W3171: Germ Cell and Embryo Development and Manipulation for the Improvement of Livestock

(Multistate Research Project)

Status: Inactive/Terminating

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SAES-422 Reports

Annual/Termination Reports:

[05/04/2015] [01/22/2016] [05/18/2017] [06/04/2018] [02/28/2019]

Date of Annual Report: 05/04/2015

Report Information

Annual Meeting Dates: 02/11/2015 - 02/12/2015
Period the Report Covers: 10/01/2013 - 09/01/2014

Participants

In attendance:
Shipka, Milan, Alaska, Administrator
Isom, Clay, Utah State University, Chair
Winger, Quinton, Colorado State University, Secretary (Ad Hoc)
White, Brett, University of Nebraska Lincoln
Schmidt, Edward, Montana State University
Rorie, Rick, University of Arkansas
Bondioli,Ken, Louisiana State University

Guest:
Pinto, Carlos, Louisiana State University

LSU Graduate Students:
Foster, Brittany, LSU
Diaz, Fabian, LSU

Brief Summary of Minutes

W3171 Meeting LSU Ag Center February 11, 2015

In attendance:
Milan Shipka, Alaska, Administrator
Clay Isom, Utah State University, Chair
Quinton Winger, Colorado State University, Secretary (Ad Hoc)
Brett White, Nebraska
Edward Schmidt, Montana
Rick Rorie, Arkansas
Ken Bondioli, Louisiana State University

Guest:
Carlos Pinto, Louisiana State University

LSU Graduate Students:
Brittany Foster, LSU
Fabian Diaz, LSU

8:20 Meeting called to order by Clay Isom

Minutes from annual meeting W2171 Reno, NV were read by Clay Isom and approved.

8:30 Video Conference Call with Mark Mirando, USDA

President’s Budget FY 2016 AFRI discretionary funding increased from 325 million to 450 million, foundational grants will use 40% of the budget.

2016 budget included an increase of 5.1% for Hatch fund dollars. A 12.5 million dollar increase put in specifically for a competitive grant program within the Hatch program. This is at the proposal level.

Dr. Mirando highlighted 2 areas in the Food Security area that have animal sections. One contains Animal Breeding in title. May be an opportunity for Animal Reproduction?

10:00 Earle Pope, ACRES Audubon Center for Research of Endangered Species presentation, “Assisted Reproductive Technologies for Conservation of Endangered Cats”

1:00 Station Reports
Arkansas
Colorado
Louisiana

3:00 Station Reports

Montana
Nebraska
Utah

Business Meeting:

Discussed the successful re-write and award of the W3171.
Discussed the importance of using the meetings as well as improving open communication within the group between meetings to foster collaborations. Money is needed to foster collaborations, therefore a strategy to improve the ability of members of the group to be co-investigators on grants with agricultural reproduction focus.
Discussed strategies to improve competitiveness of grant applications using collaborative approaches.

Clay Isom brought up the possibility of writing a peer-reviewed publication together by members of the group that would highlight the possible similarities of the groups research focus.

Discussed the use of a web based system to make resources available more easily visible to members of the group. All agreed that we should use the email address lists of the group to improve communication about research collaborations.

Collaborations: Isom and Schmidt talked about using the “embryo cradle” for nuclear transfer.

Isom and Bondioli talked about in vitro gene expression similarities.

Several talks focused on diet issues. Also, placental and fetal development due to androgen.

Selection of the date for the 2016 meeting January 22, 2016 12:00-6:00pm in Louisville, Kentucky to coincide with the IETS meeting.

Nomination for incoming secretary Rick Rorrie nominated Brett White, seconded by Clay Isom. Unanimously passed vote.

Jerry Bouma will take over the chair position 60 days after the meeting.

Discussed reaching out to new W3171 members that did not make it to the meeting.

Milan mentioned we could also reach out to other Western region universities for any researchers that might be a good fit for the W3171 to fill empty slots.

6:40pm Meeting adjourned

Accomplishments

Objective 1. Understand the biology and underlying mechanisms of gamete development, fertilization, and embryogenesis Novel findings suggest a significant role for cytoplasmic polyadenylation during bovine oocyte maturation considering changes in polyadenylated transcript levels occur without changes in total RNA abundance. Also, changes in transcript abundance are found to corresponded to differences in poly(A) tail length at specific maturation stages. Comprehensive transcriptome analyses also were conducted on single matured bovine oocytes and pre-implantation embryos and novel stage-specific modules of co-expressed genes were identified that are potential master regulators of pre-implantation development. In separate studies RNA sequencing experiments on MII oocytes matured in vitro and in vivo reveal transcripts that are impacted by the in vitro maturation process. Finally, microfluidics-based qPCR was conducted on single oocytes and their associated cumulus cells and identified candidate genes whose expression was affected by age in the mare. Similarly, relative patterns of gene expression were compared based on follicular size and oocyte competence in goat oocytes. DPPA3 is identified as a maternal factor important for correct epigenetic remodeling and normal embryonic development in cattle, and suggests that the role of DPPA3 during early development is conserved between species. In separate studies, histone methyltransferases were found to play a role in heterochromatin stabilization and potentially embryo developmental potential. Studies focused on improving in vitro maturation of oocytes and embryo culture reveal that polydimethylsiloxane well-insert systems successfully matures oocytes and culture embryos in an individually-identifiable manner, while possibly enhancing developmental potential. The endometrial transcriptome was characterized in pregnant and non-pregnant sows, and maternal pathways that could be key to embryo implantation and survival are uncovered. In addition, the cervical transcriptome was uncovered in cattle during different stages of the reproductive cycle that provide new insight into the mechanisms/factors that play a role in cervical permeability and antimicrobial activity. Ovarian responses to dietary conjugated linoleic acid uptake was studied and the observed highly regulated mechanisms involved in fatty acid uptake by ovarian components could explain the lack of ovarian responses in different cattle breeds. Metabolic studies in cultured bovine embryos were developed and data reveal that substrate balance in media greatly affects embryo metabolic pathways. Apical blebbing by epithelial cells in the epididymis was identified as a process by which proteins are transferred to sperm as they travel through the epididymis, and this process is regulated by testicular luminal factors. Objective 2. Refine methods for production of genetically modified animals to improve livestock production efficiency Laser-assisted intracytoplasmic injection was discovered to be an efficient technique to inject CRISPR/Cas9 RNA in bovine zygotes for genome editing purposes, which are a first step towards generation of genetically modified livestock. In addition, transgenic goats were generated using CRISPR/Cas9 technology to knockout myostatin, as well as using homologous recombination to knockin human K-ras oncogene. Placenta-specific gene targeting approaches have been developed by incubating sheep hatched blastocysts with lentiviral-based shRNA constructs to study stem cell factor LIN28 and transcription factor AR in placental development and function. In addition, data thus far suggest a role for LIN28 in trophoblast cell proliferation, and AR in placental angiogenesis. Separate studies were conducted to characterize trophoblast-derived stem-like cells from porcine blastocysts, and experiments are underway to assess their utility in donor nuclear transfer exeriments. A new system was developed and tested involving transduced mammary epithelial cells to evaluate vector construction that target recombinant protein expression in milk. Novel findings are reported on improved derivation efficiency and pluripotency of embryonic stem cells by supplementation of small molecules to culture media. In addition, new data reveals the importance of Akt signaling in somatic cell reprogramming. Separate experiments reveal that electromagnetic biostimulation of fibroblast cells can be used as an alternative to reprogram cells. In vitro transcribed mRNA was successfully used to transfect bovine fetal fibroblasts and induce expression of pluripotency factor OCT4, as an alternative, novel approach to generate stem cells without genetic modifications. Potential improvements were tested involving HAGM hydrogels and micromass culture systems for in vitro chondrogenic differentiation of porcine adipose derived stem cells, as well as addition of platelet-rich plasma derived growth factors to support proliferation and migration of adipose derived stem cells. Continued improvements are made to the Dracula micro-manipulator that will allow for its broad application in embryo research and cryopreservation.

