methods Mol Biol 885: 105-123.<br /> 5) Gentry, G. T., J. Lambe, W. Forbes, B. Olcott, D. Sanders, K. Bondioli, and R. A.<br /> Godke. 2012. The effect of equine chorionic gonadotropin (ecg) on pregnancy<br /> rates of white-tailed deer following fixed-timed artificial insemination.<br /> Theriogenology 77: 1894-1899.<br /> 6) Scott, B. R., D. B. Carwell, R. A. Hill, K. R. Bondioli, R. A. Godke, and G. T.<br /> Gentry. 2012. Evaluation of capsule permeability in the equine blastocyst.<br /> Journal of equine veterinary science 32: 795-798.<br /> 7) Moubarak, A. S., H. Wang, Z. B. Johnson, and C. F. Rosenkrans, Jr. 2012.<br /> Interaction of ergotamine with liver cytochrome P450 3A in rats. Agricultural<br /> Sciences 3:795-798.<br /> 8) Sales, M. A., M. J. Larson, S. T. Reiter, A. H. Brown, Jr., M. A. Brown, M. L.<br /> Looper, K. P. Coffey, and C. F. Rosenkrans, Jr. 2012. Effects of bovine<br /> cytochrome P450 single nucleotide polymorphism, forage type, and body<br /> condition on production traits in cattle. J. Anim. Physiol. Nutr. 96:545-553.<br /> 9) Smith, S. A., J. D. Caldwell, M. P. Popp, K. P. Coffey, J. A. Jennings, M. C.<br /> Savin, and C. F. Rosenkrans, Jr. 2012. Tall fescue toxicosis mitigation<br /> strategies: comparisons of cow-calf returns. J. Agricultural and Appl. Econ.<br /> 44:577592.<br /> 10) Trish Berger, Lisa M Kentfield, Jan Fay Roser, and Alan J Conley. Stimulation of<br /> Sertoli Cell Proliferation: Defining the Response Interval to an Inhibitor of Estrogen<br /> Synthesis in the Boar. Reproduction, 143: 523-9.<br /> 11) Cannovas S, Cibelli J, Ross PJ. Jumonji domain-containing protein 3 regulates<br /> histone 3 lysine 27 methylation during bovine preimplantation development.<br /> Proceedings of the National Academy of Sciences of the USA (PNAS) 2012;<br /> 109(7):2400-5<br /> 12) Bogliotti Y and Ross PJ. Mechanisms of histone H3 lysine 27 trimethylation<br /> remodeling during early mammalian development. Epigenetics. 2012 7(9):976-81.<br /> 13) Tavares, K.C.S., de Mello e Pinho, R, de Sá Carneiro, I., de Aguiar, L.H.,<br /> Calderón, C.E.M., Martins, L.T., Ambrósio, C.E., Maga, E.A., Bertolini, M., Murray,<br /> J.D. and Bertolini, L.R. (2012) Efficient RNAi-induced Protein Knockdown in<br /> Somatic Cells Using Diced or Chemically Produced Small Interfering RNAs<br /> (siRNA). Acta Scientiae Veterinariae, 40(3): In Press. Online 7/19/2012: ISSN<br /> 1679-9216.<br /> 14) Coutinho da Silva, M, Seidel G, Squires EL, Graham J, Carnevale EM. 2012.<br /> Effects of components of semen extenders on the binding of stallion<br /> spermatozoa to bovine or equine zonae pellucidae. Reproduction 43:577-585.<br /> 15) da Silveira JC, Veeramachaneni DNR, Winger QA, Carnevale EM, Bouma GJ.<br /> 2012. Cell<br /> secreted vesicles in equine ovarian follicular fluid contain miRNAs and proteins: a<br /> possible<br /> new form of cell communication within the ovarian follicle.<br /> Biol Reprod. 86(3):71.<br /> 16) Garner DL, Evans KM, Seidel GE. 2012. Sex-sorting sperm using flow<br /> cytometry/cell sorting. Methods Mol Biol 927:279-295.<br /> Patterson AL, Squires EL, Hansen TR, Bouma GJ, Bruemmer JE. 2012. Gene<br /> profiling of inflammatory genes in day 18 endometria from pregnant and nonpregnant<br /> mares. Mol<br /> Reprod Dev 79:777-784.<br /> 17) Seidel GE Jr. 2012. Several insights on evaluation of semen. Anim Reprod<br /> 9:329-332.<br /> 18) Seidel GE Jr. 2012. Assisted reproduction in horses: What can be learned from<br /> assisted reproduction in cattle? J Equine Vet Sci 32:372-375.<br /> 19) Seidel GE Jr. 2012. Sexing mammalian sperm -- Where do we go from here?<br /> J. Reprod. Dev. 58(5):505-9.<br /> 20) Tang Y, Luo Y, Jiang Z, Ma Y, Kim C, Lin CJ, Amano T, Park J, Amano M,<br /> Carter MG, Kish S, and Tian XC. 2012. Jak/Stat3 Signaling Promotes Somatic<br /> Cell Reprogramming by Epigenetic Regulation. Stem Cell 2012 Dec;30(12):2645-<br /> 56. doi: 10.1002/stem.1225.<br /> 21) Li Y; Pal R; Sung LY; Feng H; Miao W; Cheng SY; Tian C, Cheng T. 2012.