W4171: 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:

[01/30/2020] [03/04/2021] [02/27/2022] [05/05/2023] [01/17/2024]

Date of Annual Report: 01/30/2020

Report Information

Annual Meeting Dates: 01/15/2020 - 01/16/2020
Period the Report Covers: 01/01/2019 - 12/31/2019

Participants

Participants:

Kenneth White, group administrator (Utah State University), Rod Geisert (University of Missouri), Brett White (University of Nebraska Lincoln), Ken Bondioli (Louisiana State University), Kiho Lee (Virginia Tech), Jerry Bouma (Colorado State University), Curt Youngs (Iowa State University), Irina Polejaeva (Utah State University), Matt Wheeler (University of Illinois), Carol Keefer (University of Maryland), Xiuchun (Cindy) Tian (University of Connecticut), Young Tang (University of Connecticut), Pablo Ross (University of California Davis), Celina Checura (Clemson University), Jean Feugang (Mississippi State University). and Charles Looney (University of Arkansas),

Brief Summary of Minutes

Brief Minutes of the W4171 Technical Committee Meeting

Hilton Hotel, New York City, NY

January 16, 2020

The W4171 committee chair Anna Denicol (University of California, Davis), called the meeting to order at 8:10 am. In attendance were Kenneth White (Utah State University), Rod Geisert (University of Missouri), Brett White (University of Nebraska Lincoln), Ken Bondioli (Louisiana State University), Kiho Lee (Virginia Tech), Jerry Bouma (Colorado State University), Curt Youngs (Iowa State University), Irina Polejaeva (Utah State University), Matt Wheeler (University of Illinois), Carol Keefer (University of Maryland), Xiuchun (Cindy) Tian (University of Connecticut), Young Tang (University of Connecticut), Pablo Ross (University of California Davis), Celina Checura (Clemson University) and Charles Looney (University of Arkansas).

The meeting agenda and the minutes of the previous meeting were approved. The W4171 project administrator, Kenneth White (Utah State University) provided project guidance including:

Administrative process and potential options for the W4171 project team collaborator (non-PI) from institutions with no experimental station to attend the W4171 annual meeting.

The W4171 annual project report needs to be submitted to NIFA within 30 days after the annual meeting.

Discussion of the 2021 W4171 annual meeting time and location within the United States, as the 47^th IETS will be hold in Peru in 2021.

Stations started providing their annual reports. Stations that reported in the morning meeting were University of Arkansas (Looney), University of California, Davis (Ross and Denicle), Colorado State University (Bouma), University of Connecticut (Tian and Tang), and University of Illinois (Wheeler).

The meeting was paused at 12:15pm for lunch break, and continued in the afternoon at 12:35pm.

Stations that reported in the afternoon meeting were Iowa State University (Youngs), Louisiana State University (Bondioli), University of Maryland (Keefer), University of Nebraska Lincoln (White), Clemson University (Checura), Utah State University (Polejaeva), and Virginia Tech (Lee).

Discussion about potential collaborations between stations followed after the individual presentation.

Celina Checura (South Carolina) was nominated to serve as Secretary for the W4171 Technical Committee in 2020. Ken Bondioli (Louisiana State University) made a motion to approve the nomination and Irina Polejaeva (Utah State University) seconded the motion. The motion to elect Celina Checura (South Carolina) as Secretary was unanimously approved. Young Tang will serve as Chair for the W4171 Technical Committee in 2020.

The 2021 meeting of the W4171 group was discussed. It was tentatively agreed to be hold in St. Louis, Missouri in July/August 2021 and be concurrent with the 53^rd Annual Meeting of the Society for the Study of Reproduction (SSR). Other possible locations discussed were South Carolina, Reno (Nevada), and University of California, Davis. The exact meeting location and time will be determined within the following 3 - 4 months.

The meeting was adjourned at 3:00 pm.

Respectively Submitted,

Young Tang - Secretary

W4171 Technical Committee

Accomplishments

Accomplishments:   Objective 1: Understand the biology of gamete development, fertilization and embryogenesis including the underlying cellular and molecular mechanisms.   <ol> <li>Validation of antibodies against FSHR and p42/44 MAPK for use in bovine cells.</li> </ol> Characterization of gene expression in bovine PGCs.   <ol start="2"> <li>Determined the requirement of miRNA for bovine preimplantation development. Determined the effect of Magnesium in bovine preimplantation development.</li> </ol>   <ol start="3"> <li>Systematically characterized the epigenetic landscape (DNA methylome and chromatin modifiers) of the bovine in vivo developed pre-implantation embryos. Characterized the epigenetic phenomenon, X chromosome dosage compensation in the sheep.</li> </ol>   <ol start="4"> <li>The combination of concentration-exposure time to cilostamide during IVPM delayed meiotic progression in bovine after 6 and 12 h of culture. However, overall the culture period (IVPM+IVM) influenced the oocyte chromatin configuration and kinetics in both goats and cattle.</li> </ol>   <ol start="5"> <li>The bovine genome was mapped to determine specific areas where gene promoters and/or enhancers were most accessible. These regions changed in a stage specific manner and correlated with stage specific expressed genes.  Accessible chromatin regions were reveled near the transcription start sites of active genes as well as at distal regions representing transcription factor binding motifs.</li> </ol>   <ol start="6"> <li>The effect of vitrification at the blastocyst stage on gene expression in later day 14 elongated embryos was studied. Gene Ontology pathway analysis strongly suggested that mitochondrial function and energy production were impacted in the trophectoderm following vitrification and persisted in the day 14 elongated embryo stage.</li> </ol>   <ol start="7"> <li>13C-assisted metabolic flux analysis (13C MFA) is a powerful methodology to quantify intracellular fluxes through primary and intermediary metabolism. We have designed novel labeling experiments to obtain a metabolic flux map for this complex system. The methods developed by us demonstrate the extension of the MFA framework to complex mammalian systems.</li> </ol>   <ol start="8"> <li>In the pig, the patterns of protein-to-protein interactions varied throughout follicle development. Proteins such as serine protease inhibitor, clade E (SERPINE); plasminogen activator, urokinase (PLAU); and plasminogen activator, urokinase receptor (PLAUR) appeared stage-specific. The “complement and coagulation cascades” was the common major pathway. In the mare, fifty-two proteins were common to all seasons. A total of 13 proteins were unique to either season, and 25 were shared between two seasons or more.</li> <li>Magnetic nanoselection permits high throughput targeting and removal of damaged spermatozoa from semen doses, leading to rapid and effortless enrichment of semen doses with highly motile, viable, and fertile spermatozoa for artificial insemination.</li> <li>The present study utilized the CRISPR/Cas9 genomic engineering to knockout pig conceptus PTGS2 gene expression. The current results with PTGS2-/- conceptuses and our previous work with CYP19A1-/- conceptuses suggests that both PGE2 and estrogens act as maternal recognition signals, which in the absence of the other can establish pregnancy beyond 17 days. However, conceptus production of estrogen is essential for successful maintenance of pregnancy beyond day 30 as both conceptus and endometrial PGs are not successful in compensating for lost estrogen production at this time.</li> </ol>   <ol start="11"> <li>Semen from GnRHR-II KD boars had reduced sperm kinematics on Day 0, whereas littermate control sperm contained more proximal droplets. On Day 6, transgenic sperm kinetics were decreased, whereas littermate control sperm were slower. Treatment with a GnRHR antagonist (SB-75; 10 µM) decreased total motility of sperm, the percentage of morphologically normal sperm cells, and also impacted sperm kinematics.</li> </ol>   <ol start="12"> <li>Testicular tissue from GnRHR-II KD males (n = 5) produced less testosterone per gram of testicular tissue than littermate controls. At maturity, GnRHR-II KD boars had reduced penis (P < 0.05) and prostate (P < 0.10) weights than control animals. However, protein amounts for both CYP11A1 and CYP17A1 were higher in testicular tissue from GnRHR-II KD vs. control animals. Neither volume or percentage of organelles for the nucleus, cytoplasm, mitochondria, lipid and late lysosomal bodies in Leydig cells differed between GnRHR-II KD vs. control testes.</li> </ol>   <ol start="13"> <li>Design of a practical system to deliver a defined photostimulation (wavelength and intensity) protocol to cells/embryos in culture.</li> </ol> <ol> <li>The metabolomic profile analysis of bovine IVF and SCNT embryos through Raman spectroscopy was able to identify metabolomic differences between embryos of different developmental potential and embryos produced by IVF and SCNT.</li> </ol>   <ol> <li>Apoptosis as a surrogate marker for nuclear reprogramming and embryo survival post-transfer A non-invasive marker for apoptosis distinguished between high quality and low quality embryos in vitro, but not in vivo.  There were distinct and consistent differences in gene expression and DNA methylation between highly apoptotic and lowly apoptotic embryos, which may be of interest. </li> </ol>   Objective 2: Refine methods to produce animals by genetic engineering or genome editing for the improvement of livestock production efficiency and development of human biomedical models.   <ol> <li>Optimized electroporation conditions for pig zygote CRISPR/Cas9 delivery. Design of guide RNAs effective at deleting the target DNA has been achieved.</li> </ol>   <ol start="2"> <li>Established bovine ESCs from various embryo sources in alternative culture conditions.</li> </ol>   <ol start="3"> <li>Targeting shRNA constructs for KDM1A were designed, tested in vitro, and currently used to study their function in early trophectoderm development in vivo. A multiplex PCR genotyping assay has been developed to distinguish XX and XY cells/tissues in sheep.</li> </ol>   <ol start="4"> <li>Using in vitro assays, ARID3B was identified as a downstream target of LIN28-Let7 and a critical transcription factor complex and regulator of key genes necessary for placental development. LIN28B was identified as a regulator of androgen receptor in immortalized trophoblast cells through the action of Let7c.</li> </ol>   <ol start="5"> <li>LIN41 and WNT pathway are downstream targets of the NANOG and LIN28 expression in reprogramming. Modulation of the LIN41 expression and WNT signaling can greatly enhance the human iPSC reprogramming efficiency. An additional downstream target of NANOG and LIN28 is FOXH1. Proper stimulation of FOXH1 expression in reprogramming is another mechanism behind NL-stimulated reprogramming efficiency.</li> </ol>   <ol start="6"> <li>Bovine iPSCs were induced and the transcriptome profiles of the induced bovine cells and their human iPSC counterparts were generated. Allowing comparison of the reprogramming status of the bovine cells with human iPSCs.</li> </ol>   <ol start="7"> <li>Naked DNA has been shown to bind naturally to the sperm, a method called spermmediated gene transfer (SMGT). In order to determine if the liposome-DNA complex would bind 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> </ol>   <ol start="8"> <li>Incorporation of the PLA reinforcing frame does not negatively influence the osteoinductive nature of the mineralized collagen scaffold. These findings suggest a strategy to address often competing bioactivity, mechanical strength, and shape-fitting design requirements for biomaterials for craniomaxillofacial bone regeneration.</li> </ol>   <ol start="9"> <li>Using color Doppler ultrasound imaging that the highest pregnancy rate was observed in recipients with CLs with median Doppler blood flow, a palpable CL papillae, and no CL lacunae. A larger sample size is required to determine the accuracy of this measurement. Recipients with high to low CL blood flow are suitable for IVP embryo transfer programs. Alone, color Doppler imaging of the CL does not predict pregnancy rate.</li> </ol>   <ol start="10"> <li>Bovine Leukosis Virus (BLV) in embryos produced by IVF with semen from BLV seropositive bulls could not be detected. Further, no viral presence was observed in the semen or media samples after sperm selection. In conclusion, these data indicate the use of BLV seropositive bull semen for the production of in vitro fertilized embryos posed no statistically significant risk of transmitting BLV to the embryos.</li> </ol>   <ol start="11"> <li>The world’s first cria produced from a cryopreserved embryo was produced.</li> </ol>   <ol start="12"> <li>The effect of the commonly used animal derived biological semen extender supplements, egg yolk and milk on the ability to induce the acrosome reaction following capacitation treatment commonly used in in vitro fertilization was studied. The semen cryopreserved with an extender supplemented with milk led to a significantly lower response to acrosome inducting agents than when semen was cryopreserved with an extender supplemented with egg yolk.</li> </ol>   <ol start="13"> <li>An extended culture (6 hours) after vitrification-warming improved meiotic spindle recovery in bovine oocytes. Vitrified-warmed oocytes exposed to the extended culture had significantly more normal microtubular distribution. </li> </ol>   <ol start="14"> <li>The formulated liposomes can interact with boar spermatozoa, improve the proportions of motile and progressive spermatozoa, while decreasing the proportions of abnormal spermatozoa.</li> </ol>   <ol start="15"> <li>Successful in using the CRISPR/Cas 9 gene editing system to knockout expression of specific conceptus genes. Conceptus genes for IL1B2, CYP19A1 and PTGS2 have been knockout using CRISPR/Cas 9 with fibroblastic cloning in pigs.</li> </ol>   <ol start="16"> <li>Uncovered a previously unrecognized metabolic system that can help to maintain liver homeostasis under conditions of oxidative or toxic stress. The system uses catabolism of the essential sulfur amino acid methionine (Met) to generate cytosolic disulfide reducing power in the liver. This is the only NADPH-independent source of cytosolic disulfide reducing power that has, as yet, been characterized. This system consumes, in addition to Met, serine, and has broad impacts on diverse other metabolic pathways, including energetic pathways, the citric acid cycle, the folate cycle, polyamine biosynthesis pathways, methyltransferase reactions, and others.</li> <li>Developed an efficient protocol for the introduction of human specific mutations into the sheep fetal fibroblast cells using CRISPR/Cas9 RNP approach. Successfully introduced the F508del and G542X mutations in CFTR gene in SFFs. Generated biallelic NANOS2 knockout goats that served as recipients for exogenous spermatogonial stem cell transplantation.</li> </ol>   <ol start="18"> <li>Published efficiency of CRISPR/Cas9 system to introduce targeted modifications in bovine is 10-50%. Our data suggests that 100% targeting could be achieved in bovine.</li> </ol>