Publications

Publications Rorie, R.W., A. J. Davis, T. D. Lester, and J. G. Powell. 2014. Comparison of two estrous synchronization protocols for use with X sorted semen in lactating beef cows. Prof. Anim. Sci. 30:620-624. Rorie, R.W., P.A. Delgado and T.D. Lester. 2014. Variation among beef bulls in the ratio of X- to Y-chromosome bearing spermatozoa. Adv. Reprod. Sci. 2:69-75. Rowe, M.P., J.G. Powell, E.B. Kegley, T.D. Lester and R.W. Rorie. 2014. Effect of supplemental trace mineral source on bull semen quality. Prof. Anim. Sci. 30:68-73. Bakhtari A, Ross PJ. DPPA3 prevents cytosine hydroxymethylation of the maternal pronucleus and is required for normal development in bovine embryos. Epigenetics. 2014 Sep;9(9):1271-9. doi: 10.4161/epi.32087. Epub 2014 Aug 1. Berger, T., and A. Conley. 2014. Reduced endogenous estrogen and hemicastration interact synergistically to increase porcine sertoli cell proliferation. Biology of reproduction 90: 114. Reyes JM, Chitwood JL, Ross PJ. RNA-Seq profiling of single bovine oocyte transcript abundance and its modulation by cytoplasmic polyadenylation. Mol Reprod Dev. 2015 Jan 5. doi: 10.1002/mrd.22445. Campos-Chillon F, Farmerie TA, Bouma GJ, Clay CM, Carnevale EM. The effect of aging on gene expression and mitochondrial DNA in the equine oocyte and follicle. Reprod Fertil & Develop, 2015 Mar 19. doi: 10.1071/RD14472. da Silveira JC, Winger QA, Bouma GJ, Carnevale EM. Age effects on follicular fluid exosomal miRNAs and TGF? signaling during follicle development in the mare. Reprod Fertil & Develop, Accepted. Cleys ER, Halleran JL, Enriquez VA, da Silveira JC, West RC, Winger QA, Anthony RV, Bruemmer JE, Clay CM, Bouma GJ. Androgen Receptor and Histone Lysine Demethylases in Ovine Placenta. PLoS ONE 2015 Feb 12;10(2):e0117472.. Hatzel JN, Bouma GJ, Cleys ER, Bemis LT, Ehrhart EJ, McCue PM. Identification of heat shock protein 10 within the equine embryo, endometrium, and maternal peripheral blood mononuclear cells. Theriogenology 2015 Mar 15;83(5):832-9.. Nemere I, Garbi N, Winger Q. The 1,25D3 -MARRS Receptor/PDIA3/ERp57 and Lifespan. J Cell Biochem. 2014 Oct 6. doi: 10.1002/jcb.24986. [Epub ahead of print] Cleys ER, Halleran JL, McWhorter E, Hergenreder J, Enriquez VA, da Silveira JC, Bruemmer JE, Winger QA, Bouma GJ. Identification of microRNAs in exosomes isolated from serum and umbilical cord blood, as well as placentomes of gestational day 90 pregnant sheep. Mol Reprod Dev. 2014 Nov;81(11):983-93. da Silveira JC, Carnevale EM, Winger QA, Bouma GJ. Regulation of ACVR1 and ID2 by cell-secreted exosomes during follicle maturation in the mare. Reprod Biol Endocrinol. 2014 May 26;12:44. Szerlong H, Herman JA, Krause CM, Deluca JG, Skoultchi A, Winger QA, Prenni JE, Hansen JC. Proteomic characterization of the nucleolar linker histone H1 interaction network. Journal of Molecular Biology, J Mol Biol. 2015 Jan 10. pii: S0022-2836(15)00006-6. Jeckel KM, Bouma GJ, Hess AM, Petrilli EB, Frye MA. Dietary fatty acids alter left ventricular myocardial gene expression in Wistar rats. Nutr Res. 2014 Aug;34(8):694-706. Le CH, Mulligan CM, Routh MA, Bouma GJ, Frye MA, Jeckel KM, Sparagna GC, Lynch JM, Moore RL, McCune SA, Bristow M, Zarini S, Murphy RC, Chicco AJ. Delta-6-desaturase links polyunsaturated fatty acid metabolism with phospholipid remodeling and disease progression in heart failure. Circ Heart Fail. 2014 Jan;7(1):172-83. Lin, CJ; Amano, T; Tang Y; TianXC. 2014. Improved derivation efficiency and pluripotency of stem cells from the refractory inbred C57BL/6 mouse strain by small molecules. PLoSONE 9(9): e106916 published online Sept 11, 2014. Jiang Z, Sun J, Dong H, Luo O, Zheng X, Obergfell C, Tang Y, Bi J, O’Neill R, Ruan Y, Chen J, Tian XC. (2014) Complete Transcriptional Profiles of Bovine In Vivo Pre-implantation Development. BMC Genomics 15:756; published online Sept 4, 2014. Tang Y, Jiang Z, Luo Y, Zhao X, Wang L, Tian XC. (2014). Differential Effects of Akt Isoforms on Somatic Cell Reprogramming J Cell Sci 127:3998-4008. Zopf D.A., Flanagan, C.L., Wheeler, M.B., Hollister, S.J., Green, G.E. 2013. Treatment of severe porcine tracheomalacia with a 3-dimensionally printed, bioresorbable, external airway splint. JAMA Otolaryngol Head Neck Surg. 2014 Jan;140(1):66-71. Yuan, Y., Paczkowski, M., Wheeler, M.B., Krisher, R.L. 2014. Use of a novel polydimethylsiloxane well insert to successfully mature, culture and identify single porcine oocytes and embryos. Reproduction, Fertility and Development 26:375-384. Monzani, P.S., Guemra, S., Adona, P.A., Ohashi, O.M., Meirelles, F.V., Wheeler, M.B. 2014. MAC-T cells as a tool to evaluate lentiviral vector construction targeting recombinant protein expression in milk. Animal Biotechnology, 26:2, 136-142. Zopf D.A., Mitsak, A.G., Flanagan, C.L., Wheeler, M.B., Green, G.E., Hollister, S.J. 2014. Computer-Aided Designed, 3-Dimensionally Printed Porous Tissue Bioscaffolds For Craniofacial Soft Tissue Reconstruction. J Otolaryngol Head Neck Surg. Online: DOI: 10.1177/0194599814552065 Bondioli, K.R. Cryopreservation of Bovine Embryos. In: Hopper, R. (ed) Bovine Reproduction. John Wiley and Sons, 2015 pp. 718-722. Chen, H., Fu, J., Chen, H., Hu, Y., Soroka, D. N., Prigge, J. R., Schmidt, E. E., Yan, F., Major, M. B., Chen, X. and Sang, S. (2014) Ginger compound [6]-shogaol and its cysteine-conjugated metabolite (M2) activate Nrf2 in colon epithelial cells in vitro and in vivo. Chemical research in toxicology. 27, 1575-1585. Sonsteng, K. M., Prigge, J. R., Talago, E. A., June, R. K. and Schmidt, E. E. (2014) Hydrodynamic delivery of Cre protein to lineage-mark or time-stamp mouse hepatocytes in situ. PLoS ONE. 9, e91219 Gorrini, C., Gang, B. P., Bassi, C., Wakeham, A., Baniasadi, S. P., Hao, Z., Li, W. Y., Cescon, D. W., Li, Y. T., Molyneux, S., Penrod, N., Lupien, M., Schmidt, E. E., Stambolic, V., Gauthier, M. L. and Mak, T. W. (2014) Estrogen controls the survival of BRCA1-deficient cells via a PI3K-NRF2-regulated pathway. Proc Natl Acad Sci USA. 111, 4472-4477 Saunders G, Stevens JR, and Isom SC. A shortcut for multiple testing on the directed acyclic graph of Gene Ontology. BMC Bioinformatics 2014, 15:349 DOI: 10.1186/s12859-014-0349-3 Li Q, Suasnavas E, Xiao L, Heywood S, Qi X, Zhou A, Isom SC. Label-free and non-invasive monitoring of porcine trophoblast derived cells: differentiation in serum and serum-free media. J Biophotonics. 2014 Sep 22;9999(9999). doi:10.1002/jbio.201400062. Suasnavas E, Heywood S, Ward A, Cox L, O’Grady M, Zhao Y, and Isom SC. Characterization of trophoblast-derived stem-like cells from peri-implantation porcine embryos. Animal Reproduction Science. In Press. Ni W., Qiao J., Hu S., Zhao X., Regouski M., Yang M., Polejaeva I.A.*, Chen C.* Efficient gene knockout in goats using CRISPR/Cas9 system. PLoS One 2014, 9 (9), e106718. Gong J., Chen G., Wang Z., Polejaeva I., Silisga R., Chen C-T., Kao C-M., and Chen L. Activating the expression of human K-rasg12d stimulates oncogenic transformation in transgenic goat fetal fibroblast cells. PLoS ONE. 2014, 9(3): e90059. Zhou W.*, Gosch G., Guerra T., Broek D., Wu G., Walker S., Polejaeva I*. Amino acid profiles in first trimester amniotic fluids of healthy cloned pregnancies are similar to those of IVF pregnancies, but not nonviable cloned pregnancies. Theriogenology. 2014, 81 (2), 225-229

Impact Statements

Understanding the molecular mechanisms controlling oocyte maturation and epigenetic remodeling during preimplantation development have led to improvements in the success rates of in vitro embryo production, somatic cell nuclear transfer/cloning and production of genetically modified animals.
On going studies on SCNT pregnancy anomalies provide a useful screening tool for viable and nonviable SCNT pregnancies.
CRISPR/Cas9 technology is employed as a cost effective approach to edit livestock genomes for improved agricultural performance. Current studies are utilizing this approach successfully in bovine and goat.
Novel approaches are used to study gene function in ruminant placentas by infecting blastocysts with lentiviral shRNA viruses focusing on RNA binding protein and steroid hormone receptors. Findings from these studies improve our understanding of the genetic regulation of placenta development and result in methods to improve somatic cell nuclear transfer in ruminants and pregnancy losses.
Ongoing efforts also focus on studying the establishment of pluripotency in bovine stem cells through Akt mediated co-stimulation of Stat3 activity with LIF, as well as isolation and transplantation of mesenchymal stem cells. The enrichment of several functions/pathways indicate differences in therapeutic application of MSC for specific regenerative therapy.
Studies on oocyte maturation and developmental competence lead increasing embryo development rates both for in vitro embryo production and in vivo fertility in cattle breeding programs.
Markers of oocyte competence are being investigated and refined to effect the conditions for in vitro oocyte maturation and mRNA degradation pathways, which have the potential of dramatically improving the developmental competence of these embryos and enhancing the application of in vitro embryo production to cattle breeding programs.
Improved embryonic competency following in vitro production and cryopreservation stimulates the industry by lowering costs, especially those related to recipient management.

Date of Annual Report: 01/22/2016

Report Information

Annual Meeting Dates: 01/23/2016 - 01/23/2016
Period the Report Covers: 10/01/2014 - 09/30/2015

Participants

Committee Chair, Jerry Bouma (Colorado State Univ)
Secretary, Rick Rorie (University of Arkansas),
Cindy Tang (University of Connecticut),
Matt Wheeler (University of Illinois),
Curt Youngs (Iowa State University),
Ken Bondioli (Louisiana State University),
Brett White (University of Nebraska Lincoln)
Kiho Lee (Virginia Polytechnic Institute and State University)

Brief Summary of Minutes

Brief Minutes of the W3171 Technical Committee Meeting

Galt Hotel, Louisville, KY

January 22, 2016

The meeting was called to order at 1 pm by Committee Chair, Jerry Bouma (Colorado). In attendance were Rick Rorie (Arkansas), Cindy Tang (Connecticut), Matt Wheeler (Illinois), Curt Youngs (Iowa), Ken Bondioli (Louisiana), Brett White (Nebraska) and Kiho Lee (Virginia).

The meeting agenda and minutes of the previous meeting were approved as read. W3171 Administrator, Milan Shipka, addressed committee members via phone since he could not attend due to recent surgery. Milan provided guidance for submission of the project's report of accomplishments, impacts and publications. He also emphasized our cooperative/collaborative work on research projects.

Station reports were presented and discussed for Arkansas, Colorado, Connecticut, Illinois, Iowa, Louisiana, Nebraska and Virginia. Potential new collaborative projects were discussed at length. To facilitate collaborative research, Jerry Bouma agreed to develop a spreadsheet on which research personnel at each station can enter their areas of interest and expertise, capabilities and techniques. This information will be used to identify overlapping research areas for additional collaborative efforts.

A motion was made and unanimously approved to recognize Bob Godke (Louisiana) for his numerous contributions to the W171/1171/2171/3171 regional research project over many years. Bob Godke passed away during 2015.

Brett White was elected to serve Secretary for the W3171 Technical Committee for 2016. Rick Rorie will serve as Chair for the coming year. The committee voted to meet in conjunction with the IETS Annual Conference in Austin, Texas in January 2017. The meeting and location will be determined at a later date.

Meeting adjourned at 4:15 pm.

Minutes Attachment:

W3171 Technical Committee Meeting Minutes.docx

Accomplishments

Accomplishments   Objective 1:  Understand the biology and underlying mechanisms of gamete development, fertilization, and embryogenesis   Oocyte gene expression was investigated to identify markers for improved oocyte maturation in vitro.  Many deviations from normal cytoplasmic maturation were noted and maintained when maturation was delayed with Forskolin and IBMX.  Characterization of global epigenetic marks in oocytes derived from small versus large follicles suggest no difference in H3K4me3 and H3K9me3 levels, but that H3K27me3 levels may be different between oocyte from the different sized follicles.  Significant differential gene expression was noted between oocytes with different G6PDH activity that could be used as biomarkers for oocyte quality.  A system was developed for embedding and long-term culture of single porcine follicles in vitro.  Effects of follicular fluid concentrations of androstenedione on theca and granulosa cell gene expression, and the role of VEGFA isoforms in ovarian function were investigated to better understand oocyte maturation.  In vitro fertilization conditions were optimized for using sex-sorted semen for production of bovine embryos.   Investigation of sperm metabolic pathways of domestic cats demonstrated that glucose, fructose, and pyruvate are metabolized by sperm, but in a species-specific manner.   Procedures were developed for detection of GnRH-II and GnRHR-II in testicular tissue.  The role of GnRH-II in male fertility was investigated by quantifying in seminal plasma of boars, and by comparing GnRH-II receptor protein levels in the testis and epididymis, as well as the prostate, seminal vesicles and bulbourethral glands.   Viable embryos were produced in vitro, using fresh oocytes collected and transported from different countries; opening the possibility to access superior genetics and improve herds.  Also, the use of BLV seropositive donor semen for IVF showed no transmission of the virus. Progress was made in sorting embryos by an apoptotic index, then deriving meaningful gene expression and DNA methylation data.  The abundance of mRNAs for imprinted genes of embryos were determined with vast differences of these genes detected across species, suggesting these genes may play different roles in early embryo development.  Sex chromosome dosage compensation was studied in the bovine, and found that compensation occurred in both in vivo and in vitro produced embryos, as well as somatic tissues of male and female adults.    Embryo metabolites were compared between IVF and parthenogenetically activated oocytes. Parthenogenetic embryos produced more citrate than IVF embryos, indicating citrate may be a useful marker for embryo viability.  Increased acetate and formate were detected on day 3, indicating the start of glycolysis and a possible marker for embryo viability.  Substrate use and metabolic pathway fluxes were measured in embryos, using GC-mass spectrometry.  Results demonstrate substrate balance in the media has a profound influence on pathway activities and on the routes and extent that media substrates are metabolized.  Analysis of lipids by ambient ionization mass spectroscopy allowed detection of a wide range of lipid classes in single oocytes, embryos and cell pellets, and provided information on in vitro culture impact, developmental and differentiation stages.    Anti-Mullerian hormone and follicle counts as a predictor of superovulatory response and embryo production, confirm that both relative anti-Mullerian hormone concentration and number of small antral follicles positively correlate with embryo production.  In related studies, a successful superovulation protocol was developed for Boran cattle and the first pregnancy was established cryopreserved alpaca embryos.  The Dracula embryo manipulation devise was evaluated for use with a variety of embryos of different species, suggesting the technology is amendable to a variety of agriculturally important mammalian species.   Objective 2:  Refine methods for production of genetically modified animals to improve livestock production efficiency   The CRISPR/Cas9 system for gene editing is currently being utilized by several stations.  Targeting and genome editing constructs were designed to study LIN28B function in placenta in vivo.  In a study with the aim to use transgenic animals for bioreactors, results suggest that MAC-T cells may be a good system to evaluate gene editing via CRISPR for targeting recombinant protein expression in milk.   Preliminary data suggest that the combined cell transfection of gene target-specific CRISPR/Cas9 and RNAi to knockdown the NHEJ pathway is a viable and efficient approach to produce precise genetically modified goat donor cells carrying mono- and biallelic knock-ins of large size transgene constructs for use in cloning by SCNT.  Cloning procedures are underway using biallelic knock-in somatic cells to obtain live offspring, in order to produce and test a recombinant subunit vaccine against B. abortus.   The CRISPR/Cas9 system was used to disrupt up to three genes simultaneously at near 100% efficiency in vitro.  In vivo, efforts were successful in producing pigs lacking two genes simultaneously.  Other results demonstrate that the CRISPR/Cas9 system can be used to induce targeted mutations through NHEJ and/or HDR, and to induce homologous recombination during porcine embryogenesis.  In other efforts, sgRNAs have been designed specifically for HMGA2 and KDM1A for CRISPR Cas9 genome editing, and these are currently tested in in vitro assays. The CRISPR Cas9 genome editing tools were also used to generate mutations specifically in the trophectoderm that are being tested in vivo.   In studies focused on stem cells, derivation efficiency of induced pluripotent stem cells were improved by using the mouse model, and the roles of chromatin remodeling complexes in the reprogramming process were identified.  The effect of spatial location, current intensity, and time of exposure on the effect of a bioelectrical stimulus to change pluripotency gene expression levels in cultured fibroblast cells was analyzed.  The results could give a better understanding of optimal treatment conditions for inducing cell transformation and lead to improvement in efficiency of SCNT.   A novel Dynamic Impact Approach and functional enrichment analysis was used to show only a few pathways and functions were more induced during osteogenesis compared to adipogenesis. Some pathways were more inhibited during osteogenesis, such as cholesterol and protein synthesis. Up-stream transcription factor analysis indicated activation of several lipid-related transcription regulators (e.g., PPARs and CEBPα) during adipogenesis but osteogenesis was driven by inhibition of several up-stream regulators, such as MYC.  Between MSCs the data indicated an ‘adipocyte memory’ in ASC with also an apparent lower immunogenicity compared to BMSC during differentiation. Overall the analysis allowed proposing a dynamic model for the adipogenic and osteogenic differentiation in porcine ASC and BMSC.    Porcine chondrocytes and ASC were able to form cartilage matrix in a pellet culture system with chondrogenic media regardless of additive, while porcine fibroblasts and periosteal cells showed limited to no chondrogenic potential in the conditions tested. Chondrocyte pellet growth is due to extracellular matrix deposition.  Another study demonstrated a modular platform approach founded on 3D printing, for developing tissue engineering therapies and illustrated the design control process for modular implementation of two scaffold based tissue engineering therapies: airway reconstruction and bone tissue engineering based spine fusion.   Tagged versions (His, Myc or Flag tags) were generated for the most promising antigens of different zoonotic organisms including; Tuberculosis, Brucellosis, Leptospirosis, Q fever, Trypanosomiasis, Rabies and Porcine Cysticercosis. Sequences encoding these antigens were cloned into a replication defective adenoviral expression vector.  Recombinant proteins were produced in two different cell lines (HEK 293 and MAC-T), cell lysates and supernatants were examined for tagged proteins using western blot analysis. Constructs are being validated for production of recombinant proteins in the mammary gland for vaccine development.   In efforts directed toward cryopreservation, it was shown that cleavage rates post-thaw and fertilization were similar when oocytes were vitrified before or after vitrification.  The Mouse Dracula tool was used to demonstrate that mouse embryos at the early-, expanded-, or even the hatched-stage can effectively have their coleomic contents exchanged with cryoprotectant. These results could improve cryopreservation efforts and make these procedures less dependent on temperature-change schedules.  Efforts directed toward storage of ram semen demonstrated that regardless of semen extender or storage temperature, fresh ram semen was found to loose progressive motility within 24 hours of storage.

Publications

W-3171 Publications 2014 Chen H, Fu J, Chen H, Hu Y, Soroka DN, Prigge JR, Schmidt EE, Yan F, Major MB, Chen X, Sang S. 2014.  <a href="http://www.ncbi.nlm.nih.gov/pubmed/25148906">Ginger compound [6]-shogaol and its cysteine-conjugated metabolite (M2) activate Nrf2 in colon epithelial cells in vitro and in vivo.</a> Chem Res Toxicol. 2014 Sep 15;27(9):1575-85. Godke, RA, M Sansinena, and CR Youngs. 2014.  Assisted reproductive technologies and embryo culture methods for farm animals. In (CA Pinkert, Ed.) Transgenic Animal Technology: A Laboratory Handbook, 3rd Ed., Elsevier, London, pp. 581-638.  DOI: http://dx.doi.org/10.1016/B978-0-12-410490-7.00022-0 Vivanco, W, D Ponce, M Miguel, C Youngs, E Huamán, S. León, and M Asparrin.  2014.  Repeatability of the ovarian response and embryo production in superovulated Huacaya alpacas. Spermova 4(1):77-79. Youngs CR. 2014. Embryo development and survival in ruminant livestock species. In (G. Gutierrez and M Rothschild, Eds.). Proceedings of the International Seminar on Advances in Animal Production in Latin America, November 5-7, 2014, Lima Peru, pp. 40-51. 2015 Abedal-Majed, M.A., M.P. Magamage, R.J. Vinton, R.A. Cushmann, A.K. McNeel, H. Freetly, J.R. Wood, and A.S. Cupp. 2015. Vascular Endothelial Growth Factor A 165 (VEGFA165), angiogenic isoform, promotes while VEGFA165b antagonizes VEGFA165 stimulated follicular progression in bovine ovarian cortical pieces cultured from pre-pubertal heifers. Society for the Study of Reproduction 47th Annual Meeting. San Juan, Puerto Rico. p. 246. Bionaz, M., Monaco, E.  and Wheeler, M.B. 2015. Transcription Adaptation during In Vitro Adipogenesis and Osteogenesis of Porcine Mesenchymal Stem Cells: Dynamics of Pathways, Biological Processes, Up-Stream Regulators, and Gene Networks. PLoS One. 2015 Sep 23;10(9):e0137644. doi: 10.1371/journal.pone.0137644. eCollection 2015. Bondioli, K.R.  Cryopreservation of Bovine Embryos. 2015.  In: Hopper, R. (ed) Bovine Reproduction. John Wiley and Sons, pp. 718-722. Campos-Chillon F, Farmerie TA, Bouma GJ, Clay CM, Carnevale EM. 2015. The effect of aging on gene expression and mitochondrial DNA in the equine oocyte and follicle. Reprod Fertil & Develop, Mar 19. doi: 10.1071/RD14472. [Epub ahead of print]. Canovas S and Ross PJ. 2015.  Mechanisms of epigenetic remodeling during preimplantation development. Reproduction, Fertility and Development. 28(2): 25-40. Cebula M, Schmidt EE, Arnér ES.  2015.  <a href="http://www.ncbi.nlm.nih.gov/pubmed/26058897">TrxR1 as a potent regulator of the Nrf2-Keap1 response system.</a>. Antioxid Redox Signal. 23(10):823-53. Cederberg, R.A., J.E. Smith, E.A. McDonald, C. Lee, A.R. Perkins, and B.R. White. 2015. Activity of the porcine gonadotropin-releasing hormone receptor gene promoter is partially conferred by a distal gonadotrope specific element (GSE) within an upstream enhancing region, two proximal GSEs and a retinoid X receptor binding site. Reprod. Biol. Endocrinol. 13:45. Center, K., D. Dixon, and R. Rorie.  2015.  Anti-mullerian hormone and follicle counts as predictors of superovulatory response and embryo production in beef cattle. Arkansas Agri. Exp. Sta. Res. Series 628:31-33. Cleys ER, Halleran JL, Enriquez VA, da Silveira JC, West RC, Winger QA, Anthony RV, Bruemmer JE, Clay CM, Bouma GJ. 2015. Androgen Receptor and Histone Lysine Demethylases in Ovine Placenta. PLoS ONE 10(2):e0117472. [PMID: 25675430] Cox L, Hintze KJ, and ISOM SC.  June 2015.  Impact of a Total Western Diet on oocyte quality in swine.  48th Annual Meeting of the Society for the Study of Reproduction.  San Juan, PR.  Cox L, ISOM SC, and Hintze KJ.  April 2015.  Formulation of the Total Western Diet (TWD) for Pigs and Phenotypic Differences After a 12-week Feeding Trial.  29th Annual Meeting of the Federation of American Societies for Experimental Biology.  Boston, MA.  Cushman, R.A., R.G. Tait Jr., A.K. McNeel, E.D. Forbes, O.L. Amundson, C.A. 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Society for the Study of Reproduction 47th Annual Meeting. San Juan, Puerto Rico. p. 245. Rubessa, M., Ambrosi, A., Polkoff, K.M., Stewart, J.W., Herzog, K.K., Denmark, S.E. Wheeler, M.B. 2015. Metabolic Profiling of Male and Female Bovine Embryos Using Nuclear Magnetic Resonance (NMR) Imaging. Reproduction, Fertility and Development 27(1) 158–159.   Rubessa, M., Herzog, K.K., Ambrosi, A., Stewart, J.W., Polkoff, K.M., Denmark, S.E. Wheeler, M.B. 2015. Non-Invasive Analysis of Embryo Metabolites Using Nuclear Magnetic Resonance. Reproduction, Fertility and Development  27(1) 157–157. Rubessa, M., Nasser, L.F., Wheeler, M.B. 2015. Practical Applications of In Vitro Embryo Production in Latin America. Proceedings 11th Simposium Internacional de Reproduction Animal, Cordoba, AR. Rutigliano HM, Wilhelm A, Hall J, Shi B, Meng Q, Stott R, Bunch TD, White KL, Davies CJ, Polejaeva IA. Cytokine gene expression at the maternal-fetal interface after somatic cell nuclear transfer pregnancies in small ruminants. Reprod Fertil Dev. 2015 Oct 14. doi: 10.1071/RD15103. [Epub ahead of print] PubMed PMID: 26463834. Ryu J and Lee K.  2015. Complete disruption of RAG2 gene during porcine embryogenesis utilizing CRISPR/Cas9 system. Biol Reprod. (Suppl. 1): 377. Sanz-Fernandez MV, Johnson JS, Abuajamieh M, Stoakes SK, Seibert JT, Cox L, Kahl S, Elsasser TH, Ross JW, ISOM SC, Rhoads RP, Baumgard LH.  2015.  Effects of heat stress on carbohydrate and lipid metabolism in growing pigs.  Physiol Rep. 3(2) . pii: e12315. doi: 10.14814/phy2.12315. Print 2015 Feb 1.  PMID: 25716927 Sargent, K.A., R. Spuri-Gomes, J.R. Essink, S.G. Kurz, W.E. Pohlmeier, N. Ferrara, G.J. Bouma, D.J. McLean, and A.S. Cupp.  2015. Sertoli cell-specific elimination of Vegfa impairs spermatogonial stem cell (SSC) maintenance and reduces fertility in male mice. 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Impact Statements

Understanding of the roles of epigenetic regulators and metabolic pathways will provide insights into establishment of pluripotent stem cell lines and allow for stem cell therapy for biomedical applications.

Date of Annual Report: 05/18/2017

Report Information

Annual Meeting Dates: 01/13/2017 - 01/13/2017
Period the Report Covers: 10/01/2015 - 09/30/2016

Participants

Milan Shipka (Committee Administrative Advisor; Alaska)
Rick Rorie (Arkansas)
Quinton Winger (Colorado)
Cindy Tian (Connecticut)
Matt Wheeler (Illinois)
Curt Youngs (Iowa)
Ken Bondioli (Louisiana)
Carol Keefer (Maryland)
Brett White (Nebraska)

Brief Summary of Minutes

Minutes of the W3171 Technical Committee Meeting

Renaissance Austin Hotel, Austin, TX

January 13, 2017

The meeting was called to order at 1:05 pm by Committee Chair, Rick Rorie (Arkansas). In attendance were Rick Rorie (Arkansas), Quinton Winger (Colorado), Cindy Tian (Connecticut), Matt Wheeler (Illinois), Curt Youngs (Iowa), Ken Bondioli (Louisiana), Carol Keefer (Maryland) and Brett White (Nebraska). In addition, graduate student, Mohan Acharya (Arkansas), attended the meeting as a guest.