<br /> An opposite effect of the CDK inhibitor, p18INK4c on embryonic stem cells<br /> compared with tumor and adult stem cells. PLoS ONE 7(9): e45212.<br /> doi:10.1371/journal.pone.0045212.<br /> 22) Lu Y, West FD, Jordan BJ, Mumaw JL, Jordan ET, Gallegos-Cardenas A,<br /> Beckstead RB, Stice SL. 2012 Avian-Induced Pluripotent Stem Cells Derived<br /> Using Human Reprogramming Factors. Stem Cells Dev. 10;21(3):394-403<br /> 23) Mumaw J, Jordan ET, Sonnet C, Olabisi RM, Olmsted-Davis EA, Davis AR,<br /> Peroni JF, West JL, West F, Lu Y, Stice SL. 2012. Rapid Heterotrophic<br /> Ossification with Cryopreserved Poly(ethylene glycol-) Microencapsulated BMP2-<br /> Expressing MSCs. Int J Biomater. 2012;2012:861794. Epub 2012 Feb 7.<br /> 24) Jeong-Yeh Yang, Jennifer L. Mumaw, Yubing Liu, Steve L. Stice and Franklin D.<br /> West. SSEA4 2012. Positive Pig Induced Pluripotent Stem Cells Are Primed for<br /> Differentiation into Neural Cells. Cell Transplantation [Epub ahead of print 2012<br /> October 03].<br /> 25) Shirley M L Venable, A, Rao R, Boyd NL, Stice SL, Puett D., Narayan P. 2012<br /> Bone morphogenetic protein-4 affects both trophoblast and non-trophoblast<br /> lineage-associated gene expression in human embryonic stem cells. Stem Cell<br /> Discovery. Vol.2, No.4, 163-175<br /> 26) Yubing Liu, Jeong Yeh Yang, Yangqing Lu, Ping Yu, C. Robert Dove, Jessica M.<br /> Hutcheson, Jennifer L. Mumaw, Steven L. Stice and Franklin D. West. ±-1,3-<br /> Galactosyltransferase Knockout Pig Induced Pluripotent Stem Cells: A Cell<br /> Source for the Production of Xenotransplant Pig. Cellular Reprogramming.<br /> Accepted 12/17/12.<br /> 27) Wilson, S.M., Goldwasser, M.S., Clark, S.G., Monaco, E., Bionaz, M. Hurley,<br /> W.L., Rodriguez-Zas, S., Feng, L., Dymon, Z,. Wheeler, M.B. 2012. Adiposederived<br /> mesenchymal stem cells enhance healing of mandibular defects in the<br /> ramus of swine. J Oral Maxillofac Surg 70:e193-e203.<br /> 28) Monaco, E., Bionaz, M. Rodriguez-Zas, S., Hurley, W.L., Wheeler, M.B. 2012.<br /> Transcriptomics comparison between porcine adipose and bone marrow<br /> mesenchymal stem sells during in vitro osteogenic and adipogenic differentiation.<br /> PLoS ONE 7(3): e32481. doi:10.1371/journal.pone.0032481<br /> 29) Chen, K., Hawken, R., Flickinger, G.H., Rodriguez-Zas, S.L., Rund, L.A.,<br /> Wheeler, M.B., Abrahamsen, M., Rutherford, M.S., Beever J.E., Schook, L.B.<br /> 2012. Association of the Porcine Transforming Growth Factor Beta Type I<br /> Receptor (TGFBR1) Gene with Growth and Carcass Traits. Animal<br /> Biotechnology, 23: 4363, 2012, doi.org/10.1080/10495398.2011.630897<br /> 30) Yuan, Y., Wheeler, M.B., Krisher, R.L. Disrupted redox homeostasis and aberrant<br /> redox gene expression in porcine oocytes contribute to decreased developmental<br /> competence. Biol. Reprod. (2012) 87(4):78, 1<br /> 10,doi:10.1095/biolreprod.112.099952.<br /> 31) Zeng,W.X., Tang, L., Bondareva, A., Honaramooz, A., Tanco, V., Megee, S.,<br /> Modelski, M., Rodriguez-Sosa, J.R., Paczkowski, M., Silva, E., Wheeler, M.B..,<br /> Krisher , R.L., Dobrinski, I. 2012. Viral transduction of male germline stem cells<br /> results in transgene transmission after germ cell transplantation in pigs. Biol.<br /> Reprod. (December 5, 2012, doi: 10.1095/ biolreprod.112.104422).<br /> 32) Schiffmacher, A.T., Keefer, C.L. (2012). Optimization of a lipitoid-based plasmid<br /> DNA transfection protocol for bovine trophectoderm CT-1 cells. In Vitro Cellular<br /> & Developmental Biology  Animal. 48(7):403-406.<br /> 33) Lei Lei, Lei Li, Fuliang Du, Chien-Hong Chen, Huayan Wang, and Carol L.<br /> Keefer. (in press) Monitoring Bovine Fetal Fibroblast Reprogramming Utilizing A<br /> Bovine NANOG Promoter-Driven EGFP Reporter System. Mol Reprod Dev.<br /> 34) Govindaraju A, Robertson L, Atli O, Kaya A, Topper E, Uzun A, Padbury J,<br /> Memili E. Dynamics of microRNAs in bull spermatozoa. (2012) Reprod Biol<br /> Endocrinol. 