Publications

PUBLICATIONS:   Refereed journal articles Berger, T. 2019. Testicular estradiol and the pattern of Sertoli cell proliferation in prepuberal bulls. Theriogenology 136:60-65. doi: 10.1016/j.theriogenology.2019.06.031   Berger, T., P. Sidhu, S. Tang, and H. Kucera. 2019. Are testicular cortisol and WISP2 involved in estrogen-regulated Sertoli cell proliferation? Animal reproduction science 207:44-51. doi: 10.1016/j.anireprosci.2019.05.014   Kucera, H., B. Puschner, A. Conley, and T. Berger. 2019. Tissue steroid levels in response to reduced testicular estrogen synthesis in the male pig, Sus scrofa. PloS one 14(4):e0215390. doi: 10.1371/journal.pone.0215390   Candelaria, J., and A. Denicol. 2020 (accepted). Characterization of isolated bovine preantral follicles based on morphology, diameter and cell number. Zygote. doi: 10.1017/S0967199419000832   Goszczynski DE, Denicol AC, Ross PJ. 2019. Gametes from stem cells: Status and applications in animal reproduction. Reprod Domest Anim. 54 Suppl 4:22–31. doi:10.1111/rda.13503   Bogliotti YS, Chung N, Paulson E, Chitwood J, Halstead M, Kern C, Schultz RM, Ross PJ. Transcript profiling of bovine embryos implicates specific transcription factors in the maternal-to-embryo transition. Biology of Reproduction, 2019 Nov 11. pii: ioz209. doi: 10.1093/biolre/ioz209. [Epub ahead of print]   Parons Aubone AM, Bisiau CM, McCue PM, Bouma GJ. 2019. Presence of Clock genes in equine full-term placenta. J Anim Sci Submitted.   Ali A, Anthony RV, Bouma GJ, Winger QA. 2019. LIN28-let-7 axis regulates genes in immortalized human trophpblast cells by targeting the ARID3B-complex. FASEB J. Nov;33(11):12348-12363.   West RC, Russ JE, Bouma GJ, Winger QA. 2019. BRCA1 regulates HMGA2 levels in the Swan71 trophoblast cell line. Mol Reprod Dev Nov;86(11):1663-1670.   McWhorter ES, West RC, Russ JE, Winger QA, Bouma GJ. 2019. LIN28B regulates androgen receptor (AR) in human trophoblast cells. Mol Reprod Dev. Sep;86(9):1086-1093.   West RC, McWhorter ES, Ali A, Goetzman LN, Russ JE, Anthony RV, Bouma GJ, Winger QA. 2019. HMGA2 is regulated by LIN28 and BRCA1 in human placental cells. Biol Repod Jan 1;100(1):227-238.   An, L., S. L. Marjani, Z. Wang, Z. Liu, R. Liu, F. Xue, J. Xu, T. L. Nedambale, L. Yang, X. C. Tian, L. Su, and F. Du. 2019. Magnesium is a critical element for competent development of bovine embryos. Theriogenology 140:109-116. doi: 10.1016/j.theriogenology.2019.08.015   Duan, J., L. Zhu, H. Dong, X. Zheng, Z. Jiang, J. Chen, and X. C. Tian. 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):1217. doi: 10.1038/s41598-018-38083-4   Duan, J. E., K. Flock, N. Jue, M. Zhang, A. Jones, S. A. Seesi, I. Mandoiu, S. Pillai, M. Hoffman, R. O'Neill, S. Zinn, K. Govoni, S. Reed, H. Jiang, Z. C. Jiang, and X. C. Tian. 2019. Dosage Compensation and Gene Expression of the X Chromosome in Sheep. G3 (Bethesda) 9(1):305-314. doi: 10.1534/g3.118.200815   Duan, J. E., Z. C. Jiang, F. Alqahtani, I. Mandoiu, H. Dong, X. Zheng, S. L. Marjani, J. Chen, and X. C. Tian. 2019. Methylome Dynamics of Bovine Gametes and in vivo Early Embryos. Front Genet 10:512. doi: 10.3389/fgene.2019.00512   Duan, J. E., W. Shi, N. K. Jue, Z. Jiang, L. Kuo, R. O'Neill, E. Wolf, H. Dong, X. Zheng, J. Chen, and X. C. Tian. 2019. Dosage Compensation of the X Chromosomes in Bovine Germline, Early Embryos, and Somatic Tissues. Genome Biol Evol 11(1):242-252. doi: 10.1093/gbe/evy270   Tian, XC. 2019. The past, present and future of bovine pluripotent stem cells – a brief overview. Frontiers of Agricultural Science and Engineering 6(1): 3-7. https://doi.org/10.15302/J-FASE-2018247   Wang L, Su Y, Huang C, Yin Y, Zhu J, Knupp A, Chu A, Tang Y. FOXH1 Is Regulated by NANOG and LIN28 for Early-stage Reprogramming. Scientific Reports. 2019. 9:16443 | <a href="https://doi.org/10.1038/s41598-019-52861-8">https://doi.org/10.1038/s41598-019-52861-8</a>.   Wang L, Su Y, Huang C, Yin Y, Chu A, Knupp A, Tang Y. NANOG and LIN28 Dramatically Improve Human Cell Reprogramming by Modulating LIN41 and Canonical WNT Activities. Biol. Open. 2019. 8: bio047225 doi: 10.1242/bio.047225.   Rubessa, M., Lotti, S.N., Kandel, M.E., Popescu, G., Wheeler, M.B. 2019. SLIM Microscopy allows for visualization of DNA-containing liposomes designed for spermmediated gene transfer in cattle. Mol Biol Rep. 2019 Feb;46(1):695-703. doi: 10.1007/s11033-018-4525-9. doi: 10.1007/s11033-018-4525-9. [Epub ahead of print]   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. 2019. 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. [Epub ahead of print] Research in Veterinary Science 122 (2019) 22–28.   Dewey, M.J., Johnson, E.M., Weisgerber, D.W., Wheeler, M.B., Harley, B.A.C. 2019. Shapefitting collagen-PLA composite promotes osteogenic differentiation of porcine adipose stem cells. Journal of the Mechanical Behavior of Biomedical Materials 95:21–33. DOI: 10.1016/j.jmbbm.2019.03.017   Bertels, J., Rubessa, M., Kandel, M.E., Bane, T., Milner, D.J., Popescu, G., and Wheeler, M.B. 2019. Zinc’s Effect on the Differentiation of Porcine Adipose-Derived Stem Cells into Osteoblasts. Journal of Regenerative Medicine 8:2-1-7.   Stewart, J., Rubessa, M., Polkoff, K.M., Lotti, S., Wheeler, M.B. 2019. Risk of transmission of Bovine Leukosis Virus (BLV) in abattoir-derived in vitro produced embryos. International Journal of New Technology and Research (IJNTR) ISSN:2454-4116, Volume-5, Issue-9, September 2019 Pages 01-06. https://doi.org/10.31871/IJNTR.5.9.5   Rubessa, M., Kandel, M.E., Schreiber, S., Meyers, S., Beck, D.H., Popescu, G., and Wheeler, M.B. 2019. Sperm selection methods for bovine in vitro embryo production, characterized by spatial light interference microscopy. Systems Biology in Reproductive Medicine (accepted).   Kandel, M.E., Rubessa, M., Meyers, S., Szewczyk, M.J., Yu, F., Wheeler, M.B., Popescu, G. (2019). High sensitivity SLIM imaging to correlate sperm morphology and fertility outcomes. Proc.SPIE 10887, Quantative Phase Imaging V, 108871B, https://doi.org/10.1117/12.2513238.     Belala R, L Briand-Amirat, A Martinot, C Thorin, S Michaud, S Desherces, CR Youngs, and D Bencharif. 2019. A comparison of liquid and lyophilized egg yolk plasma to low density lipoproteins for freezing of canine spermatozoa. Reprod. Dom. Anim. 54(8):1131-1138 <a href="https://doi.org/10.1111/rda.13476">https://doi.org/10.1111/rda.13476</a>.   Elshazly AG and CR Youngs. 2019. Feasibility of utilizing advanced reproductive technologies for sheep breeding in Egypt. Part 1. Genetic and nutritional resources. Egyptian Journal of Sheep & Goat Sciences 14(1):39-52. <a href="https://ejsgs.journals.ekb.eg/article_33235_c2fd1cc6b968bbc2124356222fc929cf.pdf">https://ejsgs.journals.ekb.eg/article_33235_c2fd1cc6b968bbc2124356222fc929cf.pdf</a>   Gómez-Quispe OE, GA Gutierrez-Reynoso, A Gallegos-Cardenas, FG Fumuso, M Asparrin, M Asparrin-Del Carpio, CW Jara, D Salazar-Ponce, M Miguel-Gonzales, CR Youngs, HW Vivanco. 2019. Motility and recovery of alpaca (Vicugna pacos) spermatozoa after centrifugation in a density gradient solution. Iranian Journal of Veterinary Research 20(2):96-104. <a href="http://ijvr.shirazu.ac.ir/article_5257_80ac7e09a2bbc52a890720d283c01227.pdf">http://ijvr.shirazu.ac.ir/article_5257_80ac7e09a2bbc52a890720d283c01227.pdf</a>   Long JA, HD Blackburn, A Martin, FG Silversides, RL Taylor, Jr, and CR Youngs.  2019. Protecting Food Animal Gene Pools for future generations – a paper in the series on The Need for Agricultural Innovation to Sustainably Feed the World by 2050. Council on Agricultural Science and Technology (CAST) Issue Paper 65, 24 pages.   West R.C., Hao Ming*, Deirdre M. Logsdon*, Jiangwen Sun, Sandeep K. Rajput, Rebecca A. Kile, William B. Schoolcraft, R. Michael Roberts, Rebecca L. Krisher, Zongliang Jiang#, Ye Yuan#. Dynamics of trophoblast differentiation in peri-implantation stage human embryos. PNAS: Proc Natl Acad Sci U S A. 2019 Oct 21. PMID: 31636193.   Zhang M, Bener MB, Jiang Z, Wang T, Esencan E, Scott R, Horvath T, Seli E. Mitofusin 1 is required for female fertility and to maintain ovarian follicular reserve. Cell Death & Disease, 2019 Jul 22;10(8):560. PMID: 31332167.   Zhang M, Bener MB, Jiang Z, Wang T, Esencan E, Scott R, Horvath T, Seli E. <a href="https://www.ncbi.nlm.nih.gov/pubmed/31204316">Mitofusin 2 plays a role in oocyte and follicle development, and is required to maintain ovarian follicular reserve during reproductive aging.</a> Aging, 2019 Jun 16;11(12):3919-3938. PMID: 31204316.   Paes, V. M., Liao, S., Figueiredo, J. R., Willard, S. T., Ryan, P. L., Feugang, J.-M. (2019). Proteome changes of porcine follicular fluid during follicle development. Journal of animal science and biotechnology, 10, 94.   Dutra, G. A., Ishak, G. M., Pechanova, O., Pechan, T., Peterson, D. G., Jacob JCF, Willard, S. T., Ryan, P. L., Gastal, E. L., Feugang, J.-M. (2019). Seasonal variation in equine follicular fluid proteome. Reproductive biology and endocrinology: RB&E, 17(1), 29.   Durfey, C. L., Swistek, S. E., Liao, S., Crenshaw, M. A., Clemente, H. J., Thirumalai RVKG, Steadman, C. S., Ryan, P. L., Willard, S. T., Feugang, J.-M. (2019). Nanotechnology-based approach for safer enrichment of semen with best spermatozoa. Journal of animal science and biotechnology, 10, 14.   Geisert R.D., P. Sutvosky, M.C. Lucy, F.F. Bartol and A.E. Meyer. 2019. Chapter 15: Reproductive Physiology of Swine. In: Animal Agriculture: Sustainability, Challenges and Innovations.  Eds. F.W. Bazer, G.C. Lamb and G. Wu. Academic Press, Elsevier Inc. Pfeiffer C.A., A.E.Meyer, K.E. Brooks, P.R. Chen, J. Milano-Foster, L.D. Spate, J.A. Benne, R. Cecil, M.S. Samuel, L.A. Ciernia, M.F. Smith, K.D. Wells, T.E. Spencer, R.S. Prather, and R.D. Geisert. 2019. Ablation of conceptus PTGS2 provides a new understanding of early pregnancy events in the pig. Biol Reprod<a href="https://doi.org/10.1093/biolre/ioz192">  doi.org/10.1093/biolre/ioz192</a>. Meyer A.E., C.A. Pfeiffer, K.E. Brooks, L.D. Spate, J.A. Benne, R.Cecil, M.S. Samuel, C.N. Murphy, S. Behura, M.K. McLean, L.A. Ciernia, M.F. Smith, K.M. Whitworth, K.D. Wells, T.E. Spencer, R.S. Prather and R.D. Geisert. 2019. New perspective on conceptus estrogens in maternal recognition and pregnancy establishment in the pig. Biol Reprod 101: 148–161. <a href="https://doi.org/10.1093/biolre/ioz058">https://doi.org/10.1093/biolre/ioz058</a>   Dagnell, M., E. E. Schmidt, and E. S. J. Arner. 2018. The A to Z of modulated cell patterning by mammalian thioredoxin reductases. Free Radic Biol Med 115:484-496. doi: 10.1016/j.freeradbiomed.2017.12.029   Doka, E., T. Ida, M. Dagnell, Y. Abiko, N. L. Cong, N. Balog, T. Takata, B. Espinosa, A. Nishimura, Q. Cheng, Y. Funato, H. Miki, J. Fukuto, J. R. Prigge, E. E. Schmidt, E. S. J. Arnér, Y. Kumagai, T. Akaike, and P. Nagy. 2020. Control of protein function through oxidation and reduction of persulfidated states. Science Advances (in press)   McLoughlin, M. R., J. R. Prigge, D. J. Orlicky, P. Krishna, E. A. Talago, I. R. Cavigli, S. Eriksson, C. G. Miller, J. A. Kundert, V. I. Sayin, R. A. Sabol, J. Heinemann, L. O. Brandenberger, S. V. Iverson, B. Bothner, T. Papagiannakopoulos, C. T. Shearn, E. S. J. Arnér, and E. E. Schmidt. 2019. TrxR1, Gsr, and oxidative stress determine hepatocellular carcinoma malignancy. Proc Natl Acad Sci U S A. 116, 11408-11417.   Miller, C. G., A. Holmgren, E. S. J. Arner, and E. E. Schmidt. 2018. NADPH-dependent and -independent disulfide reductase systems. Free Radic Biol Med 127:248-261. doi: 10.1016/j.freeradbiomed.2018.03.051   Miller, C. G., and E. E. Schmidt. 2019. Disulfide reductase systems in liver. Br J Pharmacol 176(4):532-543. doi: 10.1111/bph.14498   Roggenbeck, B. A., E. M. Leslie, S. T. Walk, and E. E. Schmidt. 2019. Redox metabolism of ingested arsenic: Integrated activities of microbiome and host on toxicological outcomes. Curr Opin Toxicol., 13, 90-98.   Desaulniers, A.T., R.A. Cederberg, E.P. Carreiro, C.B. Gurumurthy and B.R. White. 2018. A transgenic pig model expressing a ZsGreen1 reporter across an extensive array of tissues. Anim. Biotechnol. (Under Review).   Gruhot, T.R., L.A. Rempel, B.R. White and B.E. Mote. 2019. The effect of varicocele on semen quality in boars exposed to heat stress. Translational Anim. Sci. [Epub ahead of print].   Fan, Z., M. Yang, M. Regouski, and I.A. Polejaeva, Gene Knockouts in Goats Using CRISPR/ Cas9 System and Somatic Cell Nuclear Transfer. Methods Mol Biol, 2019. 1874: p. 373-390.   Gash, K.K., M. Yang, Z. Fan, M. Regouski, H.M. Rutigliano, and I.A. Polejaeva, Assessment of microchimerism following somatic cell nuclear transfer and natural pregnancies in goats. J Anim Sci, 2019. 97(9): p. 3786-3794. PMCID: PMC6735858.   Regouski, M., O. Galenko, J. Doleac, A.L. Olsen, V. Jacobs, D. Liechty, K.L. White, T.J. Bunch, P.M. Lee, H.M. Rutigliano, I.A. Polejaeva, and M.J. Cutler, Spontaneous Atrial Fibrillation in Transgenic Goats With TGF (Transforming Growth Factor)-beta1 Induced Atrial Myopathy With Endurance Exercise. Circ Arrhythm Electrophysiol, 2019. 12(11): p. e007499.   Wu, H., Z. Fan, M. Brandsrud, Q. Meng, M. Bobbitt, M. Regouski, R. Stott, A. Sweat, J. Crabtree, R.J. Hogan, R.A. Tripp, Z. Wang, I.A. Polejaeva, and E.J. Sullivan, Generation of H7N9-specific human polyclonal antibodies from a transchromosomic goat (caprine) system. Sci Rep, 2019. 9(1): p. 366. PMCID: PMC6344498.   Carey K, Ryu J, Uh K, Lengi AJ, Clark-Deener S, Corl BA, Lee K. 2019. Frequency of off-targeting in genome edited pigs produced via direct injection of the CRISPR/Cas9 system into developing embryos. BMC Biotechnol. May 6;19(1):25. doi: 10.1186/s12896-019-0517-7.   Books, non-refereed book chapters, proceedings, instructional media, theses/dissertations   Berger, T., H. Kucera, A. Conley, and B. Puschner. 2019. Steroid concentrations in boar tissues. https://doi.org/10.25338/B85P5T   Elizabeth Johnson. 2019. Effects of intramuscularly injected plant-derived antimicrobials in the mouse model. MS thesis. University of Connecticut.   Characterizing cytoplasmic maturation and improving competence of bovine in vitro matured oocytes.  Louisiana State University Ph D Dissertation.  August 2018.   Effect of heat stress on the epigenetic profile of bovine oocytes and embryos.  Louisiana State University Ph D Dissertation.  December 2018.   Chung, J., Sriram, G., and C. Keefer.  (submitted) Magnetic Nanoparticle Technology Improves In-vitro Attachment of Cattle (Bos taurus) Trophectoderm Cells. Biotechnology Letters   Pfeiffer CA. 2019.  Ablation of Conceptus PTGS2 Provides a New Understanding of Early Pregnancy Events in the Pig.  Master of Science Thesis.   Alhojaily, Sameer M. (2019). The Effect of Lactation and Energy Status on Gene Expression in the Main Reproductive Tissues of Lactating Dairy Cattle. PhD Dissertation  #7588.  Utah State University.  <a href="https://digitalcommons.usu.edu/etd/7588">https://digitalcommons.usu.edu/etd/7588</a>   Moley, Laura A., (2019).  Epigenetic Reprogramming, Apoptosis, and Developmental Competence in Cloned Embryos.  PhD Dissertation #7571.  Utah State University.  <a href="https://digitalcommons.usu.edu/etd/7571">https://digitalcommons.usu.edu/etd/7571</a>   Lee K, Farrell K, Uh K. 2019. Application of genome editing systems to enhance available pig resources for agriculture and biomedicine. Reproduction, Fertility and Development 32(2) 40-49 <a href="https://doi.org/10.1071/RD19273">https://doi.org/10.1071/RD19273</a>   Abstracts: Candelaria, J., B. Rabaglino, and A. Denicol. 2020. Transcriptomic changes in bovine ovarian cortex in response to FSH signaling. 46th Annual Meeting of the IETS. New York, NY.   Denicol, A., B. Weldon, and L. Aguiar. 2020. Preliminary characterization of ovarian stem cells from bovine ovaries. 46th Annual Meeting of the IETS. New York, NY.   Soto DA and Ross PJ. Single-Cell RNA Sequencing Reveals Similarities Between Bovine and Human Primordial Germ Cell Development. 52nd Annual Conference of the Society for the Study of Reproduction, San Jose, CA July 18-21, 2019   Ross PJ. Transcriptional and Epigenetic Reprogramming During Bovine Preimplantation Development. 52nd Annual Conference of the Society for the Study of Reproduction, San Jose, CA July 18-21, 2019   Paulson EE, Ross PJ, Schultz RM. Expression Pattern And Role Of miRNAs During Early Development In The Cow. 52nd Annual Conference of the Society for the Study of Reproduction, San Jose, CA July 18-21, 2019   Park I, Kim SK, Ross PJ. Neurogenin 3 Disruption By CRISPR/Cas9 Electroporation Of Porcine Zygotes. 52nd Annual Conference of the Society for the Study of Reproduction, San Jose, CA July 18-21, 2019   Navarro M, Rodrigues Sangalli J. Ross PJ. Derivation and characterization of bovine embryonic stem cells from different embryo sources. Transgenic Animal Research Conference XI! (TARC XI!): Tahoe City, CA, 2019.   Gerrit J. Bouma, Asghar Ali, Erin S. McWhorter, Rachel C. West, Quinton A. Winger. Histone lysine demethylase function in placental trophoblast cells. 2019 Perinatal Biology Symposium, Aspen-Snowmass, CO, USA.   Asghar Ali, Russell V Anthony, Gerrit J. Bouma, Quinton A. Winger. LIN28-let-7 axis regulates gene expression in ovine placenta in vivo. Society for the Study of Reproduction 52nd Annual Meeting, San Jose, CA, USA.   Asghar Ali, Gina C Nay, Russell V Anthony, Gerrit J Bouma,  Quinton A Winger. Genes Governing Placental Development are Regulated by ARID3A-ARID3B-KDM4C Complex. 2019 Society for Reproductive Investigation 66thAnnual Meeting, Paris, France.   Thomas R. Hansen, Hana Van Campen, Jeanette V. Bishop, Gerrit J. Bouma, Quinton A. Winger, Leticia D.P. Sinedino, Christie E. Mayo, Richard A. Bowen. Maternal influenza A virus infection decreases the expression of T cell differentiation genes in the murine fetal thymus. Society for the Study of Reproduction 52nd Annual Meeting, San Jose, CA, USA.   Duan JE, Jiang Z, Alqahtani F, Mandoiu I, Dong H, Zheng X, Marjani SL, Chen J, and Tian XC. Whole genome bisulfite sequencing of bovine gametes and in vivo produced preimplantation embryos. Reproduction, Fertility and Development 31(1) 126. Poster at the 45th annual meeting of the International Embryo Technology Society, January 20-23, 2019, New Orleans, USA.   Silva, E. P., M. K. Sermersheim, P. V. Marchioretto, R. Della Mea, L. M. Naves, I. Rivelli, F. Rochelle, L. F. T. Nasser, M. Rubessa and M. B. Wheeler. 2019. A comparison of in vitro embryo production between heifers and lactating Holstein donors without superstimulation. Reproduction, Fertility and Development. 31(1): 195-195.   Siegel, L., T. Bane, J. Bertels, K. Ratz, M. Rubessa and M. Wheeler. 2019. Effect of selenium on the differentiation of porcine adipose-derived stem cells into osteoblasts. Reproduction, Fertility and Development. 31(1): 216-217.   Salerno, F., M. Rubessa, B. Gasparrini and M. Wheeler. 2019. Effect of deuterium oxide on bovine oocyte cryotolerance. Reproduction, Fertility and Development. 31(1): 140-140.   Padoveze, L. R., M. Rubessa, C. E. Ambrosio and M. B. Wheeler. 2019. The effects of different concentrations of MgSO4 in osteogenic differentiation. Reproduction, Fertility and Development. 31(1): 217-218.   Bane, T., L. Siegel, J. Bertels, K. Ratz, M. Rubessa and M. Wheeler. 2019. The effect of copper on the differentiation of adipose-derived stem cells into osteoblasts. Reproduction, Fertility and Development. 31(1): 229-230.   Vivanco-Mackie HW, MD Ponce-Salazar, M Miguel-Gonzales, CR Youngs, C. Jara, and M Asparrin.  2019. Comparative study between slow freezing and vitrification on the survival rate of cryopreserved alpaca embryos post-transfer. Reprod. Fertil. Dev. 31(1):182.   Gatenby, L. and KR Bondioli. 2019.  The effect of biological extenders on in vitro induction of the acrosome reaction in bovine spermatozoa.  Reprod. Fertil. Dev..   Gutierrez, EJ, Z. Jiang, KR Bondioli.  2019.  Extended culture after vitrification warming helps in spindle recovery of bovine oocytes.  Reprod. Fertil. Dev.   Jiang Z, Gutierrez E, Foster B, Ming H, Gatenby L, Mak C, Pinto C, Bondioli K. Effect of vitrification on global gene expression dynamics of bovine elongating embryos. Reprod. Fertil. Dev. 3 December 2019.   Ming, H.*, J. Sun, R. Pasquarielle, J. Herrick, Y. Yuan, E.J. Gutierrez, L. Gatenby, K. R. Bondioli, R.L. Krisher, Z Jiang#. The landscape of accessible chromatin in bovine oocytes and early embryos. Reprod. Fertil. Dev. 3 December 2019.   Rajput S, West R, Ming H*, Logsdon D, Kile R, Grimm C, Sun J, Schoolcraft W, Krisher R, Jiang Z#, Yuan Y#. Peri-implantation stage human embryos cease cell proliferation and increase metabolism to prioritize important cellular events for implantation. 52th SSR, San Jose, CA, 2019.   West R, Ming H*, Logsdon D, Kile R, Grimm C, Rajput S, Sun J, Schoolcraft W, Krisher R, Jiang Z, Yuan Y#. Single Cell RNA-Sequencing Reveals Critical Role of Interferon Signaling in Human Peri-Implantation Stage Embryos. 52th Annual conference of Society for the Study of Reproduction, San Jose, CA, 2019.   Yuan Y, Sun J, Ming H*, Logsdon D, Schoolcraft W, Krisher R, Jiang Z#. Single-cell whole genome bisulfite sequencing reveals DNA methylation dynamics of trophoblast differentiation in human peri-implantation stage embryos. Fertility Sterility. 2019, O-237.   Yuan Y, West R, Ming H*, Logsdon D, Kile R, Grimm C, Rajput S, Sun J, Schoolcraft W, Krisher R, Jiang Z#. Picturing the dynamics of trophoblast differentiation in per-implantation stage human embryos by single cell RNA sequencing. 52th SSR, San Jose, CA, 2019.   Zhang M, Bener M, Jiang Z, Wang T, Esencan E, Scott III R, Seli E. Loss of mitochondrial dynamics in oocytes results in infertility and diminished ovarian reserve. Fertility Sterility.  2019, O-217.   Zhang M, Bener M, Jiang Z, Wang T, Esencan E, Scott III R, Seli E. Loss of mitochondrial fusion protein MFN2 results in telomere shortening, reduced fertility, and accelerated depletion of follicular pool. Fertility Sterility. 2019, P-237. Chung, J., Clifford, R., Sriram, G., and C. Keefer. 2019. Flux analysis of aerobic glycolysis in bovine blastocysts and CT1 cells. Reprod. Fert. Dev. 31(1), 159.   Lugar, D., Adhikari, S., Keefer, C.L., and Sriram, G. 13C Metabolic Flux Analysis of Pre-Implantation Bovine Embryos: Surmounting Challenges Due to Small Sample Size and Complex Media, American Chemical Society National Meeting, March 22-26, 2020 Feugang, J.M., Eggert, M.W., Park, S.B., Popoola, M.A., Steadman, C.S., Arnold, R.R., Ryan, P.L. and Willard, S.T., 2019. 203 Newly designed liposome nanoparticles for drug delivery into boar spermatozoa. Reproduction, Fertility and Development, 31(1), pp.227-227. Pfeiffer C.A., A.E. Meyer., L.D. Spate, J.A. Benne, R.F. Cecil, T.E. Spencer, R.S. Prather, and R.D. Geisert. 2019. Conceptus Prostaglandin Synthase 2 is Not Essential for Early Development and the Establishment of Pregnancy in the Pig. 52nd Annual Meeting of SSR, P432   Johns D.N., C.G. Lucas, C.A. Pfeiffer, L.D. Spate, J.A. Benne, R.F. Cecil, K.M. Wells, T.E. Spencer, R.S. Prather, and R.D. Geisert. 2019. Pig Conceptus Expression of Type I Interferon Delta and Type II Interferon Gamma During Early Pregnancy.  The 16th Annual Gilbert S. Greenwald Symposium on Reproduction and Regenerative Medicine, P25.   Ross, C.E., F.H. Choat, K.N. Plager, A.T. Desaulniers, R.A. Cederberg, G.A. Mills and B.R. White. 2019. Effect of GnRH-II and its receptor on boar semen quality. Reprod. Fertil. Dev. 32:201. Ebrecht, M.A., C.E. Ross, R.A. Cederberg, K.W. Lovercamp, A.T. Desaulniers and B.R. White. 2019. Effects of GnRH-II on spermatogenic function of swine. Proceedings of the 16th Annual Gilbert S. Greenwald Symposium on Reproduction and Perinatal Research. p. 44. Desaulniers, A.T., R.A. Cederberg, G.A. Mills and B.R. White. 2019. Use of a genetically-engineered swine line to elucidate the role of GnRH-II and its receptor in gilts. J. Anim. Sci. 97(Suppl. 2):122-123.   Checura, C. M., S. L. Pratt, L. V. Campbell, K. Farmer, G. Loughlin, M. Mitchell, A. Sandford, A. M. Treske, H. Malter. 2019. The use of photostimulation to enhance oocyte cytoplasmic maturation. Reprod. Fertil. Dev. 32(2): 227. (Abstr.) doi:10.1071/RDv32n2Ab197   Perisse I.V., Z. Fan, Y. Liu, M. Regouski, A. Van Wettere, Z. Wang, A. Harris, K. L. White and I. A. Polejaeva (2019). Towards the correction of meconium ileus with cystic fibrosis transmembrane conductance regulator (CFTR) intestinal expression in CFTR knockout sheep. Reproduction, Fertility and Development 31(1) 228-228 <a href="https://doi.org/10.1071/RDv31n1Ab205">https://doi.org/10.1071/RDv31n1Ab205</a>   Fan Z., M. Regouski, M. Brandsrud, H. Wu, Y. Liu, Z. Wang, E. Sullivan and I. A. Polejaeva (2019). Towards the generation of transchromosomic goats for the production of fully human immunoglobulin. Reproduction, Fertility and Development 31(1) 228-228 <a href="https://doi.org/10.1071/RDv31n1Ab206">https://doi.org/10.1071/RDv31n1Ab206</a>   Keim J., Y. Liu and I. A. Polejaeva (2019). Increasing in vitro embryonic development through improved oocyte maturation in cattle oocytes. Reproduction, Fertility and Development 31(1) 213-213 <a href="https://doi.org/10.1071/RDv31n1Ab177">https://doi.org/10.1071/RDv31n1Ab177</a>   Silva A.C., Morgado K.P., Davies C.J., Polejaeva I.A. and Rutigliano H.M. (2019) Gene expression profiles of immune cells under the influence of bovine trophoblast cell derived extracellular vesicles. The 52 annual meeting of the Society for the Study of Reproduction. July 18-21, 2019. San Jose, CA. Cuthbert J., Russell S., Meng Q., Polejaeva I.A., White K., Benninghoff  A. (2019) Somatic cell nuclear transfer in early bovine embryo development is associated with changes in small non-coding RNA species. The 52 annual meeting of the Society for the Study of Reproduction. July 18-21, 2019. San Jose, CA. Perisse I.V., Fan Z., Wang Z., Harris A., White K.L., Polejaeva I.A. (2019) Efficient introduction of G542X mutation in the CFTR gene in sheep fetal fibroblast using CRISPR/Cas9 ribonucleoprotein. Cystic Fibrosis Foundation Research Conference. June 23-26, 2019. Stowe, VT.   Moley L.M, Jones R., Kaundal R., Thomas A., Benninghoff A., Isom S.C. (2019)  Gene expression analysis and DNA methylation patterns of porcine SCNT blastocysts with high and low incidence of apoptosis.  Reproduction, Fertility and Development 31(1), 128-128.  <a href="https://doi.org/10.1071/RDv31n1Ab5">https://doi.org/10.1071/RDv31n1Ab5</a>   Farrell K, Uh K, Haines H, Lee K. 2019. Expression patterns of PRDM family genes in porcine preimplantation embryos. Reproduction, Fertility and Development 32(2)