The meeting agenda was reviewed and adjusted as there was no report from our USDA/NIFA Administrator, Mark Mirando. Curt Youngs (Iowa) made a motion to approve the minutes from the previous meeting as read and Carol Keefer (Maryland) seconded the motion. The minutes were unanimously approved. W3171 Administrator, Milan Shipka, addressed committee members. Milan discussed some budget changes for USDA/NIFA AFRI proposals as well as provided guidance for submission of the project's report of accomplishments, impacts and publications, emphasizing our cooperative/collaborative work on research projects.

Station reports were presented and discussed for Arkansas, Colorado, Connecticut, Illinois, Iowa, Louisiana, Maryland and Nebraska. Potential new collaborative projects were discussed at length among the participating station representatives. Areas of investigation with the potential for collaboration among stations include: effects of energy sources and sex on embryo metabolism, effects of gamete/embryo culture and cryopreservation techniques on epigenetics, X-chromosome inactivation, fertility in non-traditional sheep breeds and gene knockdown approaches to evaluate reproductive mechanisms in livestock.

Kiho Lee (Virginia) was nominated to serve as Secretary for the W3171 Technical Committee in 2017. Since he was not in attendance, a subsequent conversation with Kiho Lee resulted in his acceptance of this position. Brett White (Nebraska) will serve as Chair for the coming year. The 2018 IETS Conference will be held at Jeju Island, South Korea. Therefore, the committee voted to meet in either Anchorage or Fairbanks, Alaska, which was graciously offered by our Administrator, Milan Shipka. Suggested dates for the meeting were late May/early June 2018 and will be determined at a later date.

Meeting adjourned at 4:45 pm.

Respectively Submitted,

Brett White - Secretary

W3171 Technical Committee

Accomplishments

Objective 1   Results indicated that either Anti-Mullerian Hormone (AMH) concentration at breeding or the change in AMH from weaning to breeding can identify beef heifers more likely to conceive to AI and to conceive early in the breeding season.  Anti-Mullerian hormone (AMH) also reduces the follicular growth rate of isolated goat preantral follicles in vitro without affecting follicular survival.   Generated a list of genes suitable to serve as quantitative references during embryonic development.   Developed a bioinformatics tool for cross-stage mining of genes that are co-regulated and conserved among different species. This tool is available for any scientist to use without charge.   Determined that it is possible predict the future development of in vitro produced bovine embryos after only 2 days of culture using 1H-nuclear magnetic resonance (NMR).   Evaluation of embryo metabolism by Gas Chromatogrpahy-Mass spectrometry (GC-MS) demonstrated that this approach is capable of measuring substrate use and metabolic pathway fluxes in as few as 4 embryos and that substrate balance in the media has a profound influence on pathway activities (e.g., glycolysis).   Developed a new electric channel device method to select high motility sperm without harming sperm characteristics. These results showed that our device produced the same quality of sperm obtained with discontinuous Percoll gradients and Swim-Up but in as little as 20 min.   Analysis of hormone levels in the blood of GnRH-II receptor knockdown and littermate control boars during pubertal development indicated that testosterone secretion was impaired in GnRHR-II KD males, however, LH levels were similar between swine lines.   Initial characterization of global epigenetic marks (histone modifications) in oocytes derived from small vs. large (pre-ovulatory) follicles revealed that there may be no difference in H3K4me3, but that H3K9me3, H3K27me3, and DNA methylation levels may be different between oocyte from the different sized follicles.   Objective 2   Methods for efficient delivery of solutions into livestock oocytes and zygotes as well as robust derivation of bovine embryonic stem cells were developed.   Established a reprogramming assay to study mechanisms/factors for naïve-state pluripotent iPSC generation.   Determined that novel collagen-glycosaminoglycan hydrogel (CG) scaffolds support bone and cartilage growth from mesenchymal stem cells.   Ascertained that zinc has a positive effect on bone formation of adipose stem cells in vitro.   Demonstrated that site directed insertion of a large transgene construct can be efficiently attained by using CRISPR/Cas9.   Preliminary results indicated that vitrification solutions containing glycerol in combination with ethylene glycol result in greater changes in DNA methylation levels than solutions containing DMSO in combination with ethylene glycol.   Developed and optimized smaller-sized variants of the Dracula micromanipulation system for use on embryos of other species.   Modification of the CFTR gene in sheep cells has been remarkably efficient.  Very high pregnancy rates have resulted from embryo transfers using cloned embryos derived from CFTR-modified karyoplast donor cells.

Publications

Baker, C.M., L.N. Goetzmann, J.D. Cantlon, K.M. Jeckel, Q.A. Winger, and R.V. Anthony RV. 2016. Development of ovine chorionic somatomammotropin hormone-deficient pregnancies. Am. J. Physiol. Regul. Integr. Comp. Physiol. 310:R837-R846.   Bogliotti, Y.S., M. Vilarino, and P.J. Ross. 2016. Laser-assisted cytoplasmic microinjection in livestock zygotes. J. Vis. Exp. 116.   Brauer, V.M., J.R. Wiarda-Bell, A.T. Desaulniers, R.A. Cederberg, and B.R. White. 2016. Functional activity of the Gnrhr2 gene promoter in testis-derived cells is partially conferred by nuclear factor-kkB, specificity protein 1 and 3 (SP1/3) and overlapping early growth response 1/SP1/3 binding sites. Gene 587:137-146.   Chio, I.I., S.M. Jafarnejad, M. Ponz-Sarvise, Y. Park, K. Rivera, W. Palm, J. Wilson, V. Sangar, <ol> <li>Hao, D. Öhlund, K. Wright, D. Filippini, E.J. Lee, B. Da Silva, C. Schoepfer, J.E. Wilkinson,</li> </ol> J.M. Buscaglia, G.M. DeNicola, H. Tiriac, M. Hammell, H.C. Crawford, E.E. Schmidt, C.B. Thompson, D.J. Pappin, N. Sonenberg, and D.A. Tuveson. 2016. NRF2 promotes tumor maintenance by modulating mRNA translation in pancreatic cancer. Cell 166:963-976.   Davis, K.A., K.M. Klohonatz, G.J. Bouma, and J.E. Bruemmer. 2016. Androgen receptor in the term equine placenta. J. Eq. Vet. Sci. 54:8-11.   Diaz, F., K. Bondioli, D. Pacamonti, and G.T. Gentry. 2016 .Cryopreservation of Day 8 equine embryos after blastocyst micromanipulation and vitrification Theriogenology 85:894-903.   Dóka, É., I. Pader, A. Bíró, K. Johansson, Q. Cheng, K. Ballagó, J.R. Prigge, D. Pastor-Flores, T.P. Dick, E.E. Schmidt, E.S. Arnér, and P. Nagy. 2016. A novel persulfide detection method reveals protein persulfide- and polysulfide-reducing functions of thioredoxin and glutathione systems. Science Advances. 2:e1500968.   Gladyshev, V.N., E.S. Arnér, M.J. Berry, R. Brigelius-Flohé, E.A. Bruford, R.F. Burk, B.A. Carlson, S. Castellano, L. Chavatte, M. Conrad, P.R. Copeland, A.M. Diamond, D.M. Driscoll, A. Ferreiro, L. Flohé, F.R. Green, R. Guigó, D.E. Handy, D.L. Hatfield, J. Hesketh, P.R. Hoffmann, A. Holmgren, R.J. Hondal, M.T. Howard, K. Huang, H.Y. Kim, I.Y. Kim, J. Köhrle, A. Krol, G.V. Kryukov, B.J. Lee, B.C. Lee, X.G. Lei, Q. Liu, A. Lescure, A.V. Lobanov, J. Loscalzo, M. Maiorino, M. Mariotti, P.K. Sandeep, M.P. Rayman, S. Rozovsky, G. Salinas, E.E. Schmidt, L. Schomburg, U. Schweizer, M. Simonović, R.A. Sunde, P.A. Tsuji, S. Tweedie, F. Ursini, P.D. Whanger, and Y. Zhang. 2016. Selenoprotein gene nomenclature. J. Biol. Chem. 291:24036-24040.   Hill, R., A . Canal, K. Bondioli, R. Morell, and M.D. Garcia. 2016. Molecular markers located on the DGAT1, CAST, and LEPR genes and their associations with milk production and fertility traits in Holstein cattle. Genet. Mol. Res. 15:gmr.15017794.   Hollister, S. J., C.L. Flanagan, D.A. Zopf, R.J. Morrison, H. Nasser, M.B. Wheeler, and G.E. Green. 2016. Design and quality control for translating 3D-printed scaffolds. In:  Essentials of 3D Biofabrication and Translation. Elsevier, Inc. pp. 43-59.   Hollister, S.J., C.L. Flanagan, R.J. Morrison, J.J. Patel, M.B. Wheeler, S.P. Edwards, and G.E. Green. 2016. Integrating image-based design and 3D biomaterial printing to create patient specific devices within a design control framework for clinical translation. ACS Biomater. Sci. Eng. 2:1827−1836.   Klohonatz, K.M., A.D. Cameron, J.R. Hergenreder, J.C. da Silveira, A.D. Belk, D.N.R. Veeramachaneni, G.J. Bouma, and J.E. Bruemmer. 2016. Circulating miRNAs as potential alternative cell signaling associated with maternal recognition of pregnancy in the mare. Biol. Reprod. 95:124.   Lan, A., W. Li, Y. Liu, Z. Xiong, X. Zhang, S. Zhou, O. Palko, H. Chen, M. Kapita, J.R. Prigge, E.E. Schmidt, X. Chen, Z. Sun, and X.L. Chen. 2016. Chemoprevention of oxidative stress associated oral carcinogenesis by sulforaphane depends on NRF2 and the isothiocyanate moiety. Oncotarget. 7:53502-53514.   Legacki, E.L., E.L. Scholtz, B.A. Ball, S.D. Stanley, T. Berger, and A.J. Conley. 2016. The dynamic steroid landscape of equine pregnancy mapped by mass spectrometry. Reproduction 151:421-430.   Lyman, J.T., D.K. Oh, G. Torres, R. Magin, M.B. Wheeler. 2016. The use of nuclear magnetic resonance spectroscopy for viability predictions on mouse preimplantation embryos. Int. J. New Tech. Res. 2:14-20.   Martins, L.T., S. Gaudêncio Neto, K.C.S. Tavares, C.E.M. Calderón, L.H. Aguiar, C. Lazzarotto, F.L. Ongaratto, V.H.V. Rodrigues, F.E. Lopes, L.P.R. Teixeira, I.S. Carneiro, R. Rossetto, A.P. Almeida, S.S.L. Fernandes, D. Rondina, I.A. Polejaeva, J.L. Rodrigues, F. Forell, L.R. Bertolini, and M. Bertolini. 2016. Developmental outcome and related abnormalities in goats: comparison between somatic cell nuclear transfer- and in vivo-derived concepti during pregnancy through term. Cell. Reprogram. 18:264-279.   McDonald, E.A., J.E. Smith, R. A. Cederberg, and B.R. White. 2016. Divergent activity of the gonadotropin-releasing hormone receptor gene promoter among genetic lines of pigs is partially conferred by nuclear factor (NF)-kkB, specificity protein (SP)1-like and GATA-4 binding sites. Reprod. Biol. Endocrinol. 14:36.   Mizell, S., S. Miller, K. Bondioli, and M.D. Garcia. 2016. A comparison of F1 steers sired by one of three paternal breed types for growth and carcass traits. EC Veterinary Science 2.4:177-181.   Monzani, P.S., P.R. Adona, F.V. Meirelles, O.M. Ohashi, and M.B. Wheeler. 2016. Transgenic bovine as bioreactor: challenges and perspectives. Bioengineered 7:123-131.   Newberry, H.R., T.D. Lester, B. Kegley, C. Rosenkrans, and R.W. Rorie. 2016. Use of anti-mullerian hormone to select for fertility in beef heifers. Discovery 17:79-84.   Polejaeva, I.A., H.M. Rutigliano, and K. Wells. 2016. Livestock in biomedical research: history, current status and future prospective. Reprod. Fertil. Dev. 28:112-124.   Polejaeva, I.A., R. Ranjan, C.J. Davies, M. Regouski, J. Hall, A.L. Olsen, Q. Meng, H.M. Rutigliano, D.J. Dosdall, N.A. Angel, F.B. Sachse, T. Seidel, A.J. Thomas, R. Stott, K.E. Panter, P.M. Lee, A.J. Van Wettere, J.R. Stevens, Z. Wang, R.S. MacLeod, N.F. Marrouche, and K.L. White. 2016. Increased susceptibility to atrial fibrillation secondary to myocardial fibrosis in transgenic goats with cardiac specific overexpression of transforming growth factor- B1. J. Cardiovasc. Electrophysiol. 27:1220-1229.   Rorie, R.W., C.L. Williams, and T.D. Lester. 2016. Association of osteopontin gene promoter single nucleotide polymorphisms with bull semen quality. Adv. Reprod. Sci. 4:1-7.   Rubessa, M., A. Ambrosi, D. Gonzalez-Pena, K.M. Polkoff, S.E. Denmark, and M.B. Wheeler. <ol start="2016"> <li>Non-invasive analysis of bovine embryo metabolites during in vitro embryo culture</li> </ol> using nuclear magnetic resonance. AIMS Bioengineering 3:538-551.   Rubessa, M., A. Ambrosi, S.E. Denmark, and M.B. Wheeler. 2016. Non-invasive analysis of gamete metabolites during in vitro embryo production using nuclear magnetic resonance. Int. J. New Tech. Res. 2:54-58.   Rubessa, M., A. Gaja, and M.B. Wheeler. 2016. Separation of motile bovine spermatozoa for in vitro fertilization by electrical charge. Andrology (Los Angel) 5:153.   Sun, J., Z. Jiang, X.C. Tian, and J. Bi. 2016. A cross-species bi-clustering approach to identifying conserved co-regulated genes. Bioinformatics 32:i137-i146.   Shepardson, K.M., K. Larson, R.V. Morton, J.R. Prigge, E.E. Schmidt, V.C. Huber, and A. Rynda-Apple. 2016. Differential type I interferon signaling is a master regulator of susceptibility to postinfluenza bacterial superinfection. MBio 7(3).   Twenter, H.M., A.D. Belk, K.M. Klohonatz, L.D. Bass, G.J. Bouma, and J.E. Bruemmer. 2016. An investigation into miRNAs in the equine epididymis as potential regulators of spermatozoal maturation. J. Eq. Vet. Sci. 48:61-68.   Vansandt, L.M., J.L. Livesay, J.L., M.J. Dickson, L. Li, B.S. Pukazhenthi, and C.L. Keefer. <ol start="2016"> <li>Conservation of spermatogonial stem cell marker expression in undifferentiated</li> </ol> felid spermatogonia. Theriogenology 86:1022-1035.   Yang, M., J. Hall, Z. Fan, M. Regouski, Q. Meng, H.M. Rutigliano, R. Stott, K.A. Rood, K.E. Panter, and I.A. Polejaeva. 2016. Oocytes from small and large follicles exhibit similar development competence following goat cloning despite their differences in meiotic and cytoplasmic maturation. Theriogenology 86:2302-2311.   Youngs, C.R. 2016. Modern reproductive technologies to improve cattle production. Ceiba 54:31-40.