10(1):82. PMID:22978562<br /> 35) Dogan S, Mason M, Govindaraju A, Belser EL, Kaya A, Stokes J, Rowe D,<br /> Memili E. Interrelationships between apoptosis and male fertility. (2012) Journal<br /> of Reproduction and Development. PMID: 22986927<br /> 36) Govindaraju A, Dogan S, Rodriguez-Osorio N, Grant K, Kaya A, Memili E.<br /> Delivering value from sperm proteomics for fertility. (2012) Journal of Cell and<br /> Tissue Research 349(3):783-93. PMID: 22688957<br /> 37) Rodriguez-Osorio N, Urrego R, Cibelli JB. Eilertsen K, Memili E. Reprogramming<br /> Mammalian Somatic Cell. (2012) Theriogenology S0093-691X(12)00322-6. doi:<br /> 10.1016/j.theriogenology.2012.05.030. PMID: 22979962<br /> 38) Wang H, Camargo OD and Memili E. Mycotoxin Alpha-Zearalenol Impairs the<br /> Quality of Preimplantation Porcine Embryos in vitro. (2012) Journal of<br /> Reproduction and Development. 58(3):338-43<br /> 39) Huffman, S. R., M. Almamun, and R. M. Rivera. 2012. Isolation of RNA and DNA<br /> from single preimplantation embryos and a small number of Mammalian oocytes<br /> for imprinting studies. Methods in molecular biology 925: 201-209.<br /> 40) Rivera, R. M., and P. Rinaudo. 2013. Bovine preimplantation embryo<br /> development is affected by the stiffness of the culture substrate. Molecular<br /> reproduction and development.<br /> 41) Robbins, K. M., Z. Chen, K. D. Wells, and R. M. Rivera. 2012. Expression of<br /> KCNQ1OT1, CDKN1C, H19, and PLAGL1 and the methylation patterns at the<br /> KvDMR1 and H19/IGF2 imprinting control regions is conserved between human<br /> and bovine. Journal of biomedical science 19: 95.<br /> 42) Zhao, M. T., R. M. Rivera, and R. S. Prather. 2013. Locus-Specific DNA<br /> Methylation Reprogramming During Early Porcine Embryogenesis. Biology of<br /> reproduction.<br /> 43) Yang, Z., K.A. Norwood, J.G. Kerl, and J.R. Wood. 2012. Genes involved in the<br /> immediate early response and epithelial-mesenchymal transition are regulated by<br /> adipocytokines in the female reproductive tract. Mol. Reprod. Dev. 79: 128-137.<br /> Jackson LR, Farin CE, Whisnant CS. 2012. Tumor necrosis factor alpha inhibits<br /> bovine embryo development through a prostaglandin mediated mechanism. J<br /> Anim Sci Biotech 3:7-10.<br /> 44) Sommer JR, Jackson LR, Simpson SG, Collins EB, Piedrahita JA, Petters RM.<br /> 2012. Transgenic Stra8-EYFP pigs: a model for developing male germ cell<br /> technologies. Transgenic Res 21:383-92.<br /> 45) Hall, J., Meng, Q., Sessions, B.R., Fan, Z., Stott, R., Panter, K., Rutigliano, H.,<br /> Davies, C.J., Bunch, T.D., White, K.L. and Polejaeva, I.A. 2012. Effect of Embryo<br /> Culture Length on Production of Cloned Transgenic Goats. Reproduction,<br /> Fertility and Development. 25 (1)162.

Impact Statements

1. Members of the W2171 Multistate Research Project are directing research that contributes to improved livestock production efficiency and sustainability, as well as food safety to meet food demands associated with the 10 billion by 2050 human population expansion.
2. Increased knowledge of development and genetic and epigenetic contributions to gamete and embryo production paves the way for more widespread adoption of assisted reproductive technologies that can hasten genetic progress and improve product consistency.
3. Enhanced understanding of molecular mechanisms controlling genomic reprogramming provides opportunities to understand and alleviate suboptimal reproduction in domestic animals and humans.
4. A more complete understanding of the molecular and cellular physiology of trophoblast differentiation and function will reduce early embryo and fetal mortality.
5. Developing and refining genetic technologies will lead to improved livestock disease resistance and production efficiency, and enhanced food products for human health.
6. Many of the technological advances of the W2171 multistate research group result in the production of livestock models for biomedical applications.

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