Impact Statements

IMPACTS: 1. Endogenous testicular cortisol in the juvenile interval may restrict postpuberal sperm production in the boar. Regulation of in vivo Sertoli cell proliferation did not benefit from studies with in vitro monocultures. Our data suggest reduction of endogenous estrogen will not be an effective manipulation to increase bovine sperm production. 2. New roles of FSH in the development of preantral follicles are being discovered and will be critical for the development of culture systems that promote oogenesis in vitro. 3. Understanding bovine PGC biology represents a useful resource for in-vitro differentiation of bovine pluripotent stem cells to PGCs. 4. The epigenetic profiles of the bovine embryos is the fourth species ever characterized. Similarities and differences among different mammalian species were identified. The data serve as reference base for all bovine embryos produced by assisted reproductive technologies. 5. The results filled the knowledge gap on gene dosage compensation in a new species, the domestic sheep, an understudied species in developmental epigenetics. 6. Our data demonstrated the cilostamide during IVPM delayed meiotic progression in bovine. The specific inhibitors of cGMP do not affect the lipid profiles of the resulting oocytes and embryos after IVF. 7. Determining regulatory gene expression networks driving embryo development in vivo and in vitro as well as development potentially disrupted by cryopreservation will reveal targets for improving developmental competence in embryos produced by assisted reproductive technologies such as in vitro embryo production and somatic cell nuclear transfer. 8. Unraveling the metabolic basis of normal early embryo development will provide significant benefits to human and animal reproductive health. Improved embryonic competency following in vitro production and cryopreservation would stimulate the industry by lowering costs, especially those related to recipient management. 9. This study provides extensive and functional analyses of the pFF proteome changes during folliculogenesis and offers the potential for novel biomarker discovery in pFF for oocyte quality assessment. 10. The balance between the FF contents in prothrombin, plasminogen, and coagulation factor XII proteins favoring FF fluidity may be crucial at the peak of the ovulatory season (SUM), which peak may explain the reported lower incidence of hemorrhagic anovulatory follicles during the SUM season. 11. Magnetic nanoselection allowing specific removal of abnormal spermatozoa from semen samples appears as a promising tool for improving fertility of males, particularly during periods, such as heat stress during the summer months. 12. Utilization of the CRISPR/Cas 9 gene editing system provides a powerful tool to evaluate the role of genes during early conceptus development. Research has changed the paradigm of the role of IL1B2, estrogen and PTGS2 in maternal recognition of pregnancy and maintenance of pregnancy in pigs. 13. Acquired a change in fundamental knowledge regarding LH independent steroidogenesis and postnatal testicular development, implicating GnRH-II and its receptor in this important biological mechanism. We expect to better understand how the GnRH-II receptor is regulated within extra-pituitary tissues related to reproduction. 14. New pharmacological agents may be developed to manipulate the reproductive axis, leading to enhanced fertility rates and reduced incidences of endocrine disorders impacting reproduction. Data from these experiments could lead to novel swine-specific contraceptive methods to manage feral pig populations. 15. Identification of subfertile boars at a younger age would allow producers to focus resources on reproductively superior animals and market subfertile males prior to sexual maturity, significantly increasing their value. Improved identification of fertile vs. subfertile boars would contribute more doses of semen sold per ejaculate, reducing the cost of production. 16. Novel factors may be determined to improve semen extenders in swine, extending the lifespan of sperm and decreasing the cost of semen doses. Demonstration that GnRH-II and its receptor are important to sperm function, representing a potential avenue to improve semen preservation. 17. There was an immediate impact on the undergraduate research education of ten students involved in the development and execution of the projects. We reached the scientific community, professionals, and companies involved in bovine in vitro productions systems through the publication of our results in a scientific abstract 18. Delivery of CRISPR/Cas9 by electroporation will facilitate creation of gene-edited pigs 19. Efficient production of bESC from SCNT embryos enables the generation of pluripotent stem cell lines from valuable and well characterized individuals. 20. The long-term proliferation potential of bESCs make them good candidates for introducing complex and/or multiple gene edits in high genetic merit animals for advancing genetic improvement and/or enhancing production and biotechnological traits in cattle. 21. Understanding the role of LIN28A and LIN28B in sheep placenta will improve our understanding of the genetic regulation of placenta development and result in methods to improve somatic cell nuclear transfer in ruminants. 22. Similarly establishing that androgen and estrogen signaling through their receptors are regulated by KDM1A in the placenta, and are involved in placental angiogenesis in agriculture animals will lead to new insight into factors involved in early pregnancy losses. This also can lead to development of novel strategies and/or tools to identify or prevent early embryo mortality. 23. Generated new knowledge and database to elucidate the mechanisms for enhancement of reprogramming efficiency and pluripotency establishment which are key to the successful generation of bona fide biPSCs. 24. SLIM microscopy confirmed the presence of liposomes on the sperm head and tail. 25. A PLA reinforcing frame does not negatively influence the osteoinductive nature of a mineralized collagen scaffold. 26. Alone, color Doppler imaging of the CL does not predict pregnancy rate. The use of BLV seropositive bull semen for the production of in vitro fertilized embryos posed no statistically significant risk of transmitting BLV to the embryos. 27. The successful production of live offspring from a cryopreserved alpaca embryo opens the opportunity for germplasm preservation and transport for this species. 28. The demonstration that different semen extenders used during cryopreservation can effect the way spermatozoa respond to common capacitation treatments used in in vitro embryo production highlights the importance of developing in vitro methods applicable across a wide spectrum of conditions. 29. Disruption of the meiotic spindle has been a major component of the reduced developmental potential after oocyte cryopreservation. The ability to enhance spindle recovery post vitrification-warming is major step towards improving this capacity. 30. The newly designed liposomes were effective for simple interactions with sperm plasma membrane and molecule delivery, without impairing sperm motility. Further studies are ongoing with the replacement of doxorubicin with a DNA fragment. 31. Uncovered previously unrecognized systems that support survival and function of liver cells from a redox metabolism standpoint, which, in turn, provided insights into better therapies for treating some liver diseases or toxicities. From more of a bioengineering standpoint, these studies are demonstrating the power of being able to genetically manipulate hepatic metabolism in adult animals. This promises to provide interesting new ways to genetically modify nutrient use or intermediary metabolism of animals that could improve food-production characteristics of livestock without generating genetically modified food products. 32. Our results should assist in utilizing the technology in introducing targeted modifications in bovine. Our publication indicates that unintended mutations can be introduced by CRISPR/Cas9 system, if not carefully designed. The study suggests that careful consideration should be made prior to apply the technology in genetically engineered animal production.

Date of Annual Report: 03/04/2021

Report Information

Annual Meeting Dates: 01/25/2021 - 01/25/2021
Period the Report Covers: 01/01/2020 - 12/31/2020

Participants

The W4171 committee chair Yong Tang (University of Connecticut), called the meeting to order at 11:02 am. In attendance were Charles Looney (Arkansas), Patricia J. Berger (California), Anna Denicol (California), James D. Murray (California), Quinton Winger (Colorado), X. Cindy Tian (Connecticut), Brad Daigneault (Florida), Matthew B. Wheeler (Illinois), Curt Youngs (Iowa), Carol L. Keefer (Maryland), Keith Latham (Michigan), Rodney Geisert (Missouri), Kiho Lee (Missouri), Edward Schmidt (Montana), Joao Gabriel Nascimento Moraes (Oklahoma), Celina Checura (South Carolina), Clay Isom (Utah), Irina Polejaeva (Utah), Kenneth White (Utah). Note that attendance changed during the day due to the virtual nature of the meeting.

Brief Summary of Minutes

Dr. Tang asked for permission to record the meeting and the present agreed.

Dr. Tang welcomed the group at 11:05 am.

Dr. Polejaeva requested to reverse the alphabetical order of the presentations (from Z to A for the present meeting) as it was discussed on the previous meeting. It was proposed to alternate the order of the presentations (A to Z and Z to A) in future meetings. All agreed.

Dr. Tang went over the minutes from the 2020 meeting. The minutes were approved.

Dr. Isom asked for a round of introductions and each of the participants at the time introduced her/himself.

Station reports started at 11:38 am. Dr. Polejaeva (Utah) had problems with the slide presentation, then Dr. Checura (South Carolina) started.

Dr. Jean M. Feugang (Mississippi), joined at 11:47 am and introduce himself.

Dr. Polajeava (Utah) presented her station report followed by Dr. Nascimento Moraes (Oklahoma), Dr. Schmidt (Montana), Dr. Geisert (Missouri), and Dr. Feugang (Mississippi).

There was a lunch break from 1:10 pm to 1:30 pm.

In the afternoon, the reports were presented as follows: Dr. Latham (Michigan), Dr. Keefer (Maryland), Dr. Bondioli (Louisiana), Dr. Youngs (Iowa), Dr. Wheeler (Illinois), Dr. Daigneault (Florida), Dr. Tian (Connecticut), Dr. Tang (Connecticut). Due to schedule conflicts, the alphabetical order of the presentation was altered for the rest of the afternoon. Dr. Looney (Arkansas) presented, followed by Dr. Berger (California).

At 3:42 pm there was an open floor for general discussion. Dr. Youngs commented on the USDA regulating GMO animals. The highlights for the subsequent discussion are:

There is an open comment USDA website on the topic, and it is suggested that the members submit their scientific opinions as individuals.

It is not appropriate to submit a statement to the USDA as W4171 group due to possible conflicts on the interpretation that the members may be representing their stations.

At 4:05 pm Dr. Denicol (California) presented her report.

At 4:19 pm Dr. Tang nominated Dr. Daigneault to serve as Secretary for the W4171 Technical Committee in 2022. Dr. Tian and Dr. White seconded the motion which was unanimously approved by the present members. Dr. Daigneault accepted the position as Secretary, and Dr. Checura will serve as Chair for the W4171 Technical Committee in 2022 as agreed in the 2020 committee meeting.

The 2022 meeting for the W4171 group was discussed. It was tentatively agreed to be held concurrent with the 48th annual conference of the IETS. The exact meeting format, location and time are pending, and it will be determined based on the format and dates of the annual and satellite IETS meetings.

At 4:32 pm Dr. Wheeler opened the general discussion again, but on the topic of collaborations. A brief discussion ensued with several members offering collaborative efforts to the group.

At 4:50 pm Dr. White made closing remarks and Dr. Youngs moved the motion to adjourn the meeting. The motion was accepted and the meeting adjourned.

Respectively Submitted,

Celina M. Checura - Secretary

W4171 Technical Committee

Accomplishments

ACCOMPLISHMENTS:   OBJECTIVE 1 Understand the biology of gamete development, fertilization and embryogenesis including the underlying cellular and molecular mechanisms.   1.   A series of studies over several breeding seasons at multiple sites have been conducted to optimize the ability to fixed-time AI using a 6 day CO-Synch protocol using pre-synchronization. In all replications, the CIDR pre-synch has improved FTAI conception rates, in cows and heifers.  Typically, the cows with the earliest post-partum intervals have the most improved conception   2.   a.   Determined early changes in gene expression during enhanced Sertoli cell proliferation stimulated by reduced endogenous estradiol.       b.   Identified potential receptors and ligands functioning during porcine fertilization.       c.   Successful derivation of bovine ESC from early blastocysts.   3.   Achieved the editing of the bovine ESX1 gene in somatic cells.   4.   Provided a simple and efficient protocol for extraction of DNA from bovine with high sensitivity conducive for downstream genomic and epigenomic analyses.   5.   a. Oocyte Maturation: ethanol addition augmented the follicular and oocyte degeneration rates but increased the estradiol production and the meiotic resumption. Furthermore, the follicular metabolomic profile was similar between ethanol and rbFSH treatments but different from the control treatment in sheep.       b.   Fertilization: dry mass is a valuable viability marker. A direct correlation between the presence of acrosome and the cell dry mass was observed, which can be used to evaluate the acrosome reaction. The three methods (Swim-Up, Gradient centrifugation and electro-channels) used to select viable spermatozoa for in vitro fertilization (IVF) result in very different sperm sub-populations; the dry mass of the sperm head is heavier in the spermatozoa selected with the electro-channel than the sperm selected by the other methods. These results show the potential of SLIM microscopy in reproductive biology. The tradeoff between cell viability and specificity can be circumvented by combining high-sensitivity phase imaging with deep learning. We trained a deep-convolutional neural network to perform semantic segmentation on quantitative phase maps. This approach, a form of phase imaging with computational specificity (PICS), allowed us to efficiently analyze thousands of sperm cells and identify correlations between dry-mass content and artificial-reproduction outcomes. Specifically, we found that the dry-mass content ratios between the head, midpiece, and tail of the cells can predict the percentages of success for zygote cleavage and embryo blastocyst formation. c.     Embryo Culture: The power of the GLIM microscope’s ability to perform 3D reconstruction and section of the image is similar to a CT-scan for evaluation of bovine embryos. We have also discovered an interesting “turbulence” (particle motion characterized by chaotic changes in pressure or flow velocity) within the embryo.  This phenomenon is interesting because it was more prominent in compromised embryos and almost non-existent in developing embryos.   6.   a. In vivo and in vitro heat stress was shown not to alter global levels of DNA methylation or DNA hydroxymethylation.       b.   The microtubule stabilizer taxol was shown to have some positive affect on normal chromosome distribution following vitrification of bovine oocytes but no positive effect on normal spindle configuration.       c.   The alternative microtubule stabilizer Epothilone B was detrimental for both spindle configuration and chromosome distribution after vitrification of bovine oocytes.       d.   An ultra-low-input ribosome profiling protocol was developed to systematically analyse both polysome- and nonpolysome-bound mRNA profiles of in vitro produced bovine blastocysts.       e.   Single-cell whole genome bisulfite sequencing (scWGBS) was performed to map genome-wide DNA methylation dynamics of three classes of human trophoblast cells (TB) (cytoTB, syncytioTB, and migratory TB) during human implantation using an extended embryo culture system.       f.    Single cell RNA sequencing was used to characterize developmental and transcriptomic differences between in vitro embryos produced in standard culture medium (IVC) with those produced using a culture medium with 75% reduced nutrients (carbohydrates and amino acids; IVRN).   7.   The application of 13C MFA to preimplantation bovine embryos presents unique challenges owing to the necessity to assess the metabolism of individual embryos (small sample size) and the complex medium. Novel labeling experiments had been designed to surmount these challenges and obtain a metabolic flux map for this complex system. This year (2020) was primarily focused on improving the mathematical model for analysis of flux networks and collecting samples according to the previously established design (varying substrates with varying labelled carbon atoms).  These experiments have been designed to probe the relative utilization of glycolysis and the pentose phosphate pathway, as well as the contributions of different carbon sources to metabolites originating at the pyruvate node.   8.   a.   RNAseq in oocytes of multiple species: RNA sequencing data were generated for mouse, bovine, and rhesus GV and MII stage oocytes.  Previously published and novel oocyte RNAseq from multiple species, including cow, are being used to compare transcriptome changes across species in a novel meta-analysis strategy seeking to identify conserved mechanisms driving oocyte maturation. Associated pathways and processes are being compared.       b.   SMCHD1 as a maternal transcription regulator: Maternally expressed SMCHD1 plays an important role in terminating the first wave of embryonic genome activation. Additionally, transient zygotic knockdown of SMCHD1 expression negatively impacts later development, further attesting to the importance of maternally expressed and zygotically expressed SMCHD1 in correctly regulating preimplantation gene expression in a manner consistent with high developmental potency.  Ongoing studies are assessing the maturation, quality, viability, and developmental competence of oocytes that lack SMCHD1 function.       c.   Effects of dairy cow nutrition on oocyte quality: Dairy cattle that either lose or maintain/gain body condition during lactations were compared.  Changes in serum lipid profiles were determined and oocyte transcriptomes were examined by RNA sequencing. Data are being analyzed to assess nature and mechanisms of effects.   9.  a.   Numerous up-regulated proteins in small follicles were directed to the healthy growth of both follicle and oocyte, while those in large follicles were involved in the ovulatory process. These proteins can be investigated as molecular markers of developmental competence and in vitro follicle culture. Several other proteins with potential roles during fertilization are also reported in the follicular fluid.       b.   It was shown that balanced combinations of insulin and thyroxine concentrations are beneficial to follicle survival (low doses) and development (high doses).       c.   The use of the follicle wall biopsy technique revealed the possibility of a real-time and repeated sampling of ovarian follicle cells and fluid of living mares, for effective evaluation of physiological processes occurring within the follicle. It was found that LH receptors in at least some of the layers of the follicle wall seems to trigger dominant follicles toward the anovulation process during the spring and fall transitional seasons.      d.    The label-free microscopy imaging, SLIM, was found to be more informative than the AFM for sperm dimension analyses. There were interesting correlations between different compartments and dry mass of both boar and stallion spermatozoa; and positive (boar) and negative (stallion) correlations between the sperm head dry mass and the length and width of the acrosome were found in both species.   10.  Utilization of CRISPR/Cas9 gene editing has allowed knockouts of specific conceptus genes proposed to be essential in early development and establishment of pregnancy.  Research has provided information on the role of conceptus estrogen, prostaglandins, interleukin 1B2, and interferon g in maternal conceptus elongation, recognition of pregnancy, placental attachment and development during early pregnancy.  Results indicate that conceptus expression of interleukin 1B2 is essential to rapid elongation of the pig conceptus on Day 12 of pregnancy.  It would appear that the early establishment of pregnancy does not necessarily need estrogen at least to Day 24.  Establishment of pregnancy in the pig may involve both conceptus secretion of estrogen and prostaglandin E.  The role of interferon g has not been clearly established but our current data indicate conceptus production is essential for maintenance of pregnancy.   11. A simple and practical system to deliver a defined photostimulation (wavelength and intensity) has been designed.   12. FLI cytokine supplemented maturation medium improved bovine oocyte maturation, blastocyst development and initial pregnancy rate following somatic cell nuclear transfer. We are monitoring pregnancies to determine the full-term development success.   OBJECTIVE 2 Refine methods to produce animals by genetic engineering or genome editing for the improvement of livestock production efficiency and development of human biomedical models.   1.      A series of studies over several breeding seasons at multiple sites have been conducted to optimize the ability to fixed-time AI using a 6 day CO-Synch protocol using pre-synchronization. In all replications, the CIDR pre-synch has improved FTAI conception rates, in cows and heifers.  Typically, the cows with the earliest post-partum intervals have the most improved conception rates along with the heifers that are prepuberal with low Reproductive Tracts Scores.    2.       Electroporation of CRISPR/Cas9 components into pig zygotes was used to circumvent the drawbacks of microinjection. To test viability of embryos/fetuses beyond the blastocyst stage, 2-4 cell gene deleted embryos derived from in vitro matured oocytes were transferred to oviducts of market weight gilts.  Comparison of guides used for gene deletion in fetuses recovered at 24 days was evaluated to consider if more efficient and less efficient guide sequences could be evaluated with an apparent difference noted among guides appearing equivalent in development to blastocysts. Skewed gender suggested an earlier effect of AR on gender than previously detected.   3.         The transcriptome profiles of the biPSCs were studied, and the successful reprogramming status of these biPSC lines was confirmed. The history and up-to-date progress for the studies on iPSC generation and characterization for all the major farm animals were summarized and published.   4.   a.   Transgenic Animals: There is no horizontal transmission of the transgene between transgenic and non-transgenic pigs that occurs during rearing, mating, gestation, or lactation and offer a foundation for the safe integration of transgenic animals into non-transgenic commercial production systems.       b.   Stem Cells: The addition of both platelet-rich plasma and fibrin scaffolds to autologous ASCs from liposuction improved bone healing of critical-size defects. Osteocalcin production is higher when vitamin D3, vitamin B12, and vitamin K were added to the differentiation medium compared with the standard osteogenic medium, but only 100 nM vitamin D3 treatment had a positive influence on ASC differentiation into osteoblasts. A mineralized collagen-amnion scaffold may provide a beneficial environment to aid craniomaxillofacial bone repair, especially in the course of defects presenting significant inflammatory complications.       c.   Others: There is potential for ultra-fast product design, engineering, and testing of medical devices needed for COVID-19 emergency response. A low-cost, easy-to-produce electronic sensor and alarm system for pressure-cycled ventilators have been developed. It estimates clinically useful metrics such as pressure and respiratory rate and sounds an alarm when the ventilator malfunctions during COVID-19 intubation. A point-of-care system integrated with a smartphone for detecting live virus from nasal swab media, using a panel of equine respiratory infectious diseases as a model system for corresponding human diseases such as COVID-19, has been demonstrated. Pathogen-spiked horse nasal swab samples were correctly diagnosed using the system, with a limit of detection comparable to that of the traditional lab-based test, polymerase chain reaction, with results achieved in &sim;30 minutes.   5.   The ability to obtain pregnancies and live births from transfer of fresh alpaca demi-embryos to recipients was demonstrated.    6.   a. It was shown that pretreatment of capacitated sperm with progesterone increased fertilization after intra cytoplasmic sperm injection of bovine oocytes.       b.   It was shown that while there was no effect on mitochondrial number, ATP production was significantly reduced as a result of vitrification and warming of bovine oocytes.  Vitrification at 24h of maturation followed by extended culture to complete maturation after warming did not improve ATP production compared to vitrification at 44h of maturation.         c.   An enhancing cell injection system was developed by real time confirmation of membrane penetration and embryo viability.   7.   Liver-specific genetic models have uncovered a previously unrecognized metabolic system that can help to maintain liver homeostasis under conditions of oxidative or toxic stress. The system discovered uses catabolism of the essential sulfur amino acid methionine (Met) to generate cytosolic disulfide reducing power in the liver. This is the only NADPH-independent source of cytosolic disulfide reducing power that has, as yet, been characterized.   8.   A database of Doppler measurements for normal pregnancies under different conditions (different sires; singleton vs. twins, maternal age, etc) is being developed.   9.   a. An efficient protocol for the introduction of human specific mutations into the sheep fetal fibroblast cells using CRISPR/Cas9 RNP approach has been developed.       b.   The F508del and G542X Cystic Fibrosis sheep models were successfully produced.       c.   Conditions for CF mutation correction in sheep fibroblast cells and ovine-bovine iSCNT preimplantation embryos in vitro were optimized.       d.   Preliminary results on using M3814 (a DNA-dependent protein kinase inhibitor) to increase efficiency of HDR look promising.       e.   Donor-derived spermatogenesis was sustained in NANOS2-null bucks following exogenous spermatogonial stem cell transplantation