Impact Statements

Understanding the genetic regulation of placental development may result in methods to improve the efficiency of somatic cell nuclear transfer.

Date of Annual Report: 06/04/2018

Report Information

Annual Meeting Dates: 05/31/2018 - 06/01/2018
Period the Report Covers: 10/01/2016 - 09/30/2017

Participants

Charles Rosenkrans (University of Arkansas)
Anna Denicol (University of California - Davis)
Jerry Bouma (Colorado State University)
Cindy Tian (University of Connecticut)
Ken Bondioli (Louisiana State University)
Carol Keefer (University of Maryland)
Brett White (University of Nebraska - Lincoln)
Clay Isom (Utah State University)
Irina Polejaeva (Utah State University)

Brief Summary of Minutes

Brief minutes of the W3171 Technical Committee Meeting

University of Alaska Fairbanks, Fairbanks, AK

May 31 – June 1, 2018

May 31, 2018

The meeting, held in Room 501 of the Akasofu Building (International Artic Research Center) at the University of Alaska Fairbanks, was called to order at 8:15 am on May 31^st by Committee Chair, Brett White (Nebraska). In attendance were Charles Rosenkrans (Arkansas), Anna Denicol (California), Jerry Bouma (Colorado), Cindy Tian (Connecticut), Ken Bondioli (Louisiana), Carol Keefer (Maryland), Brett White (Nebraska), Clay Isom (Utah), Irina Polejaeva (Utah) and Milan Shipka (Administrative Advisor).

The meeting agenda was reviewed and approved. Charles Rosenkrans (Arkansas) made a motion to approve the minutes from the previous meeting as read and Carol Keefer (Maryland) seconded the motion. The minutes were unanimously approved. W3171 Administrator Advisor, Milan Shipka, welcomed committee members to the University of Alaska Fairbanks and provided some historical perspectives regarding the University of Alaska Fairbanks. He also provided guidance for submission of the project's report of accomplishments, impacts and publications. Our National Institute of Food and Agriculture (NIFA) representative, Mark Mirando, joined the meeting via teleconference. Dr. Mirando provided updates from NIFA on news and personnel, the budget for the 2018 fiscal year, competitive programs, outcomes for Agriculture and Food Research Initiative (AFRI) Foundational Programs in fiscal year 2017 and interagency funding opportunities.

Station reports were presented and discussed for Arkansas, California, Colorado, Connecticut, Louisiana, Maryland and Nebraska. In addition, Charles Rosenkrans (Arkansas) initiated a discussion regarding an integrated grant proposal (Influence of sexed semen on profitability of livestock producers) previously prepared by members of the W3171 Technical Committee. Interest in submitting a NIFA integrated proposal to the AFRI Sustainable Agricultural Systems Request for Applications was gauged. Consensus of the committee was that the letter of intent deadline (6/27/2018) was too near for the current grant cycle, however, additional discussion on preparing a proposal in 2019 would take place via email.

At 3:30 pm, the meeting was adjourned for the day. The committee was provided a tour of the Robert G. White Large Animal Research Station. An interesting overview of the research being performed on muskox and reindeer was provided by Dr. Jan Rowell. Following the tour, committee members enjoyed dinner at the Alaska Salmon Bake in Pioneer Park.

June 1, 2018

The committee resumed its annual meeting at 8:30 am. We began with our final report from the Utah station. During the business meeting, Anna Denicol (California) was nominated to serve as Secretary for the W3171 Technical Committee in 2018. Charles Rosenkrans (Arkansas) made a motion to approve the nomination and Ken Bondioli (Louisiana) seconded the motion. The motion to elect Anna Denicol (California) as Secretary was unanimously approved. Kiho Lee (Virginia) will serve as Chair for the upcoming year. The 2019 annual meeting of the W3171 Technical Committee will be held in conjunction with the 2019 International Embryo Technology Society Conference at the Sheraton New Orleans Hotel in New Orleans, Louisiana. The meeting will begin at 8 am on Thursday, January 24, 2019.

The rewrite for the W3171 committee is due by January 15, 2019. A writing committee was established. Brett White (Nebraska) and Ken Bondioli (Louisiana) agreed to serve as Chair and Co-Chair of the committee, respectively. Jerry Bouma (Colorado) and Quinton Winger (Colorado) also agreed to serve on the committee. In regard to development of the outline, Cindy Tian (Connecticut) will oversee the review of Objective 1 with assistance from Clay Isom (Utah). Irina Polejaeva (Utah) will manage the review of Objective 2 with assistance from Kiho Lee (Virginia). To assist with the rewrite, monthly online meetings and establishing a Google document were recommended.

The committee thanked our Administrative Advisor, Milan Shipka, for graciously hosting the meeting and the wonderful hospitality. The meeting was adjourned at 12:10 pm.

Respectively Submitted,

Brett White – Acting Secretary

W3171 Technical Committee

Minutes Attachment:

2018W3171MtgMinutes.docx

Accomplishments

Objective 1 <ul> <li>The vaginal and fecal microbiota of beef heifers was characterized to explore a relationship between bacterial signatures and fertility.</li> <li>The roles of OCT4 and KDM6B during bovine early embryogenesis were determined.</li> <li>Preliminary characterization of bovine oogonial stem cells was accomplished.</li> <li>Systematically compared dosage compensation of the X chromosome in ovine fetuses developed under different nutrition statuses.</li> <li>RNA sequencing data comparing oocytes collected by ovum pick up (OPU) or from slaughterhouse ovaries identified differentially expressed genes in both the germinal vesicle and metaphase II stages that can be investigated as molecular markers of developmental competence.</li> <li>Analysis of embryo metabolism by gas chromatography-mass spectrometry (GC-MS) demonstrated that substrate balance in the media has a profound influence on pathway activities (e.g., glycolysis) as well as the extent that media substrates are metabolized.</li> <li>CRISPR/Cas9 gene editing was utilized to knockout pig conceptus IL1B2 gene expression and the secretion of IL1B2 during the time of conceptus elongation.</li> <li>Histological evaluation of testicular tissue revealed that testes of GnRH-II receptor knockdown males had fewer, but larger Leydig cells and seminiferous tubules than littermate control testes.</li> </ul> Objective 2 <ul> <li>Preparation of sex-sorted semen for in vitro fertilization (IVF) was optimized for maximal performance in cattle.</li> <li>Bovine embryonic stem cells were derived with high efficiency.</li> <li>Targeting and genome editing constructs for KDM1A were designed, tested in vitro, and used to study their function within the placenta in vivo.</li> <li>Different specific shRNA target constructs were designed to knockdown LIN28A and LIN28B and have been tested in vitro.</li> <li>Determined the roles of Akt3 in embryonic stem cell survival and proliferation.</li> <li>Determined that there is no positive effect of Vitamin K or B-12 on bone formation of adipose stem cells in vitro.</li> <li>Exposure of immature and mature bovine oocytes to DMSO- and glycerol-based vitrification solutions, as well as vitrification with these solutions, resulted in minor or no change in epigenetic marks such as DNA methylation and histone acetylation.</li> <li>Through using the CRISPR/Cas9 gene editing system, we disrupted up to three genes simultaneously at near 100% efficiency in vitro and successfully knocked out expression of conceptus-specific genes.</li> </ul>

Publications

Berger, T., and B. J. Nitta-Oda. 2018. Increased testicular estradiol during the neonatal interval reduces Sertoli cell numbers.  Anim. Reprod. Sci. 189:146-151. Bishop, M.O., J.R. Stevens, and S.C. Isom. 2017. Statistical methods for assessing individual oocyte viability through gene expression profiles. Proceedings of the Kansas State University Conference on Applied Statistics in Agriculture 2017. Bogliotti, Y.S., J. Wu, M. Vilarino, D.A. Soto, J.C. Izpisua Belmonte, and P.J. Ross. 2018. Efficient derivation of stable primed pluripotent embryonic stem cells from bovine blastocysts. Proc. Natl. Acad. Sci. 115:2090-2095. Bondioli, K.R., Cloning of livestock by somatic cell nuclear transfer. 2018. In: Neiman, H. and Wrenzycki, C. (eds). Animal Biotechnology 2: Emerging Breeding Technologies. Springer International Publishing AG. (In Press). Cadenas, J., C. Maside, A.C.A Ferreira, L.A. Vieira, J. Leiva-Revilla, V.M. Paes, B.G. Alves, F.Z. Brandão, A.P.R. Rodrigues, M.B. Wheeler, and J.R. Figueiredo. 2018. Relationship between follicular dynamics and oocyte maturation during in vitro culture as a non-invasive sign of caprine oocyte meiotic competence. Theriogenology 107:95-103. Center, K., D. Dixon, C. Looney, and R. Rorie<a name="_Toc415661467"></a>.  2018.  Anti-mullerian hormone and follicle counts as predictors of superovulatory response and embryo production in beef cattle.  Adv. Reprod. Sci. 6:22-33. Chung N, Y. Bogliotti, W. Ding, M. Vilarino, K. Takahashi, J. Chitwood, R. Schultz, and P.J. Ross. 2018. Active H3K27me3 demethylation by KDM6B is required for normal development of bovine preimplantation embryos. Epigenetics 16:1-9. Daigneault B.W., S. Rajput, G.W. Smith, and P.J. Ross. 2018. Embryonic OCT4 is required for expanded bovine blastocyst formation. Sci. Rep. 8:7753. da Silveira J.C., A.C.F.C.M. de Avila, H.L. Garrett, J. Bruemmer, Q. Winger, and G.J.  Bouma.  2017. Cell-secreted vesicles containing microRNAs as regulators of gamete maturation. J. Endocrinol. 236:R15-R27 Delgado, P.A., T.D. Lester, and R.W. Rorie.  2018. <a name="_Toc216171922"></a><a name="_Toc213646852"></a> Effect of a low-sodium, choline-based semen diluent on viability of bovine sperm stored at 4° C.  Adv. Reprod. Sci. 6:12-21. Desaulniers, A.T., R.A. Cederberg, C.A. Lents, and B.R. White.  2017.  Expression and role of gonadotropin-releasing hormone 2 and its receptor in mammals.  Front. Endocrinol. 8:269. Desaulniers, A.T., R.A. Cederberg, G.A. Mills, C.A. Lents, and B.R. White.  2017.  Production of a gonadotropin-releasing hormone 2 receptor knockdown (GnRHR2 KD) swine line.  Transgenic Res. 26:567-575. Diaz, F.A., E.J. Gutierrez, E. Cramer, D.L. Paccamonti, G.T. Gentry, and K.R. Bondioli. 2018. Pregnancy rates following low-temperature storage of large equine embryos prior to vitrification. J. Equine Vet. Sci. 64:12-16. Fan Z., M. Yang, M. Regouski, and I.A. Polejaeva. 2017. Effects of three different media on in vitro maturation and development, intracellular glutathione and reactive oxygen species levels, and maternal gene expression of abattoir-derived goat oocytes. Small Rumin. Res. 147:106-114. Feltrin, C., L.H. de Aguiar, C.E.M. Calderón, I. de Sá Carneiro, F. de Jesus Moraes, J. da Silva Quetz., A.A.M. Lima, M.B. Wheeler, D. Rondina, J.L. Rodrigues, J.D. Murray, E.A. Maga, L.R. Bertolini, and M. Bertolini. 2017. Effects of oocyte source, cell origin, and embryo reconstruction procedures on in vitro and in vivo embryo survival after goat cloning. Anim. Reprod. 14:1110-1123. Ferre L, J. Chitwood, C. Fresno, H. Ortega, M. Kjelland, and P.J. Ross. 2017. Effect of different mini-volume colloid centrifugation configurations on flow-cytometrically-sorted sperm recovery efficiency and quality using a computer-assisted semen analyzer. Reprod. Domest. Anim. 53:26-33. de Lima L.F., M. Rubessa, R.M.P. <a href="https://www.ncbi.nlm.nih.gov/pubmed/?term=Rocha%20RMP%5BAuthor%5D&cauthor=true&cauthor_uid=28552178">Rocha, </a>R. Winters, D.J. Milner, C.C. Campell, J.R. Figueiredo, and M.B. Wheeler. 2017. High diluted and dynamised follicle stimulating hormone modulates steroid production in isolated porcine preantral follicles cultured in vitro. Homeopathy 106:87-92. de Lima, L.F., R.M.P. Rocha, A.B.G. Duarte, I.R. Brito, G.M. Silva, G.Q. Rodrigues, A.D. Sales, C.H. Lobo, A.A. Moura, M.B. Wheeler, A.P.R. Rodrigues, C.C. Campello, and J.R. Figueiredo. 2017. Unexpected effect of the vehicle of homeopathic FSH (grain ethanol) on the in vitro survival and development of isolated ovine preantral follicles. Microsc. Res. Tech. 80:406-418. Geisert R.D., J.J. Whyte, A.E. Meyer, D.J. Mathew, M.C. Lucy, R.S. Prather, and T.E. Spencer. 2017. Rapid conceptus elongation in the pig: Interleukin 1 beta 2 and estrogen regulated phenomena. Mol. Reprod. Dev. 84:760-774. Geisert R.D. and M.C. Lucy. 2018. Comparative Mammalian Female Reproduction-Pig. In: The Encyclopedia of Reproduction Vol 2., Chapter 91. Geisert R.D., M.F. Smith, and J.A. Green. 2018. Utilizing a rat delayed implantation model to teach integrative endocrinology and reproductive biology. Adv. Physiol. Educ. 42:56-63. Huang, D., L. Wang, J. Duan, C. Huang, X. Tian, M. Zhang, and Y. Tang. 2017. LIF-activated Jak signaling determines Esrrb expression during late-stage reprogramming. Biology Open. 7:29264. Hughes, J.R., and T. Berger.  Regulation of apical blebbing in the porcine epididymis. J. Anat. 232:515-522. Lents, C.L., J.F. Thorson, A.T. Desaulniers, and B.R. White.  2017.  RFamide-related peptide 3 and gonadotropin-releasing hormone-II are autocrine-paracrine regulators of testicular function in the boar. Mol. Reprod. Dev. 84:994-1003. Lotti, S.N., K.M. Polkoff, M. Rubessa, and M.B. Wheeler. 2017. Modification of the genome of domestic animals. Anim. Biotechnol. 28:198-210. Meyer, L.R., J.G. Powell, B.R. Kutz, M.L. Looper, A.H. Brown, Jr., and C.F. Rosenkrans, Jr.  2017. Associations of single nucleotide polymorphisms in the bovine prolactin gene with phenotypic traits in beef cattle. Agri. Gene. 5:7-11. Nguyen, T.H., M.E. Kandel, M. Rubessa, M.B. Wheeler, and G. Popescu. 2017. Gradient light interference microscopy (GLIM) for 3D imaging of unlabeled specimens. Nat. Commun. 8:210. Milner, D.J., M. Bionaz, E. Monaco, J.A. Cameron, J and M.B. Wheeler. 2018. Myogenic potential of mesenchymal stem cells isolated from porcine adipose tissue. Cell Tissue Res. 372:507-522. Polkoff, K.M., and M.B. Wheeler. 2017. What’s coming: How biotechnology changes everything! 12th Simposium Internacional de Reproduction Animal Proceedings, pages 338-344. Rocha, R.M.P., L.F. Lima, I.R. Brito, G.M. Silva, H.H.V. Correia, N.A. Ribeiro de Sá, A.C.A. Ferreira, A.D. Sales, C.H. Lobo, C.C. Campello, J. Smitz, M.B. Wheeler, and J.R. Figueiredo, 2017. Anti-Müllerian hormone (AMH) reduces growth rate without altering follicular survival in isolated caprine preantral follicles cultured in vitro. Reprod. Fertil. Dev. 29:1144-1154. Rubessa, M., K. Polkoff, M. Bionaz, E. Monaco, D.J. Milner, S.J. Holllister, M.S. Goldwasser, and M.B. Wheeler. 2017. The use of the pig as a model for bone regeneration. Anim. Biotechnol. 28:275-287. Rubessa, M., and M.B. Wheeler. 2017. Advancing bovine in vitro fertilization and embryo development: New analytics. 2017. 12th Simposium Internacional de Reproduction Animal Proceedings, pages 316-323, Cordoba, AR. Rutigliano H.M., A. Wilhelm, J. Hall, B. Shi, Q. Meng, R. Stott, T.D. Bunch, K.L. White, C.J. Davies, and I.A. Polejaeva. 2017. Cytokine gene expression in the maternal-fetal interface in somatic cell nuclear transfer pregnancies in small ruminants. Reprod. Fertil. Dev. 29:646-657. Silva, G.M., I.R. Brito, A.D. Sales, F.L.N. Aguia, A.B.G. Duarte, V.R. Araújo, L.A. Vieira, D.M. Magalhães-Padilha, L.F. Lima, B.G. Alves, L.B.R. Silveira, E.G. Lo Turco, A.P. Rodrigues, C.C. Campello, M.B. Wheeler, and J.R. Figueiredo. 2017. In vitro growth and maturation of isolated caprine preantral follicles: influence of insulin and FSH concentration, culture dish, co-culture and oocyte size on meiotic resumption. Theriogenology. 90:32-41. Silva, R.F., I.R. Brito, L.F. Lima, F.L.N. Aguiar, G.Q. Rodrigues, I.L.C. Nascimento, R.M.P. Rocha, F.W.S. Cibin, M.B. Wheeler, C.C. Campello, A.P.R. Rodrigues, and J.R. Figueiredo. 2017. Platelet-derived growth factor-BB (PDGF-BB) improved in a concentration-dependent manner follicular survival as well as oocyte and follicular diameters after in vitro culture of goat preantral follicle enclosed in ovarian fragments. Anim. Reprod. 14:1095-1102. Smith M.F., R.D. Geisert, and J.J. Parrish. 2018. Reproduction in domestic ruminants during the past 50 years: Discovery to application. J. Anim. Sci. 96:2952-2970. Tribulo, P., J.I. Moss, M. Ozawa, Z. Jiang, X.C. Tian, and P.J. Hansen. 2017. WNT regulation of embryonic development likely involves pathways independent of nuclear CTNNB1. Reproduction. 153:405-419. Vilarino M, S.T. Rashid, F.P. Suchy, B.R. McNabb, T. van der Meulen, E.J. Fine, S. Ahsan, N. Mursaliyev, V. Sebastiano, S.S. Diab, M.O. Huising, H. Nakauchi, and P.J. Ross. 2017 CRISPR/Cas9 microinjection in oocytes disables pancreas development in sheep. Sci. Rep. 7:17472 Wang, L., D. Huang, Z. Jiang, Y. Luo, C. Norris, M. Zhang, X. Tian, and Y. Tang. 2017. Akt3 is responsible for the survival and proliferation of embryonic stem cells. Biol. Open. 6:850-861. Wang, L., D.L. Huang, C. Huang, Y.X. Yin, K. Vali, M. Zhang, and Y. Tang. 2017. Enhanced human somatic cell reprogramming efficiency by fusion of the MYC transactivation domain and OCT4. Stem Cell Res. 25:88-97. Wang, L., J. Zongliang, D. Huang, J. Duan, C. Huang, S. Sullivan, K. Vali, Y. Yin, M. Zhang, J. Wegrzyn, X.C. Tian, and Y. Tang. 2018. Jak/Stat3 regulated global gene expression dynamics during late-stage reprogramming process. BMC Genomics. 19:183. Weisgerber, D.W., D.J. Milner, H. Lopez-Lake, M. Rubessa, S. Lotti, K.M. Polkoff, R.A. Hortensius, C.L. Flanagan, S.J. Hollister, M.B. Wheeler, and B.A.C. Harley. 2017. A mineralized collagen-polycaprolactone composite promotes healing of a porcine mandibular ramus defect. Tissue Eng. Part A. 24:943-954. Wheeler, M. B., and M. Rubessa. 2017. Integration of microfluidics in animal in vitro embryo production. Mol. Hum. Reprod. 23:248–256. Whyte W.W., A.E. Meyer, L.D. Spate, J.A. Benne, R. Cecil, M.S. Samuel, C.N. Murphy, R.S. Prather, and R.D. Geisert. 2018. Inactivation of porcine interleukin-1β (IL1B2) results in failure of rapid conceptus elongation. Proc. Natl. Acad. Sci. 115:307-312. Xu, W., B.A. Foster, M. Richards, K.R. Bondioli, G. Shah, and C.C. Green. 2018. Characterization of prostate cancer cell progression in zebrafish xenograft model. Int. J. Oncol. 52:252-260.

Impact Statements

Utilization of the CRISPR/Cas 9 gene editing system provides a powerful tool to evaluate the role of genes during early conceptus development.