Publications

PUBLICATIONS:   <ol> <li>Refereed articles/book chapters</li> </ol>   <ul> <li>Jaques, J. & Cardoso, Rodolfo & Chachere, J. & Sinha, S. & Seery, R. & Wilkes, A. & Forrest, David & Looney, Charles. (2020). Using Anti-Müllerian Hormone (AMH) as a Predictor of Ova Production for Bovine Embryo Transfer. Advances in Reproductive Sciences. 08. 36-47. 10.4236/arsci.2020.81004. v</li> </ul> <ul> <li>Berger, T Nitta-Oda, BJ. 2020. Sertoli cell proliferation in juvenile boars and microRNA. Livestock Science, 233(March): 103954.</li> <li>Berger, T, Tang, S, Tu, L, Soto, D, Conley, A, Nitta-Oda, B. 2020. Changes in testicular gene expression following reduced estradiol synthesis: a complex pathway to increased porcine Sertoli cell proliferation. Molecular and Cellular Endocrinology.</li> <li>Candelaria JI, Rabaglino MB, Denicol AC. Ovarian preantral follicles are responsive to FSH as early as the primary stage of development. J Endocrinol. 2020; JOE-20-0126.R2. doi:10.1530/JOE-20-0126.</li> <li>The Induced Pluripotent Stem Cells from Farm Animals. Su, Y., Zhu, J., Salman, S., & Tang, Y*. (2020). Journal of animal science. <a href="https://doi.org/10.1093/jas/skaa34">https://doi.org/10.1093/jas/skaa34</a></li> <li>Daigneault, B. W. 2020. Dynamics of paternal contributions to early embryo development in large animals. Biol Reprod http://dx.doi.org/doi:10.1093/biolre/ioaa182</li> <li>Daigneault, B. W., S. K. Rajput, and G. W. Smith. 2020. Simple workflow for genome and methylation analyses of ejaculated bovine spermatozoa with low sperm input. Biotechniques 68: 155-158.http://dx.doi.org/doi:10.2144/btn-2019-0121</li> <li>Rubessa, M., Kandel, M.E., Schreiber, S., Meyers, S., Beck, D.H., Popescu, G., and</li> <li>Wheeler, M.B. 2020. Sperm selection methods for bovine in vitro embryo production, characterized by spatial light interference microscopy. Systems Biology in Reproductive Medicine. https://doi.org/10.1080/19396368.2019.1701139</li> <li>Rubessa, M., Wheeler, M.B. (2020) Label-free microscopy: a non-invasive new</li> <li>tool to assess gametes and embryo quality. Theriogenology Volume 150, 1 July 2020, Pages 241-246. https://doi.org/10.1016/j.theriogenology.2020.01.065</li> <li>Dewey, M. J., E. M. Johnson, S. T. Slater, D. J. Milner, M. B. Wheeler and B. A. C. Harley (2020). Mineralized collagen scaffolds fabricated with amniotic membrane matrix increase osteogenesis under inflammatory conditions. Regenerative Biomaterials. Online 4/8/20, doi.org/10.1093/rb/rbaa005</li> <li>William P. King, Jennifer Amos, Magdi Azer, Daniel Baker, Rashid Bashir, Catherine Best, Eliot Bethke, Steve Boppart, Elizabeth Bralts, Rachael Deitkus, Gary Durak, Stefan Elbel, Greg Elliott, Jake Fava, Nigel Goldenfeld, Mitch Goodman, Courtney Hayes, Nicole Herndon, Shandra Jamison, Harley Johnson, Mark Johnson, John Kolaczynski, Tonghun Lee, Sergei Maslov, Davis McGregor, Derek Milner, Jonathan Mosley, Ujjal Mikherjee, Andy Musser, Max Newberger, Lucas O’Bryan, Jerry O’Leary, Alex Pagano, Michael Philpott, Brian Pianfetti, Alex Pille, Marcello Rubessa, Sam Rylowicz, Stephanie Selting, Sridar Seshadr, Cliff Shipley, Brian Stewart, Rachel Switzky, Sameh Tawfick, Matthew B. Wheeler, Karen White, Sarah Womack, Eric Wood, Charles Wood, Abigail Wooldridge. (2020). Emergency Ventilator for COVID-19. PLoS ONE 15(12): e0244963. https://doi.org/10.1371/journal.pone.0244963</li> <li>Kandel, M., Y. He, M. Rubessa, M. Wheeler and G. Popescu (2020). High sensitivity SLIM imaging and deep learning to correlate sperm morphology and fertility outcomes (Conference Presentation), SPIE BIOS, vol. 11249, Quantitative Phase Imaging VI. https://doi.org/10.1117/12.2550470</li> <li>Sun, F., Ganguli, A., Nguyen, J., Brisbin, R., Shanmugam, K., Hirschberg, D. L., Wheeler, M. B., Bashir, R., Nash, D. M., Cunningham, B. T. (2020).  Smartphone-Based Multiplex 30-minute Nucleic Acid Test of Live Virus from Nasal Swab Extract. Lab Chip 20, 1621-1627 DOI: 10.1039/D0LC00304B.</li> <li>Bane T.A., Bertels J.C., Polkoff K.M., Rubessa M. and Wheeler M.B. (2020). The Effects of Vitamin D3, Vitamin B12, and Vitamin K on the Differentiation of Porcine Adipose-Derived Stem Cells (ASC) into Osteoblasts. Journal of Biology Engineering and Medicine Volume 2(1): 1-5 doi:10.31487/j.JBEM.2020.01.01</li> <li>Rubessa, M., Feugang, J.M., Kandel, M.E., Schreiber, S., Hessee, J., Salerno, F., Meyers, S., Chu, I., Popescu, G., Wheeler, M.B. (2020). High-throughput sperm assay using label free microscopy: morphometric comparison between different sperm structures in boar and stallion spermatozoa. Animal Reproduction Science 219, August 2020, 106509, online June 16, 2020, https://doi.org/10.1016/j.anireprosci.2020.10650</li> <li>Kandel, M.E., Rubessa, M., He, Y.R., Schreiber, S., Meyers, S., Naves, L.M., Sermersheim, M.K., Sell, G.S., Szewczyk, M.J., Wheeler, M.B., Popescu, G. (2020). Deep convolutional neural networks reveal slight differences in spermatozoon ultra-structure correlating with reproductive outcomes. Proceedings of the National Academy of Sciences (P.N.A.S.) 117 (31) 18302-18309; DOI:10.1073/pnas.2001754117</li> <li>Mosley, J.F., Hurley, W.L., Rodriguez-Zas, S., Wheeler, M.B. (2020). Evaluation of risks from environmental contact with transgenic livestock. J Vet Med Res 7(3): 1190. 9 pages. https://www.jscimedcentral.com/VeterinaryMedicine/veterinarymedicine-7-1190.pdf</li> <li>Maki, A.J., Rabel, R.A.C., Rubessa, M., Ercolin, A.C., Roballo, K.S., Cooper, J.J., Wheeler, M.B. (2020). Autologous Adipose-Derived Stem Cells, Platelet-rich Plasma, and Fibrin Enhance Healing of Mandibular Bone Defects in Swine. International Journal of Regenerative Medicine Volume 3(2): 2-9. DOI: 10.31487/j.RGM.2020.02.01</li> <li>Corey, R.M., Widloski, E.M., Null, D., Ricconi, B., Johnson, M.A., White, K.C.,</li> </ul> Amos, J.R., Pagano, A.,  Oelze, M.L., Switzky, R.D., Wheeler, M.B., Bethke, E.,  Shipley, C.F.,  Singer, A.C. (2020). Low-Complexity System and Algorithm for an Emergency Ventilator Sensor and Alarm. IEEE Transactions on Biomedical Circuits and Systems, doi: 10.1109/TBCAS.2020.3020702. <ul> <li>Renato Félix da Silva, Laritza Lima, Rebeca Rocha, Ivina Brito, Gerlane Silva, Hudson Correia, Giovanna Rodrigues, Anna Clara Ferreira, Diana Nunes-Pinheiro, Arlindo Moura, Larissa Silveira, Edson G. Lo Turco, Matthew B. Wheeler, Ana Paula Rodrigues, Claudio Campello. (2020). In vitro long-term culture of isolated ovine preantral follicle: influence of ethanol on steroid production, oocyte meiotic resumption and metabolomic profile. Research in Veterinary Science, accepted 30 October 2020, In Press, doi.org/10.1016/j.rvsc.2020.10.029</li> <li>Benaissa MH, Y Bentria, N Mimoune, T Kernif, A Boukhelkhal, CR Youngs, R Kaidi, B Faye, and Y Halis. Seroprevalence and risk factors for Trypanosoma evansi, the causative agent of surra, in the dromedary camel (Camelus dromedarius) population in southeastern Algeria. Onderstepoort Journal of Veterinary Research 87(1), a1891. https://doi.org/10.4102/ojvr.v87i1.1891</li> <li>Benaissa MH, N Mimoune, CR Youngs, R Kaidi, and B Faye. 2020. First report of Chlamydophila abortus infection in the dromedary camel (Camelus dromedarius) population in eastern Algeria. Comparative Immunology, Microbiology and Infectious Diseases 73:101557,  https://doi.org/10.1016/j.cimid.2020.101557</li> <li>Lutz JC, SL Johnson, KJ Duprey, PJ Taylor, HW Vivanco, DP Salazar, MM Gonzales, and CR Youngs. 2020. Birth of a live cria after transfer of a vitrified-warmed alpaca (Vicugna pacos) preimplantation embryo. Frontiers in Veterinary Science 7:581877, <a href="https://doi.org/10.3389/fvets.2020.581877">https://doi.org/10.3389/fvets.2020.581877</a></li> <li>Esencan E, Jiang Z, Wang T, Zhang M, Soylemez-Imamoglu G, Seli E. Impaired mitochondrial stress response due to CLPP deletion is associated with altered mitochondrial dynamics and increased apoptosis in cumulus cells. Reproduction Sciences. 2020 Jan 14. PMID: 31939198.</li> <li>Li Y, Sun J, Ling Y, Hao M, Chen Z, Fang F, Liu Y, Cao H, Ding J, Cao Z, Zhang X, Bondioli K, Jiang Z, Zhang Y. Transcriptional profiles of oocytes during maturation and embryos during preimplantation development in vivo in the goat. Reprod Fertil Dev. 2020 Apr;32(7):714-725. PMID: 32317096.</li> <li>Li Z, Zhao S, Nelakanti RV, Lin K, Wu TP, Alderman III MH, Guo C, Wang P, Zhang M, Wang M, Jiang Z, Wang Y, Li H, Xiao AZ. N6-methyladenine in DNA antagonizes SATB1 in early development. Nature 2020 Jul: 583, 625–630. PMID: 32669713.</li> <li>Ming H, Sun J, Pasquariello R, Gatenby L, Herrick J, Yuan Y, Pinto C, Bondioli KR, Krisher RL, Jiang Z. The landscape of accessible chromatin in bovine oocytes and early embryos. Epigenetics. 2020 Jul 14. PMID:32663104.</li> <li>Smith R, Jiang Z, Susor A, Ming Hao, Tait J, Conti M, Lin CJ. The H3.3 chaperone Hira complex orchestrates oocyte developmental competence. bioRxiv. 2020 May 26, doi: https://doi.org/10.1101/2020.05.25.114124.</li> <li>Chung J, Sriram G, Keefer CL.Nanoparticle technology improves in-vitro attachment of cattle (Bos taurus) trophectoderm cells. Biotechnol Lett. 2020 Nov;42(11):2083-2089.</li> </ul> doi: 10.1007/s10529-020-02926-w. Epub 2020 Jun 3. <ul> <li>Paes, V. M., de Figueiredo, J. R., Ryan, P. L., Willard, S. T., Feugang, J. M. (2020). Comparative analysis of porcine follicular fluid proteomes of small and large ovarian follicles. Biology, 9(5). https://api.elsevier.com/content/abstract/scopus_id/85085521478</li> <li>Paes, V. M., Lima, L. F., Ferreira, A. C.A., Lobo, C. H., Alves, B. G., Rodrigues, A. P.R., Oliveira, A. C., Figueiredo, J. R., Feugang, J. M. (2020). The subtle balance of insulin and thyroxine on survival and development of in vitro cultured caprine preantral follicles enclosed in ovarian tissue. Theriogenology, 147, 10-17.</li> </ul> https://api.elsevier.com/content/abstract/scopus_id/85079386717 <ul> <li>Ishak, G. M., Dutra, G. A., Gastal, G. D.A., Elcombe, M. E., Gastal, M. O., Park, S. B., Feugang, J. M., Gastal, E. L. (2020). Deficiency in proliferative, angiogenic, and LH receptors in the follicle wall: implications of season toward the anovulatory condition. Domestic Animal Endocrinology, 70.</li> </ul> https://api.elsevier.com/content/abstract/scopus_id/85072712691 <ul> <li>Rubessa, M., Feugang, J. M., Kandel, M. E., Schreiber, S., Hessee, J., Salerno, F., Meyers, S., Chu, I., Popescu, G., Wheeler, M. B. (2020). High-throughput sperm assay using label-free microscopy: morphometric comparison between different sperm structures of boar and stallion spermatozoa. Animal Reproduction Science, 219. <a href="https://api.elsevier.com/content/abstract/scopus_id/85086603197">https://api.elsevier.com/content/abstract/scopus_id/85086603197</a></li> <li>Geisert R. D., and T. E. Spencer. 2021. Chapter 1: Introduction. In: Placentation in Mammals: Tribute to EC Amoroso’s contributions to Viviparity. Advances in Anatomy, Embryology and Cell Biology, Springer. (In Press).</li> <li>Geisert R. D., A. E. Meyer, C. A. Pfeiffer, D. N. Johns, K. Lee, K. D. Wells, T. E. Spencer, and R. S. Prather. 2021. Gene Editing to Investigate Role of Conceptus Factors in Establishment of Pregnancy. Reproduction (In Press).</li> <li>Pfeiffer C. A., A. E. Meyer, K. E. Brooks, P. R. Chen, J. Milano-Foster, L. D. Spate, J. A. Benne, R. Cecil, M. S. Samuel, L. A. Ciernia, M. F. Smith, K. D. Wells, T. E. Spencer, R. S. Prather, and R. D. Geisert. 2020. Ablation of conceptus PTGS2 provides a new understanding of early pregnancy events in the pig. Biol Reprod 102: 475-488. doi.org/10.1093/biolre/ioz192.</li> <li>Miller CG, Schmidt EE. Sulfur Metabolism Under Stress. Antioxid Redox Signal. 2020 Dec 1;33(16):1158-1173. doi: 10.1089/ars.2020.8151. Epub 2020 Aug 14. PubMed PMID: 32799544; PubMed Central PMCID: PMC7699002.</li> <li>Dóka É, Ida T, Dagnell M, Abiko Y, Luong NC, Balog N, Takata T, Espinosa B, Nishimura A, Cheng Q, Funato Y, Miki H, Fukuto JM, Prigge JR, Schmidt EE, Arnér ESJ, Kumagai Y, Akaike T, Nagy P. Control of protein function through oxidation and reduction of persulfidated states. Sci Adv. 2020 Jan;6(1):eaax8358. doi: 10.1126/sciadv.aax8358. eCollection 2020 Jan. PubMed PMID: 31911946; PubMed Central PMCID: PMC6938701.</li> <li>Shearn CT, Fennimore B, Orlicky DJ, Gao YR, Saba LM, Battista KD, Aivazidis S, Assiri M, Harris PS, Michel C, Merrill GF, Schmidt EE, Colgan SP, Petersen DR. Cholestatic liver disease results increased production of reactive aldehydes and an atypical periportal hepatic antioxidant response. Free Radic Biol Med. 2019 Nov 1;143:101-114. doi: 10.1016/j.freeradbiomed.2019.07.036. Epub 2019 Aug 1. PubMed PMID: 31377417; PubMed Central PMCID: PMC6848778.</li> <li>Ciccarelli M., Giassetti M.I., Miao D., Oatley M.J., Robbins C., Lopez-Biladeau B., Waqas M.S., Tibary A., Whitelaw B., Lillico S., Park C.H., Park K.E., Telugu B., Fan Z., Liu Y., Regouski M., Polejaeva I.A. & Oatley J.M. (2020) Donor-derived spermatogenesis following stem cell transplantation in sterile NANOS2 knockout males. Proc Natl Acad Sci U S A 117, 24195-204.</li> <li>Perisse I.V., Fan Z., Singina,G.N., White, K.L., and Polejaeva I.A.* (2020) Improvements in Gene Editing Technology Boosts Its Applications in Livestock. Frontiers in Genetics 11,</li> </ul>   <ol start="2"> <li>Books, non-refereed book chapters, proceedings, instructional media, theses/dissertations</li> </ol>   <ul> <li>Noga, N., C. Looney. 2020. Embryo Collection and Embryo Transfer. In: Bovine Reproduction. Hopper, R. M. John Wiley & Sons.</li> <li>Berger, T. Juvenile boars and testicular microRNA. 2020. Mendeley Data.</li> <li>Berger, T Nitta-Oda, B. 2020. Alteration in porcine testicular gene expression in response to reduced testicular estradiol synthesis. NCBI Gene Expression Omnibus.</li> <li>Placentation in Mammals: Tribute to EC Amoroso’s contributions to Viviparity. 2021. R. D. Geisert and T. Spencer. Advances in Anatomy, Embryology and Cell Biology, Springer. (In Press).</li> </ul>   <ol start="3"> <li>Abstracts</li> </ol>   <ul> <li>Oosthuizen, N., Fontes, P., Filho, R., Dahlen, C.R., Grieger, D.M., Hall, J.B., Lake, S., Looney, C., Mercadante, V.R., Neville, B.W. and Perry, G., 2020. 147</li> <li>Presynchronization and delayed fixed-time artificial insemination increases pregnancy rates with sex-sorted semen in replacement beef heifers. Journal of Animal Science, 98(Supplement_4), pp.115-116.</li> <li>Vazquez, J. Berger, T. 2020. Unveiling the proteome of sperm-oocyte plasma membrane interaction using a porcine model. Society for the Study of Reproduction 2020 Annual Meeting.</li> <li>Zacanti, K, Park, I, Rowe, JD, McNabb, BR, Ross, PJ, Maga, EA, Berger, TJ. 2020. Pig androgen receptor knockout fetuses via CRISPR/ Cas9 technology. Society for the Study of Reproduction 2020 Annual Meeting</li> <li>Schefer, L., Naves, L. M., Ramires Ferreira, C., Pradebon da Silva, E., Sobreira, T. J., Vieiro Marchioretto, P., Lima Verde Leal, C., Rubessa, M., and Wheeler, M. B. (2020). Lipid profiling of bovine blastocysts produced in vitro with and without a stimulator of cyclic guanosine monophosphate synthesis by multiple reaction monitoring profiling. L. Reproduction, Fertility and Development.  32(1): 158-159.</li> <li>Schefer, L., Naves, L. M., Ramires Ferreira, C., Pradebon da Silva, E., Sobreira, T. J., Vieiro Marchioretto, P., Lima Verde Leal, C., Rubessa, M., and Wheeler, M. B. (2020). Lipid profiling of bovine oocytes matured with different stimulation of cyclic GMP synthesis by multiple reaction monitoring profiling. Reproduction, Fertility and Development.  32(1): 226.</li> <li>Sermersheim, M.K., Lindsey, B. R., Naves, L. M., Rubessa, M., and Wheeler, M. B. (2020). Relationship between corpus luteum blood flow evaluated via colour Doppler ultrasound and pregnancy rate in bovine embryo transfer recipients. Reproduction, Fertility and Development. 32(1): 176.</li> <li>Pasqual, B. M., Correa, E. E., da Silva, E. P., Sermersheim, M. K., Naves, L. M., Sell, G. S., Lindsey, B.D., Rubessa, M., and Wheeler, M.B. (2020). Relationship between oestrus manifestation and pregnancy rates on recipients used in an in vitro-produced embryo transfer program. Reproduction, Fertility and Development. 32(1): 128-129.</li> <li>Lutz JC, SL Johnson, KJ Dupree, PJ Taylor, HW Vivanco-Mackie, MD Ponce-Salazar, M Miguel-Gonzales, and CR Youngs. 2020. Pregnancy from a vitrified-warmed alpaca preimplantation embryo. Reprod. Fertil. Dev. 32(2):128. <a href="https://doi.org/10.1071/RDv32n2Ab7">https://doi.org/10.1071/RDv32n2Ab7</a></li> <li>Diaz, FA, Gutierrex, EJ, Foster, BA, Hardin PT and KR Bondioli. Effect of in vivo and in vitro heat stress on DNA methylation and DNA hydrooxymethylation of bovine oocytes and embryos. Reprod. Fertil. Dev. January 2021.</li> <li>Gatenby, L and KR Bondioli. Progesterone induced acrosome reaction and fertilization rates with bovine intracytoplasmic sperm injection.  Fertil. Dev.  January 2021</li> <li>Girka, E and KR Bondioli. Effect of taxol and epothilone B on meiotic spindle stabilization in vitrified bovine ooctyes.  Fertil. Dev.  January 2021.</li> <li>Goheen B, Rajput S, Logsdon DM, Yuan Y, Schoolcraft WB, Jiang Z, and Krisher RL. A reduced nutrient culture environment improves bovine blastocyst development and alters mitochondrial metabolism as elucidated by single cell RNA sequencing. Fertility Sterility. 2020, P-566.</li> <li>Gutierrez, EJ, Diaz, FA and KR Bondioli. Effect of vitrification of porcine oocytes on ATP production and mitochondrial copy number. Reprod. Fertil. Dev. January 2021.</li> <li>Logsdon DM, Ming H, Sun J, R. Schoolcraft WB, Krisher RL, Jiang Z, Yuan Y. Single cell multi-omics of peri-implantation stage human embryos reveals similarities between early trophoblast differentiation and neuronal behavior. 53th Annual conference of Society for the Study of Reproduction (SSR), 2020.</li> <li>Mor A, Gatenby L, Dzekunskas E, Zhu L, Bondioli KR, Jiang Z, and Seli E. Enhancing cell injection systems by real time confirmation of cytoplasmic penetration. Fertility Sterility. 2020, O-174.</li> <li>Zhu L, Ming H, Liu S, Iyyappan R, Liano ED, Dvoran M, Chen Q, Susor A, Zhou T, and Jiang Z. High-resolution ribosome profiling reveals translational selectivity in the mammalian blastocyst. Reprod, Fertil. Dev. 3 December 2020.</li> <li>Lugar D, Adhikari S, Keefer CL, and Sriram G. 13C Metabolic Flux Analysis of Pre-Implantation Bovine Embryos: Surmounting Challenges Due to Small Sample Size and Complex Media, American Chemical Society National Meeting, March 22-26, 2020</li> <li>Littlejohn, B. P., Mercer, K. B., Bowers, K. J., Messman, R. D., Contreras-Correa, Z. E., Reid, D., Feugang, J. M., Vann, R. C., Lemley, C. O. (2020). Influence of chronic melatonin supplementation on bovine testis physiology. Southern Section ASAS.</li> <li>D. Geisert, A. E. Meyer., C. A. Pfeiffer, D. N. Johns, T. E. Spencer, and R. S. Prather. 2020. Early Embryonic Loss is an Important Mechanism for Maximizing Litter Size in the Pig. Physiology and Endocrinology Symposium National ASAS Meeting, Madison, Wisconsin</li> <li>Johns D. N., C. G. Lucas, P. R. Chen, S. D. Perry, C. A. Pfeiffer, L. D. Spate, R. F. Cecil, K. M. Wells, T. E. Spencer, R. S. Prather, and R. D. Geisert. 2020. Porcine Conceptus Expression of Interferon Deltas and Gamma During Early Pregnancy. 53rd Annual Meeting of SSR.</li> <li>Johns D. N., C. G. Lucas, P. R. Chen, L. D. Spate, C. A. Pfeiffer, R. F. Cecil, M. A. Fudge, S. D. Perry, K. M. Whitworth, R. F. Cecil, K. M. Wells, T. E. Spencer, R. S. Prather, and R. D. Geisert. 2020. Conceptus Interferon Gamma is Essential for Pregnancy Maintenance in the Pig. The 17th Annual Gilbert S. Greenwald Symposium on Reproduction and Regenerative Medicine, Kansas City, MO.</li> <li>Gholson S. K., A. L. Schmelzle, J. A. Green, M. F. Smith and R. D. Geisert. 2020. Altering sexual behavior of rats through neonatal treatment with an aromatase inhibitor, estrogen receptor antagonist and testosterone. Midwest ASAS Meeting, March 2-4, 2020.</li> <li>Checura CM, Pratt SL, Campbell LV, Farmer K, Loughlin G, Mitchell M, Sanford A, Treske A, Malter H. 197 - The use of photostimulation to enhance oocyte cytoplasmic maturation. Reproduction, Fertility and Development. 2020;32(2):227.</li> <li>Checura CM, Atkinson K, Boulos N, Crowfoot E, Friend H, Jackson S, Lubowicki S, Silva C, Walker E. 124 - Utilization of Doppler Ultrasonography for the Study of Fetal Development in Sheep. FOCI meeting; Clemson University; 2020, 55.</li> <li>Goldberg M, Mitchell M, Pratt SL, Malter H, Checura CM. The Use of Photostimulation to Improve In Vitro Fertilization. HSC Showcase 2020; Health Sciences Center at Prisma Health; Greenville, SC, USA: 2020; 57.</li> <li>Fan Z., Regouski M., Liu Y., Keim J., Perisse I.V., Oatley J.M., Polejaeva I.A. (2020) Generation of NANOS2 knockout goats using CRISPR/Cas9 and SCNT techniques. Reproduction, Fertility and Development 32 (2) 193 - 194.</li> <li>Perisse I.V., Fan Z., Van Wettere A., Wang Z., Harris A., White K., Polejaeva I.A. (2020) Introduction of F508del human mutation into the CFTR gene of sheep fetal fibroblasts using CRISPR/Cas9 ribonucleoprotein. Reproduction, Fertility and Development 32 (2) 192 - 193.</li> <li>Singina G.N.*, Shedova E.N., Polejaeva I.A. and Taradajnic T.E. (2020) Effect of cytokines during in vitro maturation of bovine oocytes on the development potential of partheno-genetic embryos. Reproduction, Fertility and Development 32 (2) 222.</li> <li>Keim J., Zhang W., Liu Y., Rutigliano H., Zhou A., Polejaeva I.A. (2020) Analyzing metabolomic profile of bovine IVF and SCNT embryos through Raman spectroscopy. Reproduction, Fertility and Development 32 (2) 169 - 170.</li> <li>Bunch K., Perisse I.V., Fan Z., Wang Z., Harris A., White K.L., Polejaeva I.A. (2020) Construction of candidate vectors for correction of the intestinal CFTR gene expression in Cystic Fibrosis sheep fetal fibroblast cells. The 14th Utah Conference on Undergraduate Research, Utah State University, Logan, Utah, USA, February 7, 2020.</li> </ul>   <ol start="4"> <li>Miscellaneous publications (semi-technical/lay publications)</li> </ol>   <ul> <li>Hahn, M., C. Looney. 2020. Precision Agriculture in Dairy. In: Arkansas Cattle Business.</li> <li>Noga, N., C. Looney. 2020. Defined Breeding Season. In: Arkansas Cattle Business.</li> <li>Looney, C., N. Noga. 2020. Ultrasound Scanning to Measure Body Composition in Beef Cattle. FSA 3106. University of Arkansas, Fayetteville, AR.</li> <li>Sep 14, 2020 “Surrogate sires' could create specially bred livestock” The Gardian.</li> <li>Sep 14, 2020 “Gene editing to produce ‘super dad’ livestock” BBC News.</li> <li>Sep 14, 2020 “Scientists create gene-edited animals as 'surrogate sires' to boost food production”</li> <li>Daily Mail.</li> <li>Sep 14, 2020 “Gene-edited livestock 'surrogate sires' successfully made fertile”</li> <li>Science Daily.</li> <li>Sep 14, 2020 “First gene-edited livestock created that can serve as ‘surrogate sires’” Irish examiner.</li> <li>Sep 15, 2020 “Scientists create ‘surrogate sire’ animals to pass on genetic traits of donor males.</li> <li>Yorkshire Post, UK.</li> <li>Sep 15, 2020 “GM ‘super livestock’ to meet food shortage” The Daily Telegraph.</li> <li>Sep 15, 2020 “Stem cell hopes of beefing up livestock” Daily Mail.</li> <li>Sep 15, 2020 “Gene-edited livestock could boost food production in growing world” The Scotsman.</li> <li>Sep 15, 2020 “CRISPR-Cas9 Used to Generate “Surrogate Sire” Livestock that Produce Only Donor Sperm” Genetic Engineering & Biotechnology News.</li> <li>Sep 16, 2020 “Gene-edited livestock surrogate sires promise to transform selective breeding”</li> <li></li> </ul>