Date of Annual Report: 02/28/2019

Report Information

Annual Meeting Dates: 01/19/2019 - 01/20/2019
Period the Report Covers: 10/01/2017 - 09/30/2018

Participants

Milan Shipka – University of Alaska Fairbanks (group administrator)
Rod Geisert - University of Missouri
Brett White - University of Nebraska Lincoln
Carl Jiang - Louisiana State University
Kiho Lee - Virginia Tech
Quint Winger - Colorado State University
Curt Youngs - Iowa State University
Young Tang - University of Connecticut
Irina Poleajava - Utah State University
Clay Isom - Utah State University
Matt Wheeler - University of Illinois
Anna Denicol - University of California Davis
Xiuchun (Cindy) Tian - University of Connecticut
Pablo Ross - University of California Davis
Jean Feugang - Mississippi State University
Charles Rosenbanks - University of Arkansas

Brief Summary of Minutes

Minutes Attachment:

Brief minutes of the 2019 meeting W3171 USDA Hatch and Multistate Group.pdf

Accomplishments

OBJECTIVE 1: Understand the biology and underlying mechanisms of gamete development, fertilization, and embryogenesis. <ol> <li>Relationships between anti-mullerian hormone (AMH) and first exposure pregnancy rate (PR) to spring or fall breeding, number of lambs born to first lambing, age at first lambing (using lifetime records), and estimated breeding values (EBVs) for number of lambs born (NLB), number of lambs weaned (NLW), maternal weaning weight (MWT), weaning weight (WWT), and maternal index were determined. While AMH did not appear to consistently predict fertility in ewes, higher EBVs (NLB, NLW, Index) were associated with younger ewes and greater prolificacy at first lambing.</li> <li>Pregnancy rate was greater in fall than spring bred Katahdin ewes. Ewes that became pregnant compared with non-pregnant had a greater concentration of AMH regardless of season bred but tended to have a lower concentration of prolactin. The overall lower prolactin in spring suggests a reduction associated with fescue toxins.  Selecting ewes more tolerant to fescue toxins may improve success of out-of-season breeding.</li> <li>The vaginal and fecal microbiota of beef heifers was characterized to explore a relationship between bacterial signatures and fertility. Our study shows that bovine vaginal and fecal microbiome could be used as biomarkers of bovine reproduction.</li> <li>ATAC-seq was used to identify changes in chromatin structure genomewide at the time of bovine genome activation.</li> <li>Increased testicular cortisol decreased Sertoli cell numbers in juvenile boar testis.</li> <li>Addition of specific concentrations of the fatty acid transport inhibitor FABP3-I to oocyte maturation medium reduces lipid accumulation in bovine oocytes and embryos without impairing embryonic development.</li> <li>The DNA methylome of bovine in vivo developed pre-implantation embryos was systematically characterized.</li> <li>Previous confusions of different types of stem cells in the porcine testis were clarified.</li> <li>In vitro follicle development can be paired with in vitro oocyte maturation with specific individual follicles. Using this approach, it was possible to stablish a non-invasive marker for the efficiency of IVFC based on follicle daily growth rate and diameter. In addition, 18 days seems to be the more suitable culture time for caprine early antral follicles (EAFs).</li> <li>Pretreatment with low concentrations (3%) of D2O improved the cleavage rate of bovine vitrified-warmed oocytes, suggesting a potential beneficial effect, whereas deleterious effects were observed using the higher concentrations. Therefore, further studies are required to assess a potential use of D2O to improve oocyte cryotolerance, likely testing different incubation times.</li> <li>Higher numbers of total and viable COCs were aspirated from Holstein lactating cows than heifers. Furthermore, cleavage and blastocyst rates were higher in lactating cows than in the heifers. Conventional semen gave more embryos than did sexed semen in heifers but not in cows. Overall, oocytes from Holstein lactating donors are more suitable for in vitro embryo production than are those from heifer donors.</li> <li>Using NMR, it was confirmed that sugar metabolism is different between sexes. The new information obtained identifies markers that can be used to predict the embryo sex. These results open a new, non-invasive method to effectively evaluate sex of the embryos before transfer.</li> <li>A method for isolation of spermatozoa from the highly viscous alpaca ejaculate was developed.</li> <li>It was confirmed that stimulation with FSH prior to ovum pick up (OPU) results in greater developmental competence compared to non-stimulated OPU or slaughterhouse derived oocytes. RNA seq data identified differentially expressed genes in both the germinal vesicle and metaphase II stages of oocytes retrieved by these methods and could serve as molecular markers of developmental competence.</li> <li>RNA seq analysis identified genes differentially expressed as a result of heat stress at the germinal vesicle (GV) stage and metaphase II (MII) stages of oocyte development. This information provides new insight into the molecular mechanisms of heat stress on bovine oocytes which may help to reveal master regulators controlling oocyte competence.</li> <li>Effects of heat stress on DNA methylation and DNA hydroxymethylation of bovine oocytes at the GV and MII stage. In this study there was no difference between cold and hot months in DNA methylation and DNA hydroxymethylation of GV stage and MII stage oocytes.</li> <li>Using both single cell/embryo reduced representation bisulfite sequencing (RRBS) and single-cell/embryo whole genome bisulfite sequencing (WGBS), stage-specific genome-wide dynamics of DNA methylation was characterized in bovine sperm, immature, in vivo and in vitro matured oocyte as well as in vivo developed early embryo stages. This study provides the first comprehensive analysis of the global dynamics of DNA methylation in bovine gametes and single early embryos. These data will serve as an important reference base for embryos produced by assisted reproduction such as in vitro fertilization and cloning as well as models for human early embryo development. </li> <li>Metabolites expelled into the medium were accurately monitored and identified from a single blastocyst thru metabolic flux analysis of heavy isotope labeling. This is tremendously lower number of embryos than the hundreds of embryos used in more traditional metabolic approaches. Measurement of mass isotopomer distributions of metabolites, chiefly pyruvate, lactate, citrate and amino acids, followed by correction for natural abundances and metabolic modeling revealed several metabolic insights.</li> <li>CRISPR/Cas 9 gene editing was utilized to knockout pig conceptus IL1B2 expression and the secretion of IL1B2 during the time of conceptus elongation. This study demonstrates that IL1B2 is involved with the rapid transformation of pig conceptuses from spherical to long filamentous form.</li> <li>CRISPR/Cas 9 gene editing was utilized to knockout pig conceptus CYP19A1 gene expression. Estrogen production by CYP19A1-/- conceptuses was inhibited on Day 14 and 17 of pregnancy. Trophoblast elongation and attachment occurred normally in both Day 14 CYP19A1+/+ and CYP19A1-/- conceptuses indicating that conceptus estrogen production was not essential to early development and attachment in the pig. CYP19A1-/- conceptuses maintained functional CL and progesterone production beyond Day 15 and embryo development continued to Day 25 of pregnancy. However, gilts carrying CYP19A1-/- embryos abort before or after Day 30 of pregnancy. Endometrial RNA-Seq analysis indicated that gene expression was altered in recipient gilts containing CYP19A1-/- conceptuses on Day 14 and 17. Co-transfer of IVF wild type conceptuses rescued the pregnancy of CYP19A1-/- conceptuses.</li> <li>CRISPR/Cas 9 gene editing was utilized to knockout pig conceptus PTGS2 gene expression. Results indicate that conceptus PTGS2 expression is not essential for blastocyst development, uterine migration, conceptus elongation, and establishment and maintenance of pregnancy.</li> <li>Serum testosterone concentrations responded differently to hCG treatment in GnRHR-II KD compared to littermate control boars; hCG-stimulated testosterone levels were significantly lower in GnRHR-II KD vs. control males.</li> <li>Treatment with a GnRH analogue that simultaneously agonizes GnRHR-II and antagonizes GnRHR-I revealed that circulating testosterone levels in control males tended to increase above baseline concentrations, whereas testosterone secretion was not stimulated in GnRHR-II KD animals.</li> <li>Concentrations of 10 different gonadal steroid hormones were either significantly lower or tended to be lower in serum from GnRHR-II KD compared to littermate control males.</li> <li>Mass spectrometry analysis of serum from mature GnRHR-II KD and littermate control boars indicated that concentrations of 10 different gonadal steroids did not differ between lines following castration.</li> <li>After castration, concentrations of all 10 gonadal steroids plummeted in control barrows, whereas only DHEA-S, testosterone and androstenedione production were decreased in GnRHR-II KD castrates.</li> <li>Semen analysis of mature males from each line suggested that number of artificial insemination doses and total motility were reduced in GnRHR-II KD boars, whereas total sperm per ejaculate, progressive motility and percentage of rapid cells tended to be reduced.</li> <li>On Day 10 of the estrous cycle, circulating progesterone concentrations were lower in GnRHR-II KD vs. littermate control gilts.</li> <li>Ovulation rate was reduced in GnRHR-II KD compared to control gilts, despite no difference in weight of the ovaries between lines.</li> <li>The active transposition into active chromatin (ATAC-seq) protocol was optimized to allow for mapping of open chromatin regions genome-wide in small numbers of cells. To reveal differences in chromatin status between in vitro fertilized (IVF) and SCNT embryos genome-wide, we have generated high-quality open chromatin maps from stage-specific preimplantation embryos generated from IVF or from SCNT embryos.</li> </ol> OBJECTIVE 2: Refine methods for production of genetically modified animals to improve livestock production efficiency. <ol> <li>A method to corroborate genotype/phenotype changes in bovine CRISRP mutated embryos was developed.</li> <li>CRISPR induced SOCS2-KO was optimized for sheep embryos.</li> <li>Embryo biopsy followed by embryo transfer identified issues of mutation chimerism after injection of CRISPR/Cas9 in sheep embryos.</li> <li>Targeting and genome editing constructs for KDM1A and FATP4 were designed, tested in vitro, and used to study their function in placenta in vivo.</li> <li>LIN28A and LIN28B have been knocked down in vivo and tissues collected. RNA and protein have been collected from the trophectoderm samples and knockdown confirmed. Let-7 miRNA levels have been measured and are confirmed to be altered in the knockdown samples and several let-7 target genes have been investigated.</li> <li>Preliminary data targeting FATP4 in the trophectoderm suggest significantly increased fetal muscle and fatty acid oxidation capacity. Several transfers have been completed during the past breeding season to establish pregnancies.</li> <li>Based on preliminary data generated involving KDM1A, a previously submitted NIH/USDA Dual Purpose award was funded (provided by USDA), and is starting January 2019. This 5-year grant proposal focuses on the role of KDM1A in regulating AR and ESR1 signaling in placental development and function, and involves both in vitro and in vivo experimental approaches.</li> <li>We generated lentiviral GFP fluorescent reporters controlled by bovine OCT4 and NANOG full-length enhancers, OCT4 distal enhancer, and OCT4 proximal enhancer regions, respectively. These reporters will help us to identify and enrich the reprogrammed cells during bovine iPSC generation. We also developed a reprogramming system that increases the TRA-1-60 positive colony formation ~50-100x at day 10 of reprogramming for human iPSC induction. The successful development of bovine fluorescent reporters and the highly efficient reprogramming system we developed provided the much needed tools to help identify the bovine-specific events that are under-regulated in reprogramming process compared with human iPSC induction.</li> <li>Naked DNA has been shown to bind naturally to the sperm, a method called sperm-mediated gene transfer (SMGT). In order to determine if the liposome-DNA complex had bound to sperm, real time PCR was used to detect GFP DNA and images of the sperm were analyzed using the Spatial Light Interference Microscopy (SLIM). SLIM confirmed the presence of liposomes on the sperm head and tail.</li> <li>Transgenic cows, one for the production of human proinsulin and another for factor IX of human blood coagulation were produced and are being submitted to induction of hormonal lactation for the evaluation of the expression of recombinant proteins in milk.</li> <li>Tissue-engineered cartilage was successfully produced on 3D-printed bioresorbable scaffolds using an adipose-derived stem cell and chondrocyte co-culture technique. This potentiates co-culture as a solution for several key barriers to a clinically translatable cartilage tissue engineering process.</li> <li>Co-culturing GFP-positive adipose-derived mesenchymal stem cells (ASC) with porcine satellite cells demonstrated enhanced myogenic capability of ASC, as the percentage of labeled myotubes increased compared to mouse co-cultures. Enhancing myogenic potential of ASC through soluble factor treatment or expansion of ASC with innate myogenic capacity should allow for their therapeutic use to regenerate muscle tissue lost to disease or injury.</li> <li>Novel collagen-glycosaminoglycan hydrogel (CG) scaffolds have shown promise for supporting bone and cartilage growth from mesenchymal stem cells. We have shown that there is a differential ability of ASC and BMSC to break down and metabolize the scaffold matrix and may indicate that one cell type may be preferable to the other for repairing osteogenic defects using these scaffolds. Current experiments underway will analyze expression of matrix-degrading enzymes to determine the source of the difference between cell types in scaffold shrinkage during differentiation.</li> <li>Higher concentrations of magnesium can improve nodule formation into osteogenic differentiation in vitro; the 2 mM concentration showed the highest nodule formation compared with the other groups. Low concentrations of copper (0.1 and 1 mM) and selenium (0.1, 0.5, and 1.0 mM) have positive effects on bone formation in vitro. These results showed the value of magnesium, copper and selenium in bone physiology.</li> <li>Monoallelic and biallelic targeted cells were used for cloning by somatic cell nuclear transfer (SCNT), resulting in 55% pregnancies per embryo transfer (30/55 recipients). Our findings demonstrate that site directed insertion of a large transgene construct can be efficiently attained by using CRISPR/Cas9 along with the suppression of the NHEJ-dedicated Ku70 protein.</li> <li>A tissue engineering approach to address craniofacial defects requires a biomaterial that balances macro-scale mechanical strength with the micron-scale features that promote cell expansion and tissue biosynthesis. We report an analytical approach to quantify radial, angular, and depth bone infill from micro-computed tomography data. The collagen-PCL composite showed improved overall infill, and significantly increased radial and angular bone infill versus the PCL cage alone. Bone infill was further enhanced in the composite for defects that penetrated the medullary cavity, suggesting recruitment of marrow-derived cells. These results indicate a multi-scale mineralized collagen-PCL composite offers strategic advantages for regenerative repair of craniofacial bone defects.</li> <li>Pregnancies were established from frozen-thawed and from vitrified-warmed alpaca preimplantation embryos.</li> <li>It was shown that oocytes collected by ovum pick-up (OPU) had decreased mitochondrial density, and increased incidence of irregularly shaped mitochondria as well as decreased vesicle density. FSH oocytes had a decreased density of cortical granules, especially noticeable following maturation. Oocytes recovered by conventional OPU had the poorest developmental rates, beginning with poor cleavage rates. Follicles collected from OPU are a heterogenous population of follicular sizes potentially resulting in a high variability in oocyte competence.</li> <li>Successfully knocked-out 5 Ig genes in goat fetal fibroblasts using CRISPR/Cas9 and SCNT</li> <li>Confirmed that human artificial chromosome (HAC) is functional in goats. HAC was introduced into goat fetal fibroblasts that were consecutively used for cloning. Human IgG were produced in the transchromosomal goats.</li> <li>A CFTR-/- sheep model was produced using CRISPR/Cas9 manipulation of sheep fetal fibroblasts that were subsequently reprogrammed and used to clone the CFTR null animals.</li> <li>A novel genetically modified line of sheep with an inactivated type I interferon receptor (IFNAR) was produced. These sheep: (1) should be more susceptible to viral infections than wildtype sheep and will make an excellent model for studying viral pathogenesis, and the effectiveness of viral vaccines and antivirals; and (2) female, IFNAR deficient sheep should be sterile due to an inability to respond to interferon-tau, the pregnancy recognition factor in ruminants.</li> <li>Immunodeficient pigs were used for human stem cell transplantation. The pigs could support proliferation and differentiation of lineage-specific organoids derived from human. These immunodeficient pigs were produced without SCNT or breeding step.</li> </ol>