Impact Statements

IMPACTS: 1. Continue to perfect Beef Heifer Replacement from weaning to breeding. We have managed a total of 1500 heifers from reproductive tract scores and pelvic measurements at 14-16 months following breeding and pregnancy checks. Pregnancy rates using fixed timed AI with conventional semen is 52% (n=600) and with female sexed semen, 46% (n=250). By using this data we can fully demonstrate to beef producers the methods and procedures and display the economic factors of heifer development. Our novel pre-synchronization protocols improve success rates by 15% in beef heifers and 10% in beef cows. The greatest impact of this new protocol is with heifers and cows that have not begun to cycle prior to the synchrony program. It can be easily administered to young prepuberal heifers 13 months of age and early post-partum cows less than 40 days from calving with low body condition scores (< 5 BCS). Current impacts in beef cattle research are being made with first utilization of precision agriculture in Animal Science using sensors that record temperature, activity, rumination, and fertility. Herd owners can realize immediate returns in efficiency by saving time and money on cattle especially in feed lots, predicting sickness. This technology is complimentary using the RFID tags that we have adopted at SWREC. The utilization of Anti-Mullerian Hormone (AMH) as a predictor of oocyte production for advanced reproductive technologies was confirmed and has been successfully used in commercial herds to select heifers or cows to produce more total ova but not production of viable embryos. 2. The identification of additional membrane proteins involved in early porcine gamete interaction provides a number of targets that might be used to evaluate fertility/fertilizing ability of respective gametes and males. The finding that FSH plays a role in preantral follicle development has a potential impact on the use of the hormone in cattle and human ovarian stimulation protocols. It will be critical to test this hypothesis in vivo to have a better understanding of whether we are unintentionally manipulating preantral follicles when performing FSH stimulation, and whether this could be used strategically to improve current protocols. 3. Our study confirmed the successful reprogramming status of the biPSCs established in our laboratory, and generated transcriptome datasets of these biPSCs in order to help elucidate the key events and mechanisms to further improve the reprogramming efficiency and pluripotency of biPSCs. These biPSCs could greatly facilitate the studies on cattle embryo development, cell differentiation, and development of transgenic cows for biomedical and agricultural purposes. 4. The complete understanding of X chromosome inactivation, an important epigenetic event during early embryo development, paves the way to identify abnormalities of XCI in embryos from biotechnology. Strategies can therefore be developed to influence epigenetic modifications such that such anomalies can be alleviated. 5. Provided sperm DNA isolation techniques applicable to human biomedical sciences and livestock agriculture for fertility, genomic and disease indications. Provided a reference resource of paternal contributions to embryo development in large animals to advance models for human biomedical research. 6. a. Oocyte Maturation: Ethanol augments follicular and oocyte degeneration rates but increased the estradiol production and meiotic resumption. Ethanol has the same effect on follicular metabolites as rbFSH treatment. b. Fertilization: The use of GLIM microscopy to determine dry-mass content ratios between the head, midpiece, and tail of sperm to predict the percentages of success for zygote cleavage and embryo blastocyst formation may fundamentally change how sperm are evaluated for ARTs. c. Embryo Culture: The discovery of particle-motion characterized by chaotic changes in pressure or flow velocity within the embryo being more prominent in compromised embryos and almost non-existent in developing embryos could provide an objective metric for embryo quality. d. Transgenic Animals: The demonstration of the lack of horizontal transmissions of transgenes in swine offers a foundation for the safe integration of transgenic animals into non-transgenic commercial production systems. e. Stem Cells: Platelet-rich plasma and fibrin scaffolds along with autologous ASCs improved bone healing of critical-size defects. These materials can be added to the therapies to regenerate large bone defects. f. Other: an ultra-fast product design, engineering, and testing system for an emergency ventilator needed for COVID-19 emergency response have been used. A low-cost, easy-to-produce electronic sensor and alarm system for pressure-cycled ventilators was produced. A point-of-care system integrated with a smartphone for detecting live virus from nasal swab media that could be useful to detect Covid-19 virus was developed. 7. The successful production of live offspring from a bisected alpaca embryo provides a new opportunity to increase the number of offspring from genetically valuable alpaca donor females. 8. a. Understanding how epigenetic effects are induced by environmental influences is essential for development of strategies to optimize animal production systems in the face of changing climates. b. Oocyte vitrification and intra cytoplasmic sperm injection are a potentially valuable technology for animal breeding, conservation of species and application of gene editing technologies to domestic animals. The accomplishments reported here represent incremental progress towards making these technologies available. c. High-resolution ribosome profiling reveals the “Bona Fide” active translating mRNAs in the mammalian blastocyst. The data presented here set an example and open future avenues for detailed ribosome-fraction based translatome analyses to reveal novel cellular/embryonic functional regulators beyond transcriptomic data. d. Genome-wide DNA methylation analysis of human trophoblast differentiation suggest DNA methylation is an important driving force for directing TB lineage emergence during implantation and that there are analogies between early trophoblast differentiation and neuronal behavior. e. A reduced nutrient culture environment improves bovine blastocyst development and alters mitochondrial metabolism. This provides novel insights into bovine metabolic strategies that support blastocyst development, as well as potential approaches to manipulate metabolism to improve both the quantity and quality of embryos produced in vitro. f. Intra cytoplasmic sperm injection and oocyte vitrification are potentially valuable technologies for application of gene modification to animal production systems. g. When attempting zygote cytoplasmic microinjection, electrical resistance increase can serve as a reliable tool to confirm successful cell penetration and embryo viability, independent of optical visualization. This technology can potentially be integrated into a manual or robotic cell injection system. 9. The methods and advanced mathematical models developed will help extend the application of MFA framework to complex mammalian systems. Unraveling the metabolic basis of normal early embryo development will provide significant benefits to human and animal reproductive health. Improved embryonic competency following in vitro production and cryopreservation would stimulate the industry by lowering costs, especially those related to recipient management. 10. a. RNAseq in oocytes of 4 species: Should provide a new and unifying understanding of core developmental mechanisms applicable across mammalian species that establish high quality oocytes, and how genotype and species can impact oocyte characteristics. b. SMCHD1 as a maternal transcription regulator: Should reveal fundamental mechanisms that establish female fertility by enabling the oocyte to regulate early embryonic gene expression correctly. c. Effects of dairy cow nutrition on oocyte quality: Seeks ultimately to mitigate negative impacts of weight loss and nutrition deficits on dairy cow fertility. This could lead to changes in education/extension/outreach to dairy industry and improved methods of animal and herd management. 11. a. The dynamic of the follicular fluid composition having evolutive function during follicle growth is demonstrated in pigs, and subsets of proteins are identified as potential candidates to improve oocyte in vitro maturation or in vitro follicle culture. b. The combination of insulin and thyroxine at lower concentrations is beneficial for follicular survival, while higher levels provide better follicle development. c. The novel follicle wall biopsy technique is revealed as a reliable tool to conduct in situ investigation of intrafollicular events in living mares. Both follicular fluids and follicle cells can be harvested for longitudinal studies in the same animal, reducing individual variations and number of animals to use at each experimental time-point. d. The label-free microscopy system, SLIM, is demonstrated as a potential tool for rapid and high-throughput evaluation of sperm viability that may be useful during in vitro fertilization procedures. 12. Understanding the roles of pig conceptus genes expressed during early pregnancy will help in determining how to improve survival and growth of the fetus during pregnancy as well as improve survivability at birth. The pig model also provides information concerning establishment and maintenance of pregnancy in women. The large loss of pig embryo clones could be result of failure to properly elongate. Understanding lack of elongation in these clones could be related to improper/adequate expression of interleukin 1B2. Improving culture conditions which increase interleukin 1B2 production may increase the efficiency of cloning. 13. From a redox metabolism standpoint, current studies are uncovering previously unrecognized systems that support survival and function of liver cells, which, in turn, is providing insights into better therapies for treating some liver diseases or toxicities. From more of a bioengineering standpoint, these studies are demonstrating the power of being able to genetically manipulate hepatic metabolism in adult animals. This promises to provide interesting new ways to genetically modify nutrient use or intermediary metabolism of animals that could improve food-production characteristics of livestock without generating genetically modified food products.

Date of Annual Report: 02/27/2022

Report Information

Annual Meeting Dates: 01/09/2022 - 01/09/2022
Period the Report Covers: 01/01/2021 - 12/31/2021

Participants

Participants
In attendance were Charles Looney (Arkansas),
Patricia J. Berger (California),
Anna Denicol (California),
Jerry Bouma (Colorado)
X. Cindy Tian (Connecticut),
Young Tang (Connecticut),
Brad Daigneault (Florida),
Matthew B. Wheeler (Illinois),
Curt Youngs (Iowa),
Kenneth Bondioli (Louisiana),
Zongliang Jiang (Louisiana),
Carol L. Keefer (Maryland),
Jean Feugang (Mississippi),
Kiho Lee (Missouri),
Brett White (Nebraska),
Joao Gabriel Nascimento Moraes (Oklahoma),
Celina Checura (South Carolina),
Irina Polejaeva (Utah)
Du Min (Washington).

Note that attendance was fluid during the day due to the virtual nature of the meeting.

In person attendance
Dr. Checura,
Dr. Daigneault,
Dr. Bondioli,
Dr. Brett White,
Dr. Looney,
Dr. Nascimento,
Dr. Feugang,
Dr. Tian,
Dr. Wheeler

Zoom attendance
Dr. Du,
Dr. Keefer,
Dr. Jiang,
Dr. Polejaeva,
Dr. Ken White,
Yot07001,
Dr. Li,
Dr. Young,
Dr. Berger

Brief Summary of Minutes

Brief Summary of Minutes of Annual Meeting

Hyatt Regency, Savannah, GA

January 9^th, 2022

11:08 – Dr. Checura asked for permission to record the meeting over Zoom and

was agreed.

11:10 – Motion to approve 2021 minutes -Dr. Daigneault, approved Dr. Bondioli

11:10 – Station reports:

- Arkansas rep: Dr. Looney: > 645 cows for GnRH CIDR studies on dominant follicle development studies; sexed semen and Sexing Technologies with improved preg rates (Brangus 74% conception at D70); precision agriculture studies to reduce labor around working cattle – virtual CIDR

- Missouri 11:20AM: rep Dr. Lee; Dr. Geisert still part of multi-state, Dr. Kiho Lee will represent. Research on activation of oocytes focused on zinc studies based on porcine work with relevance to bovine translation. Gene KO strategies to understand specific genes important for oogenesis and folliculogenesis. Efficiency of KO studies and optimizations using different CRISPR strategies.

- Connecticut 11:40AM: – Dr. Tang – bovine induced pluripotent stem cells with focus on transgene silencing; refinement of media compatible for experimentation; Dr. Cindy Tian – 11:48 – plant derived antimicrobials to reduce antibiotic use in embryo and cell culture; in-vivo studies of oocyte maturation to understand dichotomy of in vitro vs in vivo;

- Iowa – Dr. Young – 12:02 PM – Ethiopian oocyte/embryo research; microbiome research with sheep;

- Florida – 12:15PM – sperm studies to improve shelf life at ambient temperature; roles of PPAR-gamma in sperm function; sheep characterization studies (female cyclicity and sperm); effects of TBT on sperm; embryo PPAR gamma functions;

- Louisiana – 12:28PM – Dr. Bondioli; lipids in porcine oocytes (identification); glutathione and vitrification studies with cultures; 12:47PM - Dr. Jiang – gene profiling and epigenetic regulation of bovine embryos at multiple stages of preimplantation development; approaches to obtain higher single- cell coverage;

- Maryland – Keefer – 1:11PM – moonlighting proteins in bovine embryos; glucose studies at different developmental stages of embryo development;

- Michigan – Not Present

- Mississippi 1:25PM – Dr. Feugang – equine follicular fluid characterization at different developmental stages; shotgun proteomic approaches to identify stage specific proteins; microvessicle content;

1:50 PM – Biological Break; decided to reconvene at 2:30PM

2:35PM – Continue Station Reports

- Colorado – 2:35PM – Dr. Bouma – transfection of ovine trophoblast cells for androgen receptor studies

- Montana – not present

- Nebraska - Brett White – 2:52PM – GnRH receptor studies in transgenic porcine; endocrine profiles

- Illinois – Dr. Wheeler – 3:08 PM – Oocyte maturation in cross bred cattle; practical application of embryo transfer; pig follicle in vitro maturation; sheep oocyte maturation; long bone models for non union defects in pigs;

- Oklahoma – 3:30 PM – Dr. Nascimento – Regulation of conceptus elongation in bovine (transcriptomic, miRNA and open chromatin)

- South Carolina – 3:39PM – Dr. Checura – oocyte maturation with focus on mitochondrial function and ATP; sheep as twin biomedical model for humans; role of mitochondria in developmental programming; mitochondrial studies with light

- Utah – 4:07PM – Dr. Polejaeva – effect if cytokine supplemented maturation medium on bovine SCNT; mitochondrial studies on interspecies SCNT; small ruminant models of gene edited animals; modeling sickle cell disease – in utero gene therapy using sheep; improve efficiency of knock-in and point mutation introduction using different oligos; compounds that influence NHEJ and HR for DNA repair;

- Washington State – 4:41PM – Dr. Du – Early embryo and fetal development; using mice and then beef cattle for later stages

- California – 4:44PM – Dr. Berger – Sertoli cell research in testis; reduction of testicular estrogen in boars promotes Sertoli cell production; aromatase treatments to sustain estradiol levels;

4:57PM -Dr. Checura opened up the floor for further collaborative discussion;

- Dr. Checura opened discussion for arrangements of 2023 meeting – decision on location will be dependent upon IETS decision of annual meeting next year. AETA in Orlando identified as a possible location.

- Motion by Dr. Nascimento, Seconded by Dr. Checura. Date is in October.

- Dr. Checura nominated Dr. Nascimento for Secretary of 2023 meeting. Seconded by Dr. Daigneault. All agreed – 5:10PM

- 5:12 PM- Dr. Checura motioned to adjourn. Dr. Tian seconded. All agreed. End minutes.