Publications

Bai Y, Zhu C, Feng M, Wei H, Li L, Zhao Z, Liu S, Ma N, Zhang X, Shi R, Fu C, Tian X, Wu Z, Zhang S 2018. Previously claimed male germline stem cells from porcine testis are actually progenitor Leydig cells. Stem Cell Research & Therapy 9:200; https://doi.org/10.1186/s13287-018-0931-0. Behluli B, Musliu A, Sherifi K, Youngs CR, and Rexhepi A 2017. Risk factors for occurrence of displaced abomasum and their relation to nutritional management of Holstein dairy cattle. Veterinarski Arhiv 87(4):419-430. DOI: 10.24099/vet.arhiv.a60216 Benaissa MH, S Ansel, A Mohamed-Cherif, K Benfodil, D Khelef, CR Youngs, R Kaidi, and K Ait-Oudhia. 2017. Seroprevalence and risk factors for Coxiella burnetii, the causative agent of Q fever in the dromedary camel (Camelus dromedarius) population in Algeria. Onderstepoort Journal of Veterinary Research 84(1):a1461. https://doi.org/10.4102/ojvr.v84i1.1461 Benaissa MH, Faye B, Youngs CR, and Kaidi R  2016.  Slaughterhouse survey of culled female camels (Camelus dromedaries) in southeast Algeria: Fetal wastage and pregnancy characteristics. Emirates Journal of Food and Agriculture 28(11):805-812. 10.9755/ejfa.2016-06-735 Correia, H.H.V., Vieira, L.A., Mielgo, C.M., Paes, V.M., Alves, B.G., Viana, J.R., Wheeler, M.B., Rodrigues, A.P.R., Figueiredo, J.R. 2018. Cilostamide affects in a concentration and exposure time-dependent manner the viability and the kinetics of in vitro maturation of caprine and bovine oocytes. Research in Veterinary Science 2018 Nov 12;122:22-28. doi: 10.1016/j.rvsc.2018.11.002. CR Youngs 2018. Embryo transfer technologies in pigs In (H. Niemann and C. Wryzenski, Eds.) Animal Biotechnology 1: Reproductive Biotechnologies, Springer, pp 167- 178. Daigneault BW, Vilarino M, Rajput S, Frum  T, Smith GW, Ross PJ 2018. CRISPR editing validation, immunostaining and DNA sequencing of individual fixed bovine embryos. Biotechniques 65(5):281-283. doi: 10.2144/btn-2018-0051. Degefa T, A Lemma, E Demissie, S Ali, A Funga, and CR Youngs 2018. Superovulation response and in vivo embryo production potential of Boran cows in Ethiopia. Ethiop. J. Agric. Sci. 28(1):71-80. Degefa T, Lemma A, Jemal J, Mamo G, Tegegne A, and Youngs CR 2016.  Ovarian follicular dynamics in purebred and crossbred cows in Ethiopia. African Journal of Biotechnology 15(33):1763-1770. DOI: 10.5897/AJB2016.15267 Degefa T, Lemma A, Tegegne A, and Youngs CR 2016.  Effects of genotype and FSH dose on estrous and ovarian response of Boran and Boran X Holstein Friesian cows in Ethiopia. Livestock Research for Rural Development Vol. 28, article 169. http://www.lrrd.org/lrrd28/9/dege28169.html Duan JE, Flock K, Jue N, Zhang M, Jones A, Seesi S, Mandoiu I, Pillai S, Hoffman M, O’Neill R, Zinn S, Govoni K, Reed S, Jiang H, Jiang Z, Tian XC 2018. Dosage compensation and gene expression of the X chromosome in sheep. G3:Genes, Genomes, Genetics. 9(1):305-314. doi: 10.1534/g3.118.200815. Duan JE, Zhang M, Flock K, Seesi SA, Mandoiu I, Jones A, Johnson E, Pillai S, Hoffman M, McFadden K, Jiang H, Reed S, Govoni K, Zinn S, Jiang Z, Tian XC 2018. Effects of Maternal Nutrition on the Expression of Genomic Imprinted Genes in Ovine Fetuses. Epigenetics 13(8):793-807. doi: 10.1080/15592294.2018.1503489. Duan J, Zhu L, Dong H, Zheng X, Jiang Z, Chen J, Tian XC 2019. Analysis of mRNA abundance for histone variants, histone- and DNA-modifiers in bovine in vivo and in vitro oocytes and embryos. Sci Rep 9(1):217. doi: 10.1038/s41598-018-38083-4. Duan JE, Shi W, Jue N, Jiang Z, Kuo L, O’Neill R, Wolf E, Dong H, Zheng X, Chen J, Tian XC 2019. Dosage Compensation of the X Chromosomes in Bovine Germline, Early Embryos and Somatic Tissues. Genome Biology and Evolution 11(1):242-252. doi: 10.1093/gbe/evy270. Fan, Z., Perisse, I. V., Cotton, C. U., Regouski, M., Meng, Q., Domb, C., Van Wettere, A., Wang, Z., Harris, A., White, K. L., & Polejaeva, I. 2018. A sheep model of cystic fibrosis generated by CRISPR/Cas9 disruption of the CFTR gene. JCI insight, 3(19). Huang D, Wang L, Talbot NC, Huang C, Pu L, Zhao X, Tian X, Zhang M, Tang Y 2018. Analyzing bovine OCT4 and NANOG enhancer activity in pluripotent stem cells using fluorescent protein reporters. PLoS ONE 13(10):e0203923. doi: 10.1371/journal.pone.0203923. eCollection 2018. Jeckel KM, Boyarko AC, Bouma GJ, Winger QA, Anthony RV 2018. Chorionic somatomammotropin impacts early fetal growth and placental gene expression. J Endocrinol. 237(3):301-310. Jiang Z, Lin J, Dong H, Zheng X, Marjani SL, Duan JE, Ouyang Z, Chen J, and Tian XC 2018. DNA methylomes of bovine gametes and in vivo produced preimplantation embryos. Biol Reprod. 99(5):949-959. doi: 10.1093/biolre/ioy138 Koroghli, J., Floyd, E., Regouski, M., Rood, K., Gash, K., Panter, K., Stott, R. D., Davies, C., Polejaeva, I., & Rutigliano, H 2018. Gene expression and lymphocyte population at the fetal-maternal interface in sheep pregnancies established by somatic cell nuclear transfer. Reproduction, Fertility and Development. doi: 10.1071/RD17224. Liu, L., Kandel, M., Rubessa, M., Schreiber, S., Wheeler, M.B., Popescu, G. 2018. Topography and refractometry of sperm cells using SLIM. Journal of Biomedical Optics 23(2):1-6. doi: 10.1117/1.JBO.23.2.025003. Manca, S., B. Upadhyaya, E. Mutai, A.T. Desaulniers, R.A. Cederberg, B.R. White and J. Zempleni. 2018. Milk exosomes are bioavailable and distinct microRNA cargos have unique tissue distribution patterns. Sci. Rep. 8:11321. McWhorter ES, Russ JE, Winger QA, Bouma GJ. 2018. Androgen and estrogen receptors in placental physiology and dysfunction. Front Biol 13(5):315-326. Milner, D. J., M. Bionaz, E. Monaco, J. A. Cameron and M. B. Wheeler. 2018. Myogenic potential of mesenchymal stem cells isolated from porcine adipose tissue. Cell and Tissue Research 372(3): 507-522. Morrison, R.J., Nasser, H.B., Kashlan, K.N., Zopf, D.A., Flanangan, C.L., Wheeler, M.B., Green, G.E., Hollister, S.J. 2018. Co-Culture of Adipose-Derived Stem Cells and Chondrocytes on Three-Dimensionally Printed Bioscaffolds for Craniofacial Cartilage Engineering. Laryngoscope 128:E251-E257. Page, R., T. Lester, R. Rorie, and C. Rosenkrans, Jr.  2018.  Ergot Alkaloid Effects on Bovine Sperm Motility In Vitro.  Adv. Reprod. Sci. (accepted). Ross PJ, Sampaio RV 2018. Epigenetic remodeling in preimplantation embryos: cows are not big mice. Animal Reproduction 15(3):204-2014. doi: 10.21451/1984-3143-AR2018-0068 Rubessa, M., Ambrosi, A., Gonzalez-Pena, D., Polkoff, K.M., Wheeler, M.B.  2018. Non-Invasive Nuclear Magnetic Resonance Analysis of Male and Female Embryo Metabolites During In Vitro Embryo Culture. Metabolomics 14: 113. https://doi.org/10.1007/s11306-018-1414-0. Rubessa, M., Lotti, S.N., Kandel, M.E., Popescu, G., Wheeler, M.B. 2018. SLIM Microscopy allows for visualization of DNA-containing liposomes designed for sperm-mediated gene transfer in cattle. Molecular Biology Report. doi: 10.1007/s11033-018-4525-9. [Epub ahead of print] Vilarino M, Rashid ST, Suchy FP, Lindsay H, Reyes J, McNabb BR, van der Meulen T, Huising MO, Nakauchi H, Ross PJ 2018. Mosaicism diminishes the value of pre-implantation embryo biopsies for detecting CRISPR/Cas9 induced mutations in sheep. Transgenic Research. doi: 10.1007/s11248-018-0094-x. Wang T, Babayev E, Jiang Z, Li G, Zhang M, Esencan E, Horvath T, Seli E 2018. Mitochondrial unfolded protein response gene Clpp is required to maintain ovarian follicular reserve during aging, for oocyte competence, and development of pre-implantation embryos. Aging Cell. e12784. PMID: 29851234 West RC, McWhorter ES, Ali A, Goetzman LN, Russ JE, Anthony RV, Bouma GJ, Winger QA 2018. HMGA2 is regulated by LIN28 and BRCA1 in human placental cells. 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Impact Statements

Through introducing CRISPR/Cas9 system during embryogenesis and using a highly efficient approach, an efficient way to generate genetically modified animals without the need of cloning can be developed.