Accomplishments

<h3>Accomplishments</h3> Objective 1 Understand the biology of gamete development, fertilization and embryogenesis including the underlying cellular and molecular mechanisms. <ol> <li>We have determined the 6-day CO-Synch protocol with GnRh and AI, 72 hrs. post CIDR removal + PGF is very effective in producing acceptable Fixed-Timed AI pregnancy rates in cows. Research and Workshops with ES and AI have improved our adoption of these technologies in Arkansas. Research on Pre-Synchronization and utilization of Gender-Selected semen has proven competitive commercially feasible results.</li> <li>Reduction in aromatase activity with peripubertal and post pubertal development of the boar testis is associated with increased methylation.</li> <li>Letrozole treatment has a prolonged effect on aromatase activity without increasing DNA methylation.</li> <li>Follicle growth after ovarian cortex cryopreservation indicates that this can be a viable method to preserve follicles that could later be retrieved from the tissue for further culture.</li> <li>We identified H3K9me3 as a reprogramming barrier for the generation of bovine iPSCs, and successfully established multiple primed- and naïve-like biPSC lines by targeting this epigenetic mark in reprogramming.</li> <li>We completed collection of samples for future studies of follicular fluid dynamics during oocyte maturation. The analysis of the follicular fluid for electrolytes, hormones (progesterone and LH), metabolites, acid-base parameters have been completed.  Proteomics analysis has also been completed and data analysis is underway.  Metabolomics analysis is being conducted at Metabolon. </li> <li>Developed a modified semen extender compatible for long-term storage of frozen-thawed bovine sperm at ambient temperature.</li> <li>Characterized the expression and localization of PPARʏ in bovine sperm.</li> <li>Optimized pre-cooling conditions to increase storage time of cooled stallion sperm.</li> <li>Characterized expression and function of nuclear proteins important for bovine in vitro embryo development.</li> <li>Demonstrated impacts of environmental toxicants on post-ejaculatory sperm function.</li> <li>Results show that performing DFR in half-blood Bos taurus X Bos indicus cattle is beneficial for the development of higher quality COCs and a shorter interval of OPU following DFR in Bos taurus X Bos indicus cattle increases the number of higher quality COCs.</li> <li>The addition of AMH and/or FSH did not affect follicular survival and growth in pigs. α-MEM is a more effective base medium than NCSU23 for the in vitro follicular development of pig preantral follicles and AMH reduces the steroid production induced by FSH.</li> <li>These results have shown that ethanol addition augmented the follicular and oocyte degeneration rates but increased the estradiol production and the meiotic resumption. Furthermore, the follicular metabolomic profile was similar between ethanol and rbFSH treatments but different from the control treatment in sheep.</li> <li>The results have shown a beneficial effect of conjugated linoleic acid (CLA) supplementation on bull semen quality.</li> <li>The porcine radius segmental defect model provides a viable platform for testing printed scaffolds and bioengineered composite scaffold constructs for the efficacy of healing segmental defects and other forms of non-union in long bones.</li> <li>We conducted a preliminary study on the feasibility of producing bovine embryos in vitro in Ethiopia and demonstrated that abattoir-derived ovaries can yield suitable cumulus-oocyte complexes.</li> <li>Using an improved Scarce Sample Polysomal-profiling (SSP-profiling) approach, we systematically and comparatively analyzed the transcriptome, polysome- and nonpolysome-bound mRNA profiles of bovine oocytes and early embryos at 2-, 8-cell, morula, and blastocyst stage, and presented the first translatome dynamics during mammalian preimplantation embryo development.</li> <li>Using a hybrid network architecture, a mixture of convolutional neural network (CNN) and recurrent neural network (RNN), the missing CpGs sites by WGBS were predicted. With this approach, we provide a completed DNA methylome dynamics in bovine preimplantation embryo development and reveal critical features of the bovine embryo methylomes that are not characterized by the scWGBS technology alone.</li> <li>The effects of maternal gestational restricted- and over-nutrition on semen parameters and sperm epigenetics (small RNAs) in F1 generations were tested. Semen analysis did not detect statistically differences in semen characteristics (volume, pH, sperm concentration and motility) between RES, OVER and CON groups. However, both sperm small RNA composition and expression levels were significantly altered in responses to poor maternal gestational nutrition in sheep.</li> <li>Using matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry the lipid profiles of individual porcine oocytes was profiled during in vitro </li> <li>Accumulation of cytoplasmic glutathione (GSH) was increased in bovine oocytes by addition of a cell permeable substrate, GSH ethyl ester, during in vitro maturation.</li> <li>Tests are ongoing with standards to develop new protocols for analyzing the spent media to analyze metabolic flux.</li> <li>RNA sequencing data were generated for mouse, bovine, and rhesus GV and MII stage oocytes.</li> <li>Dairy cattle that either lose or maintain/gain body condition during lactations were compared. Changes in serum lipid profiles were identified and oocyte transcriptomes were examined by RNA sequencing. Changes in pathways and functions detected at the transcriptome level were mechanistically linked to serum lipid changes using Ingenuity Pathway Analysis. Manuscript has been submitted.</li> <li>Numerous differentially regulated proteins (up and down) were directed to the equine follicle deviation (26), development (13), and ovulation (4). In addition, the 53 uniquely classified high abundant proteins across developmental follicle stages showed differential detection levels.</li> <li>Zn chelators possess specific effects on pig and cow oocytes.</li> <li>Production of GDF9 knockout pigs. Detailed analysis at puberty should expand our knowledge on the involvement of GDF9 on oogenesis.</li> <li>Results are confirming a role for transsulfuration and de novo glutathione synthesis, as was predicted from our earlier studies. However, we are also now beginning to uncover many other metabolic realignments in sulfur utilization and in amino acid metabolism that is providing unexpected insights into how liver responds to and survives oxidative and toxic stresses.</li> <li>During the follicular phase of the estrous cycle, serum from GnRHR-II KD gilts tended to have approximately 20% lower concentrations of 17ß-estradiol compared to littermate control gilts.</li> <li>Throughout the follicular phase, transgenic females secreted less LH than counterpart pubertal control gilts. Although numerical reductions were observed, average circulating concentrations for FSH, LH and progesterone were not different between GnRHR-II KD and littermate control gilts. The duration of FSH pulses in GnRHR-II KD animals was significantly reduced vs. littermate control gilts. The frequency of FSH pulses tended to be higher in control gilts compared to GnRHR-II KD females. Antral follicles tended to be larger in GnRHR-II KD gilts; however, total antral follicle counts were not different between lines. Neither body weight nor ovarian, uterine or oviduct weights differed between GnRHR-II KD and littermate control females.</li> <li>A defined photobiomodulation system (time, wavelength, and intensity) that affect oocyte competence has been developed.</li> <li>FLI cytokine supplemented maturation medium improved bovine oocyte maturation, blastocyst development and pregnancy rate. Importatntly, a four-fold greater full-term development rate using this treatment was achievd following somatic cell nuclear transfer.</li> <li>We developed a method allowing significantly decrease the mtDNA copy numbers in oocytes P<0.001 that might be beneficial for iSCNT.</li> <li>Established techniques for ooplasmic biopsies followed by successful parthenogenote development in vitro and quantification of mRNA content from biopsies. Micromanipulation techniques were used to sample oocyte cytoplasm for molecular analysis but leaving enough cytoplasm to support early development. EmbryoArray and well-of-the-well (WoW) systems were used to track development of biopsied oocytes.  Micro-scale RNA extraction and cDNA synthesis, followed by nano-scale qPCR analysis of biopsy-derived cDNA were perfected to allow for molecular analysis of representative subsamples of MII-arrested bovine oocytes AND concurrent development of the biopsied oocytes. </li> <li>Long noncoding RNA, Dio3os, via inhibiting D3 activity, promotes local thyroid hormone signaling and prenatal brown adipose tissue development. Dio3os expression is regulated by DNA methylation, which is enhanced due to maternal obesity, suppressing its expression. Elevation of DNA methylation of the Dio3os promoter has an oocyte origin.</li> </ol> Objective 2 Refine methods to produce animals by genetic engineering or genome editing for the improvement of livestock production efficiency and development of human biomedical models. <ol> <li>No off-target editing was detected in blastocysts or conceptuses following CRISPR editing.</li> <li>A review paper was submitted and published providing an overview on the role androgen in placental development and pregnancy (Life (Basel). 2021 Jul 1;11(7):644.)</li> <li>A book chapter was contributed and published in Reproductive Hormones describing the importance of androgens in pregnancy. (Chapter 4 Androgen Signaling in the Placenta).</li> <li>The genetic modification of cattle is a powerful tool for biotechnology. It allows for the generation of new or modified products and functionality that are not currently available in this species.</li> <li>ASC can be successfully edited to express GFP, and results suggest that utilizing a dual fluorescent label sorting strategy does not enhance the number of GFP ASC colonies isolated.</li> <li>Development of a culture condition enables the long-term culture of bovine TSC without altered morphology and differentiation from bovine IVF embryos. The generated bovine TSCs were fully characterized for its self-renewal, pluripotency, molecular features, and in vitro developmental potential.</li> <li>Utilization of an extended incubation time after warming of vitrified bovine oocytes resulted in ATP concentration of oocytes remaining the same, however, oocytes that underwent 4 hours of culture after warming were similar to the none vitrified control group in regards with mitochondrial membrane potential.</li> <li>Secured CRISPR/Cas9 systems resulting in different off-target activities.</li> <li>A database of Doppler measurements for pregnancies under different conditions (singleton vs. twins, different sires; maternal feed restriction, etc) is under development.</li> <li>We developed an efficient protocol for the introduction of human specific mutations into the sheep fetal fibroblast cells using CRISPR/Cas9 RNP + ssODN approach.</li> <li>Conditions for CF mutation correction in ovine-bovine iSCNT preimplantation embryos in vitro were optimized.</li> <li>Preliminary results on using a combination of M3814 (a DNA-dependent protein kinase inhibitor) and RS-1 (enhancer of hRAD51 binding) to increase efficiency of HDR look promising.</li> </ol>

Publications

Publications <ol> <li> Refereed articles/book chapters</li> </ol> Berger, T., J. Vanselow, A. Conley, T. J. Almand, and B. S. Nitta-Oda. 2021. Multifaceted epigenetic regulation of porcine testicular aromatase. Mol Cell Endocrinol 541:111526. doi: 10.1016/j.mce.2021.111526 Vanselow, J., A. J. Conley, and T. Berger. 2021a. Aromatase and the three little paralogs. Biol Reprod 105(1):5-6. doi: 10.1093/biolre/ioab112 Vanselow, J., A. J. Conley, C. J. Corbin, and T. Berger. 2021b. Genomic Structure of the Porcine CYP19 Locus and Expression of the CYP19A3 Paralog. Genes (Basel) 12(4)doi: 10.3390/genes12040533 Parsons AM, Bouma GJ. A Potential Role and Contribution of Androgens in Placental Development and Pregnancy. Life (Basel). 2021 Jul 1;11(7):644. Dailey DD, Hess AM, Bouma GJ, Duval DL. 2021. MicroRNA Expression Changes and Integrated Pathways Associated With Poor Outcome in Canine Osteosarcoma. Front Vet Sci 8:637622 Ali A, Hadlich F, Abbas MW, Iqbal MA, Tesfaye D, Bouma GJ, Winger QA, Ponsuksili S. 2021. MicroRNA-mRNA Networks in Pregnancy Complications: A Comprehensive Downstream Analysis of Potential Biomarkers. Int J Mol Sci. 2021 Feb 25;22(5):2313. Parsons Aubone, AP, Evans R, Bouma GJ. 2021. “Androgen Signaling in the Placenta”, in Reproductive Hormones, InTech Open, Editor: Marsh C. Chapter 4. Ranjitkar S, Zhang D, Sun F, Salman S, He W, Venkitanarayanan K, Tulman ER, and Tian X. 2021. Cytotoxic effects on cancerous and non-cancerous cells of trans-cinnamaldehyde, carvacrol, and eugenol. Sci Rep: 11:16281. doi:10.1038/s41598-021-95394-9. Su Y, Wang L, Fan Z, Liu Y, Zhu J, Kaback D, Oudiz J, Patrick T, Yee SP, Tian XC, Polejaeva I, Tang Y. 2021. Establishment of Bovine-Induced Pluripotent Stem Cells. Int J Mol Sci. 22(19), 10489; https://doi.org/10.3390/ijms221910489. Sang L, Xiao Y, Jiang Z, Forde N, Tian XC, Lonergan P, Hansen PJ. 2021. Atlas of receptor genes expressed by the bovine morula and corresponding ligand-related genes expressed by uterine endometrium. Mol Reprod Dev. 88(10):694-704. doi: 10.1002/mrd.23534. Daigneault BW. Dynamics of paternal contributions to embryo development in large animals. Biol Reprod. 2021 Feb 11;104(2):274-281. doi: 10.1093/biolre/ioaa182.   Daigneault BW and Miller DJ. Transient receptor potential polycystin-2 (TRPP2) regulates motility and intracellular calcium of porcine sperm. Andrologia, 2021 Sep;53(8) :e14124. doi: 10.1111/and.14124   Renato Félix da Silva, Laritza Lima, Rebeca Rocha, Ivina Brito, Gerlane Silva, Hudson Correia, Giovanna Rodrigues, Anna Clara Ferreira, Diana Nunes-Pinheiro, Arlindo Moura, Larissa Silveira, Edson G. Lo Turco, Matthew B. Wheeler, Ana Paula Rodrigues, Claudio Campello, José R. Figueiredo. (2021). In vitro long term culture of isolated ovine preantral follicle: influence of ethanol on steroid production, oocyte meiotic resumption and metabolomic profile. Research in Veterinary Science, 135:432-441 doi.org/10.1016/j.rvsc.2020.10.029   Liman, M.S., Cardoso, C.L, Franco, V., Longobardi, V., Gasparrini, B., Wheeler, M.B., Rubessa, M. and Esposito, G. (2021). Effects of dietary supplementation ofconjugated linoleic acids and their inclusion in semen extenders on bovine sperm quality. Animals (2021), 11, 483. https://doi.org/10.3390/ani11020483   Milner, D.J., Long, S.A., Flanagan, C.L., Hollister, S.J., Gurtler, R., Bane, R., Zimmerman, J., Cameron, J.A, Gutierrez-Nibeyro, S.D., Wheeler, M.B. A Porcine Model for Repair of Long Bone Non-Union Defects Using Three-Dimensionally Printed Scaffolds. J Regenerative Medicine & Biology Res. 2021;2 (2):1-10. http://dx.doi.org/10.46889/JRMBR.2021.2203   Dewey, M.J., Milner, D.J., Weisgerber, D., Flanagan, C.L., Rubessa, M., Lotti, S., Polkoff, K.M., Crotts, S., Hollister, S.J., Wheeler, M.B., Harley, B.A.C. 2021. Repair of critical-size porcine craniofacial bone defects using a collagen-         polycaprolactone composite biomaterial. Biofabrication 2021 Oct 18. doi: 10.1088/1758-5090/ac30d5. Epub ahead of print. PMID: 34663761.   Rocha, R. M. P., Rubessa, M., Lima, L. F. de , Silva, A. F. B. da, Winters, R., Polkoff, K., Milner, D., Campello, C. C., Figueiredo, J. R. de, Wheeler, M. B. 2021. Effect of base media, FSH and anti-Müllerian hormone (AMH) alone or in combination on the growth of pig preantral follicles in vitro. Research, Society and Development, [S. l.], v. 10, n. 15, p. e53101522488DOI: 10.33448/rsd-v10i15.22488. https://rsdjournal.org/index.php/rsd/article/view/22488. Accessible: 20 nov. 2021.   Monzani, O.S., Adona, P.R., Long, A.S., Wheeler, M.B. (2021). Cows as bioreactors for the production of nutritionally and biomedically significant proteins. G. Wu (ed.), Recent Advances in Animal Nutrition and Metabolism, Advances in Experimental Medicine and Biology 1354,gs. 299-314. https://doi.org/10.1007/978-3-030-85686-1_15   Wilson, S.M., Goldwasser, M.S., Clark, S.G., Monaco, E., Rodriguez-Zas, S., Hurley, W.L., Wheeler, M.B. (2021). Adipose-Derived Stem Migration in the Vascular System after Transplantation and the Potential Colonization of Ectopic Sites in Swine. J Regenerative Medicine & Biology Res. 2021;2(3):1-26. DOI:http://dx.doi.org/10.46889/JRMBR.2021.2302   Ali, S, T Degefa, A Lemma, and CR Youngs. 2021. Presence of CL influence on the quantity and quality of COC in slaughterhouse-derived ovaries of Boran heifers. World News of Natural Sciences 34:29-37, http://www.worldnewsnaturalsciences.com/wp-content/uploads/2020/10/WNOFNS-34-2021-29-37-1.pdf Benaissa, MH, N Mimoune, B Faye, Y Bentria, FZ Mimouni, CR Youngs, and R Kaidi. 2021. First serological evidence of BHV-1 virus in Algerian dromedary camels: Seroprevalence and associated risk factors.  Comparative Immunology, Microbiology, and Infectious Diseases 76:101638, <a href="https://doi.org/10.1016/j.cimid.2021.101638">https://doi.org/10.1016/j.cimid.2021.101638</a>   Koester LR, AL Petry, CR Youngs, and S Schmitz-Esser. 2021. Ewe vaginal microbiota: associations with pregnancy outcome and changes during gestation. Frontiers in Microbiology 12:745884. <a href="https://doi.org/10.3389/fmicb.2021.745884">https://doi.org/10.3389/fmicb.2021.745884</a>   Whitaker BD, WR Lamberson, MF Smith, BS Walters, and CR Youngs. 2021. What best animal science teachers do. Translational Animal Science 5:1-6,txaa223, <a href="https://doi.org/10.1093/tas/txaa223">https://doi.org/10.1093/tas/txaa223</a>   Diaz FA, Gutierrez-Castillo EJ, Foster BA, Hardin PT, Bondioli, KR, Jiang Z. Evaluation of seasonal heat stress on transcriptomic profiles and global DNA methylation of bovine oocytes. Front Genet. 2021. PMID: 34777457.   Sang L, Xiao Y, Jiang Z, Forde N, Tian XC, Lonergan P, Hansen PJ. Atlas of receptor genes expressed by the bovine morula and corresponding ligand‐related genes expressed by uterine endometrium. Mol Reprod Dev. 2021 Oct. PMID: 34596291.   Li C, Sun J, Liu Q, Ming H, Wang L, Li Y, Li R, Jiang Z, Francis J, Fu X. The landscape of accessible chromatin in quiescent and post-myocardial infarction cardiac fibroblasts. Epigenetics 2021. PMID: 34551670.   Iyyappan R, Aleshkina D, Zhu L, Jiang Z, Kinterova V, Susor A. Oocyte specific LncRNA variant Rose influences oocyte and embryo development. Non-coding RNA research. 2021 June. PMID: 34278057.   Zhu L, Marjani SL, Jiang Z. The epigenetics of gametes and early embryos and potential long-range consequences in livestock species - filling in the picture with epigenomic analyses. Front Genet. 2021 Mar 2. PMID: 33747031.   Gutierrez-Castillo, E., Hao, M., Foster, G., Gatenby, L., Chun, M., Pinto, C., Bondioli, K., Jiang, Z.  2021. "Effect of vitrification on global gene expression dynamics of bovine elongating embryos." Reproduction, Fertility and Development 33(5): 338-348.   Schall PZ, Latham KE. Essential shared and species-specific features of mammalian oocyte maturation-associated transcriptome changes impacting oocyte physiology.  Am J Physiol Cell Physiol. 2021 Jul 1;321(1):C3-C16. doi: 10.1152/ajpcell.00105.2021. Epub 2021 Apr 21. PMID: 33881934   Ruebel ML, Zambelli F, Schall PZ, Barragan M, VandeVoort CA, Vassena R, Latham KE. Shared aspects of mRNA expression associated with oocyte maturation failure in humans and rhesus monkeys indicating compromised oocyte quality.  Physiol Genomics. 2021 Apr 1;53(4):137-149. doi: 10.1152/physiolgenomics.00155.2020. Epub 2021 Feb 8. PMID: 33554756   Placentation in Mammals: Tribute to EC Amoroso’s contributions to Viviparity. 2021.  Eds. R.D. Geisert and T. Spencer. Advances in Anatomy, Embryology and Cell Biology, Springer.  Print ISBN 978-3-030-77359-5   Geisert R.D. and T.E. Spencer. 2021. Chapter 1: Introduction. In: Placentation in Mammals: Tribute to EC Amoroso’s contributions to Viviparity. Advances in Anatomy, Embryology and Cell Biology, Springer.   Green J.A., R.D. Geisert, G.A. Johnson and T.E. Spencer. 2021. Chapter 7: Implantation and Placentation in Ruminants. In: Placentation in Mammals: Tribute to EC Amoroso’s contributions to Viviparity. Advances in Anatomy, Embryology and Cell Biology, Springer.   Hay A., Farrell K., Leeth, C.M., Lee K. 2022. Use of genome editing techniques to produce transgenic farm animals. Adv Exp Med Biol. 1354:279-297. doi: 10.1007/978-3-030-85686-1_14.   Johns D.N., C.G. Lucas, C.A. Pfeiffer, A.E. Meyer, P.R. Chen, S.D. Perry, R.M. Sullivan, L.D. Spate, R.F. Cecil, M.A. Fudge, M.S. Samuel, K.M. Whitworth, C.M. Spinka, H. Liu, M.F. Smith, K.D. Wells, R.S. Prather, T.E. Spencer, and R.D. Geisert. 2021. Conceptus Interferon Gamma is Essential for Pregnancy in the Pig. Biol Reprod 105: 1577–1590. https://doi.org/10.1093/biolre/ioab186 https://doi.org/10.1093/biolre/ioab186   Chen P.R., C.G. Lucas, R.F. Cecil., C.A. Pfeiffer, M.A. Fudge, M.S. Samuel, M. Zigo, H. Seo, L.D. Spate, K.M. Whitworth, P. Sutovsky, K.D. Wells, R.D. Geisert, and R.S. Prather. 2021. Disrupting porcine glutaminase does not impair preimplantation development and elongation nor decrease mTORC1 activation. Biol Reprod 105:1104–1113. https://doi.org/10.1093/biolre/ioab165   Geisert R.D., A.E. Meyer, C.A. Pfeiffer, D.N. Johns, K. Lee, K.D. Wells, T.E. Spencer, and R.S. Prather. 2021. Gene Editing to Investigate Role of Conceptus Factors in Establishment of Pregnancy. Reproduction 161: R79-R88.   Timlin C.L., Lynn A., Wooldridge L.K., Uh K., Ealy A.D., White R.R., Lee K., Mercadante V.R.G. 2021. Physical parameters of bovine activated oocytes and zygotes as predictors of development success. Zygote. Mar 19;1-7. doi: 10.1017/S0967199421000058. Online ahead of print.   Uh K., Hay A., Chen P., Reese E., Lee K. 2021. Design of novel oocyte activation methods: The role of zinc. Biol Reprod. Dec 22; ioab235. doi: 10.1093/biolre/ioab235. Online ahead of print.   Miller CG, Kundert JA, Prigge JR, Amato JA, Perez AE, Coppo L, Rizzo GN, Kavanaugh MP, Orlicky DJ, Shearn CT, Schmidt EE. Supplemental Ascorbate Diminishes DNA Damage Yet Depletes Glutathione and Increases Acute Liver Failure in a Mouse Model of Hepatic Antioxidant System Disruption. Antioxidants (Basel). 2021 Feb 27;10(3). doi: 10.3390/antiox10030359. PubMed PMID: 33673577; PubMed Central PMCID: PMC7997133.   Gauthier AG, Wu J, Lin M, Sitapara R, Kulkarni A, Thakur GA, Schmidt EE, Perron JC, Ashby CR Jr, Mantell LL. The Positive Allosteric Modulation of alpha7-Nicotinic Cholinergic Receptors by GAT107 Increases Bacterial Lung Clearance in Hyperoxic Mice by Decreasing Oxidative Stress in Macrophages. Antioxidants (Basel). 2021 Jan 19;10(1). doi: 10.3390/antiox10010135. PubMed PMID: 33477969; PubMed Central PMCID: PMC7835977.   Desaulniers, A.T., R.A. Cederberg, E.A. Carreiro, C.B. Gurumurthy and B.R. White. 2021. A transgenic pig model expressing a CMV-ZsGreen1 reporter across an extensive array of tissues. J. Biomed. Res. 35(2):163-173.   Viotti Perisse I., Fan Z., Van Wettere A., Liu Y., Leir SH., Keim J., Regouski M, Wilson M.D., Cholewa K.M., Mansbach S.N., Kelley T.J., Wang Z., Harris A.*, White K.L., Polejaeva I.A.* (2021). Sheep models of F508del and G542X cystic fibrosis mutations show cellular responses to human therapeutics. FASEB Bioadv. 3(10):841-54. PubMed PMID: 34632318; PMCID: PMC8493969.   Polejaeva I.A.* (2021) 25th ANNIVERSARY OF CLONING BY SOMATIC CELL NUCLEAR TRANSFER: Generation of genetically engineered livestock using somatic cell nuclear transfer. Reprod Suppl. 162(1):F11-F22. PubMed PMID: 34042607.   Cuthbert, J. M., Russell, S. J., Polejaeva, I.A., Meng, Q., White, K. L., & Benninghoff, A.* (2021). Comparing mRNA and sncRNA profiles during the maternal-to-embryonic transition in bovine IVF and scNT embryos. Biology of Reproduction. PubMed PMID: 34514499.   Cuthbert, J. M., Russell, S. J., Polejaeva, I. A., Meng, Q., White, K. L., & Benninghoff, A. D.* (2021). Dynamics of small non-coding RNAs in bovine scNT embryos through the maternal-to-embryonic transition. Biology of Reproduction. PubMed PMID: 34086842.   Su Y., Wang L., Fan Z., Liu Y., Zhu J., Kaback D., Oudiz J., Patrick T., Yee SP, Tian X(C), Polejaeva I.* and Tang Y.* (2021). Establishment of Bovine-Induced Pluripotent Stem Cells. Int. J. Mol. Sci. 22(19), 10489; https://doi.org/10.3390/ijms221910489   Perisse, I., Fan, Z., Singina, G. N., White, K. L., & Polejaeva, I.A.* (2021). Improvements in Gene Editing Technology Boost Its Applications in Livestock. Frontiers in Genetics, 11: 614688. PubMed PMID: 33603767; PMCID: PMC7885404.   Rose, M. F., Waldron, B. L., Isom, S. C., Peel, M. D., Thornton-Kurth, K., Miller, R. L., Rood, K. A., Hadfield, J. A., Long, J., Henderson, B., and Creech, J. E. (2021). “The effects of organic grass and grass-birdsfoot trefoil pastures on Jersey heifer development: Herbage characteristics affecting intake.” Journal of Dairy Science, USA, 104(10), 10879-10895.   Hadfield, J., Waldron, B. L., Isom, S.C., Creech, J. E., Rose, M. F., Long, J., Miller, R. L., Rood, K. A., Young, A., Stott, R. D., Sweat, A., and Thornton-Kurth, K. (2021). “The effects of grass and grass-legume pastures on organic dairy heifer development: Heifer growth and performance.” Journal of Dairy Science, USA, 104(10), 10863-10878.   Zhao, Y., Bunch, T. D., and Isom, S.C. (2021). “Effects of electrical biostimulation and silver ions on porcine fibroblast cells. PLoS One. 16(2):e0246847. doi: 10.1371/journal.pone.0246847. PMID: 33566869; PMCID: PMC7875371.   Chen, Y.T., Q.Y. Yang, Y. Hu, X.D. Liu, J.M. de Avila, M.J. Zhu, P.W. Nathanielsz, and M. Du. 2021.  Imprinted LncRNA Dio3os preprograms intergenerational brown fat thermogenesis and obese resistance. Nat. Commun. 12: 6845. <ol start="2"> <li> Books, non-refereed book chapters, proceedings, instructional media, theses/dissertations</li> </ol> Hahn, Matthew D. Methods to Reducing Weaning Stress in Early Weaned Spring Beef Calves. A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Animal Science. University of Arkansas Bachelor of Science in Animal Science, 2019 May 2021. Jahnke M and CR Youngs. 2021. Superovulation in cattle. In (RM Hopper, Ed) Bovine Reproduction, 2nd Ed., pages 1032-1040. MM Jahnke, JK West, and CR Youngs. 2020. Evaluation of in vivo-derived bovine embryos. In (RM Hopper, Ed) Bovine Reproduction, 2nd Ed., pages 1073-1090. Destiney N. Johns. 2021. Conceptus Interferon Gamma is Essential for Pregnancy Maintenance in the Pig. MS Thesis Fonnesbeck, Sawyer (2021).  The Influence of Dairy Breed and Forage Type on Organic Dairy Heifer Performance.  MS Thesis #8340.  Utah State University. <a href="https://digitalcommons.usu.edu/etd/8340/">https://digitalcommons.usu.edu/etd/8340/</a>   <ol start="3"> <li> Abstracts</li> </ol> Comparison of two ultrasound systems for estimation of carcass attributes in feedlot steers. N. Jesko*, T. L. Perkins*, J. Richeson*, T. E. Lawrence, and C. Looney⸭ *West Texas A&M University, Canyon, Texas 79016; ⸭ University of Arkansas, Hope, Beef Improvement Federation, June 22-25,2021, Des Moines, IA. Evaluation of fixed-time artificial insemination and estrus expression in Angus-cross heifers with female sex-sorted semen using 6-day and 7-day CIDR protocols at multiple time intervals. W. E. Weber1, J. G. Powell1, B. P. Littlejohn1, E. B. Kegley1, N. N. Noga1, C. R. Looney21Department of Animal Science, Division of Agriculture, University of Arkansas System, Fayetteville, AR2Department of Animal Science, Division of Agriculture, University of Arkansas System, Hope, AR. Comparison of 6-day versus 7-day CIDR protocol with GnRH and PGFα to synchronize crossbred beef cows for fixed-timed AI with sexed semen at two different times.  Nicole N. Noga1, Jeremy Powell1, Brittni Littlejohn1, Whitney Rook2, Martin L. Mussard3, Alvaro Garcia Guerra3, Charles R. Looney21Department of Animal Science, Division of Agriculture, University of Arkansas, Fayetteville, AR, 2Department of Animal Science, Division of Agriculture, University of Arkansas, Hope, AR, 3Department of Animal Sciences, The Ohio State University, Columbus, OH.   Pre-synchronization using an intravaginal progesterone device improves fertility in suckled beef cows submitted to fixed time artificial insemination.  A.E. Crist1; J. C. L. Motta1; C. Hayden1; C. Rykaczewski1; M. Mussard1; C.R. Looney2; A. Garcia-Guerra11Department of Animal Sciences, The Ohio State University, Columbus, OH, USA.2Department of Animal Science, University of Arkansas, Hope, AR, USA. International Congress on Animal Reproduction, June, 2033, Bologna, Italy Influence of prenatal exposure to endophyte-infected tall fescue on glucose metabolism in weaned steer calves. R. F. Benefield1, D. S. Hubbell2, D. L. Galloway1, C. R. Looney3, J. G. Powell1, E. B. Kegley1, B. P. Littlejohn11Department of Animal Science, Division of Agriculture, University of Arkansas, Fayetteville, AR2Livestock and Forestry Research Station, Division of Agriculture, University of Arkansas, Batesville, AR 3Department of Animal Science, Division of Agriculture, University of Arkansas, Hope, AR, Journal of Animal Science, submitted. Candelaria, JI; Denicol, AC. Culturing bovine ovarian cortex may rescue damages caused by cryopreservation on the extracellular matrix, follicle, and stromal morphology. Submitted to the 2022 International Congress in Animal Reproduction, Bologna, Italy. Guiltinan, C; Candelaria, JI; Rabaglino, MB; Smith, JM; Denicol, AC. Derivation and evaluation of bovine embryonic stem cells from early and full blastocyst stage embryos. Reprod, Fert Dev 34(1-2), 2022. <a href="https://doi.org/10.1071/RDv34n2abs">https://doi.org/10.1071/RDv34n2abs</a> Weldon, BM; Denicol AC. Single cell transcriptomic analysis of the bovine adult ovarian cortex reveals remodeling characteristics of stromal cells. Submitted to the 2022 International Congress in Animal Reproduction, Bologna, Italy. Gerrit J. Bouma, Agata M. Parsons, Jeremy D. Cantlon, Fiona K. Hollinshead, Quinton A. Winger, Jason E. Bruemmer. Androgen Receptor Signaling in Placenta. Society for the Study of Reproduction 54th Annual Meeting, St. Louis, MO, USA. 2021 Agata M. Parsons, Gerrit J. Bouma, Adam J. Chicco. Androgen receptor – A potential novel player in placental mitochondria function. Society for the Study of Reproduction 54th Annual Meeting, St. Louis, MO, USA. 2021 “Cloning & Gene Editing Technologies: Potential use in a breeding program?”, Multi-purpose Canine Workshop, C. Wayne McIlwraith Translational Medicine Institute, Fort Collins, CO. 2021 “Manipulating genes during placental development in sheep”, Emerging Topics in Reproduction 2021, University of Sao Paulo & Texas A & M, Webinar. Gungor OF, Salman S, Ranjitkar S, Zhang D, Tian XC. 2022. Metabolic and Acid-base Parameters in Blood, Follicular, Oviductal and Uterine fluids during in vivo Maturation of Bovine Oocytes. Fertility and Development 33(1) ???. Poster at the 48th annual meeting of the International Embryo Technology Society, January 10-13, 2021, Savannah, GA, USA. Losano JDA, Parks J, Bromfield JJ, Daigneault BW. Rosiglitazone extends maintenance of frozen–thawed bull sperm for 24 hours at ambient temperature. Proc. Society for the Study of Reproduction 48h Annual Meeting 2021, RFD https://doi.org/10.1071/RDv34n2abs McGraw M, Daigneault BW. Functional characterization of PPAR

Impact Statements

Impacts (Objective 1) Demonstrations and research have made impact on improving the conception rate of fixed-timed AI and natural service. AI workshops have increasing enrollments in the state and our pregnancy determination demonstrations using ultrasound has been well attended. Pre-synchronization protocols have increased conception rates by 10% (n=645). The long-term effects of letrozole on aromatase activity through post-translational modification (microRNA, etc.) may mean responses mediated by alternative genes and that the prolonged effect on the Sertoli cell population is not specific to reduced estrogens. Single-cell RNA sequencing of the adult bovine ovary allows the detailed study of the different cells that comprise the ovary and a better understanding of ovarian biology. Ovarian cortex cryopreservation can be a valuable alternative to preserve gametes and ultimately fertility. Our study for the first time demonstrated that the established biPSCs can actively silence the transgene expression, and can self-renewal for long-term with endogenous pluripotent gene circuitry. These biPSCs could greatly facilitate the studies on cattle embryo development, in vitro differentiation, and the development of agricultural biotechnologies. In vitro oocyte maturation is the cause of abnormal in vitro embryo development. The proper condition during maturation of oocytes in vivo has not been established. Our study will be provide the first comprehensive environmental profile during this important process through multi-omics approach. The application of the data in maturation cultures in vitro has the potential to allow us to reproduce a natural environment for oocytes to properly mature. Developed a semen extender compatible for assisted reproductive biotechnologies including sex sorted sperm, reverse sex-sorting, artificial insemination protocols, research and potential use in other species. Provided evidence for environmental contaminant exposure on post-ejaculatory sperm function and embryo development useful for clinical considerations that include idiopathic subfertility. Optimized protocols for managing cooled stallion sperm that could be applied to industry for increased flexibility of managing stallions and mares including but not limited to flexibility of semen collection schedules, shipment and asynchronous ovulation. Dominant follicle removal (DFR) and a shorter interval of OPU following DFR in Bos taurus X Bos indicus cattle increases the number of higher quality cumulus-oocyte-complexes (COCs). This allows for the optimization of in vitro embryo production (IVP) from Bos indicus crossbred cattle. α-MEM is a more effective base medium than NCSU23 for the in vitro follicular development of pig preantral follicles and AMH reduces the steroid production induced by FSH. This may allow for improved in vitro follicle development that could result in more in vitro-produced pig embryos available for embryo transfer. Low levels of ethanol have the same effect on follicular metabolites as rbFSH treatment. Ethanol may serve as a substitute for FSH in follicular maturation in sheep during times of FSH shortages such as the present situation due to Covid-19 labor shortages. The results have shown a beneficial effect of conjugated linoleic acid (CLA) supplementation on bull semen quality. Improved semen quality after freezing will increase the number of calves born from artificial insemination. The porcine radius segmental defect model provides a viable platform for testing printed scaffolds and bioengineered composite scaffold constructs for non-union healing in long bones. This could rapidly accelerate the testing and use of artificial scaffolds for bone tissue regeneration. We demonstrated the feasibility of the laboratory production of bovine embryos in Ethiopia. The established the mRNA translation landscape in bovine early embryos suggests genome-wide translational selectivity mechanism regulating preimplantation embryo development, and provides insights for further mechanistic studies for improving developmental competence in embryos produced by assisted reproductive technologies. The development of training deep neural networks approach and the application of transfer learning approach enable imputing single-cell DNA methylome profiles to predict methylation in scarce material samples, such as oocytes, and embryos. The altered small RNA code by maternal gestational malnutrition could be essential in programming male reproduction and mediating induced epigenetic inheritance in sheep. The underlying mechanisms would infer the development of promising technologies or management interventions to counteract adverse conditions or to program reproductive performance, that would enhance reproductive efficiency and productivity in livestock species. Understanding the molecular characteristics of oocytes following in vitro maturation under various conditions will provide insight into improving the developmental competence of in vitro produced bovine embryos. Cryopreservation of bovine oocytes could be an important technique for genetic selection however cryopreservation of bovine oocytes using methods developed in other species leads to sub optimum results. These studies provide methods to improve those results. We acquired a change in fundamental knowledge regarding how the GnRH-II/GnRHR-II system regulates 17ß-estradiol levels and follicular dynamics in porcine females, representing a potential avenue for future reproductive therapies including litter size in pigs. New pharmacological agents may be developed to manipulate the reproductive axis, leading to enhanced fertility rates and reduced incidences of endocrine disorders impacting reproduction. Data from these experiments could lead to novel swine-specific contraceptive methods to manage feral pig populations. A genetic test to identify fertile and/or subfertile female gametes could enhance traditional selection procedures and novel pharmacological agents could improve swine production practices. Ultimately, this will lead to a change in condition, increasing profitability and therefore, sustainability for pork producers. Photobiomodulation will ease most ethical concerns in humans concerns with current therapies proposed to treat oocyte mitochondrial dysfunction (mitochondrial transfer, GV transfer, etc). Significant improvement in bovine oocyte maturation, embryo development and full-term development using FLI cytokine supplemented maturation medium could have a significant impact on improving calving rates following IVF and SCNT. Reduction of mtDNA copy number using handmade cloning approach should decrease the incidence of mitochondrial heteroplasmy in the reconstructed embryos, promoting better embryonic and fetal development. Supplementation with mitochondrial extract from the somatic cell donor cell may be essential to achieve proper embryonic development. Ooplasm biopsies plus WoW culture system and nano-scale qPCR will be a powerful format to continue to analyze the molecular constitution of in vitro matured oocytes used for ART in large domestic species. Maternal obesity and other adverse physiological conditions negatively affect fetal development, which generating long-term negative effects on offspring health.Maternal obesity not only affects embryonic and fetal development, but also impairs oocyte quality, which exerts negative effects on subsequent pre- and postnatal development of progeny. (Objective 2) Although small in scope, the absence of off-target responses in blastocysts and conceptuses supports the viability of CRISPR editing in livestock. Understanding the role of LIN28A and LIN28B in sheep placenta will improve our understanding of the genetic regulation of placenta development and result in methods to improve somatic cell nuclear transfer in ruminants. Similarly establishing that androgen and estrogen signaling through their receptors are regulated by KDM1A in the placenta, and are involved in placental angiogenesis in agriculture animals will lead to new insight into factors involved in early pregnancy losses. This also can lead to development of novel strategies and/or tools to identify or prevent early embryo mortality. AR shRNA targeting vectors are now used to knockdown AR and study its role during in placental development and angiogenesis in pregnancy. Genetic modification of cow’s milk allows for the (1) the change of nutritional components, including proteins, amino acids and lipids for human nutrition; (2) the removal allergic proteins in milk; (3) the production of cows resistant to disease; or (4) the production of essential proteins used in biomedicine (biomedical proteins) in milk and blood plasma. Fluorescent stem cells can facilitate the tracing and visualization of stem cell migration, fusion, and participation in tissue regeneration after stem cell injection therapy, and represent a useful tool for tissue engineering research. The production of stem cells containing eGFP by homology-directed repair of Cas9-cleaved DNA at the ROSA 26 locus can be used to label stem cells for in vivo tracking. Systemic administration of ASC can be a successful method of cell transplantation for tissue regeneration. The bovine TSC established in this study will provide a powerful in vitro model to study bovine early placental establishment and early pregnancy failure. Unraveling the metabolic basis of normal early embryo development will provide significant benefits to human and animal reproductive health. Improved embryonic competency following in vitro production and cryopreservation would stimulate the industry by lowering costs, especially those related to recipient management. Provided a new and unifying understanding of core developmental mechanisms applicable across mammalian species that establish high quality oocytes, and how genotype and species can impact oocyte characteristics. The dynamic of the follicular fluid composition having evolutive function during follicle growth is demonstrated in the mare, and subsets of proteins are identified as potential candidates to improve in vitro oocyte maturation or in vitro follicle culture. MicroRNA expression profiling reveals miRNAs in microvesicles isolated from follicular fluid and which biological impacts on ovarian granulosa and cumulus cells may be influenced by the follicle size and the seasonal variation. The differential effect of Zn chelators leads us to investigate mechanistic pathway of oocytes activation between pigs and cows as a comparative study. The importance of Zn on the resumption of meiosis and oocytes activation will be investigated using pigs and cows. Analyzing GDF9 knockout pigs will expand our knowledge on oogenesis and folliculogenesis. Ability to reduce off-targeting events, caused by the genome editing systems, will expand application of genome editing systems in livestock. These studies are uncovering previously unrecognized systems that support survival and function of liver cells, which, in turn, is providing insights into better therapies for treating some liver diseases or toxicities. These studies are demonstrating the power of being able to genetically manipulate hepatic metabolism in adult animals. This promises to provide interesting new ways to genetically modify nutrient use or intermediary metabolism of animals that could improve food-production characteristics of livestock without generating genetically modified food products. A combination of M3814 and RS-1 compounds would likely lead to a greater improvements in KI efficiency in livestock cells and embryos. The human SCD mutation site is present in sheep HBB gene, suggesting that introduction of the mutation in sheep may result in red blood cell (RBC) sickling and a SCD phenotype similar to that seen in human patients. We generated sheet fetal fibroblasts that will be used to produce animals by SCNT. We anticipate that the development of a novel domestic sheep model of SCD will be highly impactful as it will provide a critically needed, translationally relevant platform for the development of gene therapies, and thereby accelerate the advancement of such therapies into the clinic.

Date of Annual Report: 05/05/2023

Report Information

Annual Meeting Dates: 03/03/2023 - 03/03/2023
Period the Report Covers: 01/01/2022 - 12/31/2022

Participants

Participants
In attendance were
Anna Denicol (California),
Quinton Winger (Colorado),
X. Cindy Tian (Connecticut),
Brad Daigneault (Florida),
Zongliang (Carl) Jiang (Florida),
Matthew B. Wheeler (Illinois),
Curt Youngs (Iowa),
Kenneth Bondioli (Louisiana),
Carol L. Keefer (Maryland),
Jean Feugang (Mississippi),
Kiho Lee (Missouri),
Brett White (Nebraska),
Jingyue (Ellie) Duan (New York),
Joao Gabriel Nascimento Moraes (Oklahoma),
Celina Checura (South Carolina),
Irina Polejaeva (Utah),
Chris Davies (Utah).

In person attendance
Dr. Daigneault,
Dr. Moraes,
Dr. White,
Dr. Winger,
Dr. Checura,
Dr. Polejaeva,
Dr. Davies,
Dr. Youngs,
Dr. Jiang,
Dr. Feugang,
Dr. Duan

Zoom attendance
Dr. Tian,
Dr. Wheeler,
Dr. Denicol,
Dr. Lee,
Dr. Keefer,
Dr. Bondioli

Brief Summary of Minutes

Brief Summary of Minutes of 2023 Annual Meeting

University of Florida, Gainesville, FL

March 3^th, 2023

09:03: Dr. Daigneault started the meeting and welcomed the participants.

09:04: Dr. Daigneault presented the agenda.

There was a discussion about PIs that have left W4171 multistate group, and whether we could remove those that left from the current report.

Checura pointed out that we were not supposed to remove people from the report without USDA approval.

Daigneault offered to reach out to Kenneth White, Agriculture Experiment Station coordinator, to verify possible PIs to be excluded from the current group.

A new participant from the University of Cornell, Dr. Jingyue (Ellie) Duan, introduced herself as a new member of the group.

09:18: Dr. Checura proposed a motion to approve the minutes from last meeting. Dr. Daigneault seconded.

09:18: Dr. Davies reminded everyone that this multistate project approval extends through September, 2024. Therefore, there is a need to restructure the current project and resubmit.

Davies also pointed out that the length of the meeting could be increased so participants would have more time to discuss their projects and make collaborations.

Winger reminded the group that the next IETS meeting will be in the Denver, CO. Dr. Winger offered to organize with the IETS committee so that participants of the W4171 multistate group would have a room for 1 or 2 extra days to be able to participate in our 2024 annual meeting.

Tian proposed that instead of having a longer group meeting (2 days) we could do a mid-year Zoom meeting to discuss collaborations. Therefore, we would keep a traditional 1-day annual meeting (in person) generally during the IETS annual meeting.

Daigneault suggested that we make the decision regarding the duration of our annual meeting at the end of today’s meeting.

09:20: Everyone introduced themselves including the participants attending the meeting in person and the ones that joined via Zoom.

09:36: Dr. Davies explained that the summary report should submitted through the NIMSS system. Dr. Checura mentioned that, in the previous year, she submitted the final report directly to Kenneth White.

09:37: Discussion of individual station reports started. It was decided that, in the current year, station reports will be discussed in alphabetical order. For the 2024 annual meeting, it was decided that station reports will be discussed in reverse alphabetical order.

09:40: Connecticut - Dr. Tian started discussing her work on improving maturation conditions. Then she moved to her work generating naïve bovine embryonic stem cells.

Data mining project: showed widespread intergenic transcription in forms of read-throughs and read-ins in bovine pre-implantation embryos.

10:05: California - Dr. Denicol: Characterization of the bovine ovary by single cell sequencing. Compared ovary from pre-ovulatory and mid-luteal state. Compared fetal and adult bovine ovary using single cell RNA-seq. Provided an update on her work in pre-antral folliculogenesis.

10:25: Colorado - Dr. Winger: Informed everyone that Dr. Jennifer Barfield (Colorado State University) plans on being part of the group. Dr. Barfield works on embryology and bison.

Dr. Winger: presented his model for gene knock down in the trophectoderm of blastocyst using lentivirus. Dr. Winger gave updates on his work with T4 transfer from the mother to the fetus.

11:08: Florida - Dr. Daigneault briefly reported on his work for the characterization of reproductive seasonality in Florida Native sheep, and sperm work on stallions. One of the focuses of Dr. Daigneault lab is to explore how the environment affects post ejaculatory sperm function.

11:30 Dr. Jiang: Discussed his work studying the molecular mechanism of early embryonic development, particularly during pre-implantation development. Highlighted his work aiming to profile the bovine pre-implantation embryo using single cell sequencing. He also presented data on his work on bovine blastoids derived from trophoblast stem cells.

12:00: Illinois – Dr. Wheeler: Dominant follicle removal in Bos indicus cattle and half-blood Gir x Holstein crosses. Dr. Wheeler reported results of a trial feeding rumen-protected methionine on the endometrium and embryos in Holsteins.

Regarding the biomedical model, Dr. Wheeler reported on the development of a 3D printer cage to be used for fusing damaged spines. This work will move on to clinical trials.

13:35: Iowa - Dr. Youngs: presented some of his work overseas including word done with alpacas in Peru and dairy cattle in Ethiopia.

14:00: Louisiana – Dr. Bondioli: Localization of key proteins involved in meiotic spindle formation. Occyte cryopreservation. Improved methods developed in his lab for ICSI.

14:35: Maryland – Dr. Keefer: Reported on her work with energy metabolism in embryos.

14:45: Mississippi – Dr. Feugang: Reported on studies exploring seasonal variations in extracellular vesicles miRNA in follicular fluid of horses. Characterization of seminal extracellular vesicles of divergent sperm quality.

15:00: Missouri – Dr. Lee: Reported on his work generating GDF9 knockout pigs and work on the identification of off-targeted events introduced by the CRISPR-Cas9 system in gene-edited pigs.

15:21: Nebraska – Dr. White: Transgenic work on GnRH2 using a porcine model.

15:30: Oklahoma – Dr. Moraes: Provided updates on his work investigating the genetic regulation of conceptus elongation in cattle. Collaborative work with Matt. Lucy (University of Missouri) investigating disease programing of dairy cattle infertility. Provided an update on future projects.

15:50: South Carolina – Dr. Checura: Provided updates on her work on photobiomodulation on oocyte competence.

16:00: Utah – Dr. Polejaeva: Laparoscopic OPU derived oocytes associated with improved blastocyst development following SCNT. Development of sheep model for sickle cell disease.

16:20: Dr. Davies reported novel data on the establishment of pregnancy in IFNAR2 knockout sheep.

16:43: New York - Dr. Duan: Provided an update on her transition from graduate student to assistant professor and setting up her new lab at Cornell. Dr. Duan also reviewed some of the work she performed as a PhD student and during her postdoc training.

16:50: Dr. Daigneault nominated Dr. Moraes for Chair of the 2024 meeting. Dr. Youngs seconded.

16:53: Dr. Daigneault nominated Dr. Duan for secretary of the 2024 meeting. Dr. Polejaeva seconded.

16:54: Possible dates for the 2024 annual meeting are January 8^th, 9^th or 13^th, depending on the schedule of activities of IETS.

Once the schedule of activities for IETS has been released, there will be a Doodle Pool to decide the time for the 2024 annual meeting.

17:00: Discussions regarding resubmitting a new project to give continuation to the efforts of the current group. The current project expires in September 2024.

Daigneault proposed to create a group in Teams to work on the new project.

White agreed to take the lead coordinating the draft for the new project.

It was decided that members of this group will work as a team to write the new project.

For citation, we decided to use DOI to facilitate retrieval of references.

Daigneault will send out an e-mail to start organizing preparation for next report.

17:25: Dr. Youngs - motion to end the meeting. Dr. Polejaeva seconded. End minutes.

Accomplishments

Accomplishments Objective 1 Understand the biology of gamete development, fertilization and embryogenesis including the underlying cellular and molecular mechanisms. <ol> <li>Research on Pre-Synchronization, extended progesterone treatments with 14-day intra-vaginal CIDRs (7&&) and utilization of Gender-Selected semen has proven competitive commercially feasible results.</li> <li>Recent results with the 14-sday CIDR with GnRh at 7 and 6 days had the greatest estrus expression rates and conception rates ever achieved at our experiment stations.</li> <li>Developed an efficient method to isolate preantral follicles from the adult bovine ovary without the need of tissue digestion with enzymes.</li> <li>Demonstrated that the reprogramming of aromatase required for maintenance of an expanded Sertoli cell population is not due to DNA methylation although changes in DNA methylation are related to age-associated changes in aromatase activity.</li> <li>Alteration in expression of CSF1, AR, or SRD5A2 do not appear to be involved in signaling the increase in population size of Sertoli cells. However, macrophages may be involved in the tissue machinery that leads to the enlarged population size following reduced endogenous estrogen signaling.</li> <li>To investigate the impact of CRISPR-cas9-mediated knockout of Keap1 gene in modulating NRF2 activity, three guide RNAs targeting the exon 2 of the Keap1 gene was tested for their efficiency in inducing targeted knockout of the gene in zygotes electroporated with RNP at 10 h post IVF.</li> <li>Supplementation of EVs derived from thermal stress granulosa cells can induce tolerance to oocytes subjected to heat stress by reducing the accumulation of ROS, improving mitochondrial activity, reducing the expression of stress associated genes thereby reducing the severity of stress in oocytes and developing blastocysts.</li> <li>Conducted the first analysis of intergenic transcription activities in natural samples and also the first study on embryos.</li> <li>Established the detailed profiles of hormonal events in follicular fluid in pre-ovulatory follicles, correlated their patterns to other ovarian structures.</li> <li>Characterized the bioenergetic status of bovine sperm when maintained for 24 hr.</li> <li>Optimized bovine sperm conditions for compatibility with SeaHorse assay.</li> <li>Developed a modified semen extender compatible for prolonged storage of fresh and frozen bovine sperm with industry application.</li> <li>Prolonged the maintenance of cooled stallion sperm for up to 6 days</li> <li>Characterized the reproductive parameters of the Florida Native Sheep</li> <li>High-resolution ribosome fractionation and low-input ribosome profiling of bovine oocytes and preimplantation embryos has enabled us to define the translational landscapes of bovine early embryo development.</li> <li>The use of transfer learning together with Kullback-Leibler (KL) divergence to train predictive models and provides completing methylome profiles of bovine early embryos datasets with very low coverage.</li> <li>The H3.3 chaperone Hira complex has a maternal effect function in oocyte developmental competence and embryogenesis, through modulating chromatin condensation and transcriptional quiescence.</li> <li>Understanding the roles of SGK1 in mouse oocytes and establishment of new insights into the regulation of MPF via SGK1 in mammalian oocyte.</li> <li>Oocyte maturation: Results show that performing DFR in half-blood Bos taurus X Bos indicus cattle is beneficial for the development of higher quality COCs and a shorter interval of OPU following DFR in Bos taurus X Bos indicus cattle increases the number of higher quality COCs. Further, new findings suggest that DFR promotes growth of SF into MF, possibly by removing the suppression exerted on SF by the dominant follicle.</li> <li>Embryogenesis: Cows fed supplemental methionine had higher polyunsaturated lipid concentrations in embryos than cows on the control diet. Cows fed supplemental methionine had lower concentrations of un/monounsaturated lipids in uterine tissue at 15 days in milk (DIM) than cows on control diets. Specific lipid classes and lipid unsaturation level of preimplantation embryos and endometrial tissue of cows fed rumen-protected methionine were altered.</li> <li>Using immune staining two key proteins associated with micro tubule organizing center mediated spindle formation were localized in in vitro matured bovine oocytes. Differential localization of these two proteins was observed which is different than observed in other species.</li> <li>Studies looking at the effects of glucose and fructose on lipid droplets and mitochondrial activity in bovine embryos have been completed and the analysis almost finalized. While there appear to be shifts in size and numbers of lipid droplets, these shifts are subtle.</li> <li>Studies involving inhibitors of the mitochondrial pyruvate carrier (UK5099) and the carnitine palmitoyltransferase-1 (CPT-1) (etomoxir) have also been completed and demonstrate that bovine embryos are very metabolically flexible, capable of shifting metabolic pathways depending on substrate availability.</li> <li>MiRNA derived from follicular fluid-extracellular vesicles (FF-EVs) provide greater insights into the stage-specific expression dynamics of the FF EV-miRNAs during equine follicular development, which may play a role during folliculogenesis in mares.</li> <li>Isolated FF-EVs of pre-ovulatory-sized follicles provide greater insight into the seasonal variations of miRNA pools affecting the ovarian activity of mares as seasonal breeders.</li> <li>Near-Infra-Red Spectroscopy (NIRS), seminal plasma-derived EVs, transcriptomics, and proteomics tools were used to define molecular differences between Good and Poor quality semen of commercial boars.</li> <li>Proteome analyses of human sperm and corresponding seminal plasma indicated a rich repertoire of several hundred proteins. Many showed dynamic changes during aging, potentially impacting sperm physiology, structure, motility, metabolism, and fertilization.</li> <li>Production of GDF9 knockout pigs. Detailed analysis indicates GDF9 is essential for folliculogenesis in pigs.</li> <li>Supplementation of IVM medium with E-64 significantly improved the developmental competence of OPU-derived immature oocytes.</li> <li>CTSL supplementation to the IVM medium or IVC medium improved oocyte and embryo developmental competence under normal and heat stress conditions.</li> <li>During the follicular phase of the estrous cycle, concentrations of GnRH-II in follicular fluid did not differ between lines (GnRHR-II knock down (KD) and control).</li> <li>At 24 hours prior to the onset of estrus, GnRH-II levels in the blood were diminished in GnRHR-II KD compared to littermate control females.</li> <li>There was a strong positive correlation between circulating concentrations of 17ß-estradiol and GnRH-II.</li> <li>Protein for GnRHR-II was detected in both porcine granulosa and theca cells.</li> <li>Theca cells from GnRHR-II KD females expressed 40% less GnRHR-II protein than control theca cells or transgenic granulosa cells.</li> <li>Levels of GnRHR-II protein in granulosa cells were similar between lines.</li> <li>Generated spherical, ovoid, tubular, and filamentous bovine conceptuses for sequencing.</li> <li>Generated novel data contributing to the current understanding of how uterine disease postpartum programs long-term infertility in dairy cattle.</li> <li>Generated data on the effects of FGF2, LIF, and IGF1 supplementation on pregnancy success following embryo transfer of bovine IVP embryos.</li> <li>Developed a photobiomodulation system (time, wavelength, and intensity) that affect oocyte competence. The treatment alters ATP concentrations and mitochondria membrane potential by the end of oocyte maturation with lasting impacts into the blastocyst stage.</li> <li>Preliminary data indicate improvement in oocyte maturation and blastocyst development following SCNT using L-OPU in sheep.</li> </ol>     Objective 2 Refine methods to produce animals by genetic engineering or genome editing for the improvement of livestock production efficiency and development of human biomedical models. <ol> <li>Added to data evaluating the likelihood of off-target edits following CRISPR/Cas9 gene editing in porcine embryos.</li> <li>Created a new area of research – investigation of thyroid hormone physiology in growth restricted fetuses induced by knockdown of chorionic somatomammotropin (CSH) using placenta specific RNAi.</li> <li>Established putative naïve bovine embryonic stem cells that have shown in vitro and in vivo differentiation capabilities. Cells are amenable to gene-editing through single cell colony expansion. In vivo same species chimer work in underway.</li> <li>Achieved 100% editing efficiency for zygotic delivery of CRISPR/Cas9 in bovine embryos</li> <li>Establishment of bovine trophoblast stem cells (TSCs). Bovine TSCs exhibit transcriptomic and epigenetic features characteristic of trophectoderm cells from bovine embryos and retain developmental potency to differentiate into mature trophoblast cells.</li> <li>Development of an efficient method to generate bovine blastocyst-like structures (termed blastoids) via the assembly of trophoblast stem cells and expanded potential stem cells. Bovine blastoids resemble blastocysts in morphology, cell composition, single-cell transcriptomes, and represent an accessible in vitro model for studying bovine embryogenesis.</li> <li>Development of Human Biomedical Models: A topology-optimized polycaprolactone cage with BMP-2 is capable of resulting in an intervertebral fusion, similar to a conventional ring-based design of the same bioresorbable material.</li> <li>It was demonstrated that submitting vitrified-warmed, partially denuded bovine oocytes to a post-warming recovery period supplemented with 1µM resveratrol improves vitrification outcomes.</li> <li>A modification in the method used for bovine intra cytoplasmic sperm injection, referred to as vigorous injection, improved subsequent embryo injection.</li> <li>Successfully identified off-targeting events potentially introduced by the CRISPR/Cas9 system in the genome of gene-edited pigs.</li> <li>Generated novel data and insights into how liver responds to and survives oxidative and toxic stresses, using transgenic mice models.</li> <li>Acquisition of a NEPA21 electroporator to knock out genes of interest in bovine embryos using the CRISPR-Cas9 system.</li> <li>Developed an efficient protocol for the introduction of human specific mutations into the sheep fetal fibroblast cells using CRISPR/Cas9 RNP + ssODN</li> <li>Developed a novel sheep model of sickle cell disease, which reproduces human clinical and laboratory parameters.</li> <li>Created a line of type I interferon receptor (IFNAR) KO sheep and are using them to study pregnancy recognition in ruminants.</li> <li>Cas-CLOVER gene editing tool could introduce KO in livestock fetal fibroblasts.</li> </ol>

Publications

Publications <ol> <li>Refereed articles/book chapters</li> </ol> McDonnell SP, Candelaria JI, Morton AJ, Denicol AC. Isolation of Small Preantral Follicles from the Bovine Ovary Using a Combination of Fragmentation, Homogenization, and Serial Filtration. J Vis Exp. 2022 Sep 27;(187). doi: 10.3791/64423. Pedroza GH, Lanzon LF, Rabaglino MB, Walker WL, Vahmani P, Denicol AC. Exposure to non-esterified fatty acids in vitro results in changes in the ovarian and follicular environment in cattle. Anim Reprod Sci. 2022 Mar;238:106937. doi: 10.1016/j.anireprosci.2022.106937.  Berger T, Vanselow J, Conley A, Almand TJ, Nitta-Oda, BS. Multifaceted Epigenetic Regulation of Porcine Testicular Aromatase. Molecular and Cellular Endocrinology. 2022 541: 111526. Katleba K, Legacki E, Berger T. Expression of CSF1, AR, and SRD5A2 during postnatal development of the boar reproductive tract. Animals 2022  12: 2167. Kang S, Berger T. Macrophages in juvenile pig testis: Link with increase in Sertoli cells induced by estradiol suppression. Reproduction in Domestic Animals 2023 doi: 10.1111/rda.14322. Lynch CS, Kennedy VC, Tanner AR, Ali A, Winger QA, Rozance PJ, Anthony RV. Impact of Placental SLC2A3 Deficiency during the First-Half of Gestation. Int J Mol Sci. 2022 Oct 19;23(20):12530 Ali A, Iqbal MA, Abbas MW, Bouma GJ, Anthony RV, Spencer TE, Winger QA. Trophectoderm transcriptome analysis in LIN28 knockdown ovine conceptuses suggests diverse roles of LIN28-let-7 axis in placental and fetal development. Cells. 2022 Apr 5;11(7):1234. Tanner AR, Kennedy VC, Lynch CS, Hord TK, Winger QA, Rozance PJ and Anthony RV. In Vivo Investigation of Ruminant Placenta Function and Physiology – A Review. J Anim Sci. 2022 Jun 1;100(6). Sohel MM, Hoelker M, Schellander K, Tesfaye D (2022): The extent of the abundance of exosomal and non-exosomal extracellular miRNAs in the bovine follicular fluid.  Reproduction in Domestic Animals, Vol 57: 1208-1217. Miskel D, Poirier M, Beunink L, Rings F, Held E, Tholen E, Tesfaye D, Schellander K, Salilew-Wondim D, Blaschka C, Große-Brinkhaus C, Bertram B, Hoelker M (2022): The cell cycle stage of bovine zygotes electroporated with CRISPR/Cas9-RNP affects frequency of Loss-of-heterozygosity editing event. Nature Scientific Report 12 (1), 1-3. Zhang R, Neuhoff C, Yang Q, Cinar M, Uddin MJ, Tholen E, Schellander K, Tesfaye D (2022): Suforaphane Enhanced proliferation of porcine satelite celkls via epigenetic augumentation of SMAD7. Animals 12 (11), 1365 Ranjitkar S, Duan JE, Srirattana K, Alqahtani F, Tulman ER, Mandoiu I, Venkitanarayanan K, and *Tian XC. 2022. Transcriptomic Responses of Mycoplasma bovis upon Treatments of Plant-derived Antimicrobials. Frontiers in Microbiology, published on June 2, 2022. https://doi.org/10.3389/fmicb.2022.888433.   Liu Z, Cui L, Wang W, Li M, Wang Z, Presicce GA, Tian XC, An L, Du F. 2022. Dynamic and aberrant patterns of H3K4me3, H3K9me3, and H3K27me3 during early zygotic genome activation in cloned mouse embryos. Zygote 30(6): 903-9. DOI: https://doi.org/10.1017/S0967199422000454   Johnson EJ, Duan JE, Srirattana K, Venkitanarayanan K, Tulman ER, *Tian XC. 2022. Effects of intramuscularly injected plant-derived antimicrobials in the mouse model. Scientific Reports 12:5937. doi:10.1038/s41598-022-09705-9 Losano JDA, Daigneault BW. Pharmacological perturbation of peroxisome-proliferator-activated receptor gamma alters motility and mitochondrial function of bovine sperm. Andrology. 2022 Oct 5;. doi: 10.1111/andr.13308. 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Synergistic effect of standardized extract of Asparagus officinalis stem and heat shock on progesterone synthesis with lipid droplets and mitochondrial function in bovine granulosa cells, J Steroid Biochem Mol Biol, 20:106181. doi:10.1016/j.jsbmb.2022.106181.   London-Vasquez D., A. Jurkevich, A.Z. Balboula. 2022. Multi-photon laser ablation of cytoplasmic MTOCs in mouse oocytes, Journal of Visualized Experiments, 11 (189). doi: 10.3791/64439. Feng J., E.J. Soto-Moreno, A. Prakash, A.Z. Balboula, H. Qiao. 2022. Adverse PFAS effects on mouse oocyte in vitro maturation are associated with carbon chain length and sulfonate group, Cell Proliferation, e13353, doi: 10.1111/cpr.13353. Vasquez, D.L., K. Rodriguez-Lukey, S.K. Behura, A.Z. Balboula. 2022. Microtubule organizing centers regulate meiotic spindle positioning in mouse oocyte. Developmental Cell, 57 (2), pp. 197-211. Spooner-Harris, M., K. Kerns, M. Zigo, P. Sutovsky, A. Balboula, A.L., Patterson. 2022. A re-appraisal of mesenchymal-epithelial transition (MET) in endometrial epithelial remodeling. Cell and Tissue Research. 2022 Nov 19. doi: 10.1007/s00441-022-03711-z. Lenis, YY., J.W., George, S. Lind, A. Balboula, J.M. Teixeira, A.L. Patterson. 2022. The Effects of Periostin Expression on Fibroid-Like Transition of Myometrial Cells. Reproductive Sciences. 2022 Nov 23. doi: 10.1007/s43032-022-01128-1. Online ahead of print. Rakha, S., M. Elmetwally, H. El-Sheikh, A.Z. Balboula, A. Montaser, S.M. Zaabel. 2022. Lycopene Reduces the In Vitro Aging Phenotypes of Mouse Oocytes by Improving Their Oxidative Status. Veterinary Sciences, 9 (7): 336. Rakha, S.I., M.A. Elmetwally, A.H. El-Sheikh, A.Z. Balboula, A.M. Mahmoud, S.M. Zaabel. 2022. Importance of Antioxidant Supplementation during In Vitro Maturation of Mammalian Oocytes. Veterinary Sciences, 9 (8): 439. Chen, P.R., K. Uh, K. Monarch, L.D. Spate, E.D. Reese, R.S. Prather, K. Lee K. Inactivation of growth differentiation factor 9 blocks folliculogenesis in pigs. Biol Reprod. 2023 Jan 16;ioad005. doi: 10.1093/biolre/ioad005. Online ahead of print. Chen, P., K. Uh, B.K. Bedel, E.D. Reese, R.S. Prather, K. Lee. Production of Pigs From Porcine Embryos Generated in vitro. Front. Anim. Sci., 16 March 2022; https://doi.org/10.3389/fanim.2022.826324 Shearn CT, Anderson AL, Devereux MW, Orlicky DJ, Michel C, Petersen DR, Miller CG, Harpavat S, Schmidt EE, Sokol RJ. The autophagic protein p62 is a target of reactive aldehydes in human and murine cholestatic liver disease. PLoS One. 2022;17(11):e0276879. doi: 10.1371/journal.pone.0276879. eCollection 2022. PubMed PMID: 36378690; PubMed Central PMCID: PMC9665405. Stancill JS, Hansen PA, Mathison AJ, Schmidt EE, Corbett JA. Deletion of Thioredoxin Reductase Disrupts Redox Homeostasis and Impairs β-Cell Function. Function (Oxf). 2022;3(4):zqac034. doi: 10.1093/function/zqac034. eCollection 2022. PubMed PMID: 35873655; PubMed Central PMCID: PMC9301323. Le Gal K, Schmidt EE, Sayin VI. Cellular Redox Homeostasis. Antioxidants (Basel). 2021 Aug 28;10(9). doi: 10.3390/antiox10091377. PubMed PMID: 34573009; PubMed Central PMCID: PMC8469889.   White, B.R., R.A. Cederberg, D.H. Elsken, C.E. Ross, C.A. Lents and A.T. Desaulniers. 2022. Role of gonadotropin-releasing hormone-II and its receptor in swine reproduction. Mol. Reprod. Dev. 1–11. <a href="https://doi.org/10.1002/mrd.23662">https://doi.org/10.1002/mrd.23662</a>. [Online ahead of print].   Desaulniers, A.T., R.A. Cederberg, C.A. Lents and B.R. White. 2022. Gonadotropin-releasing hormone II and its receptor regulate motility, morphology, and kinetics of porcine spermatozoa. Gen. Comp. Endocrinol. (Submitted).   Davies C.J.*, Z. Fan, K.P. Morgado, Y. Liu, M. Regouski, A.J. Thomas, S-I. Yun, B.-H. Song, J.C. Frank, I. V. Perisse, A. Van Wettere, Y-M. Lee, I.A. Polejaeva* (2022). Development and Characterization of Type I Interferon Receptor Knockout Sheep: A Model for Viral Immunology and Reproductive Signaling. Frontiers in Genetics 13: 986316. PMID: 36246651. Adams L., Y. Liu, B. Durrant, E. Ruggeri, C. Young, R. Krisher, I. Polejaeva* (2022) Use of Bisection to Reduce Mitochondrial DNA in the Bovine Oocyte. JoVE. PMID: 35876541. Gao, Y., L. Zhao, J.S. Son, X. Liu, Y. Chen, M. J. Deavilla, M. J. Zhu, G. Murdoch, and M. Du. 2022. Maternal exercise before and during pregnancy facilitates embryonic myogenesis by enhancing thyroid hormone signaling. Thyroid. 32: 581-593. Zhao, L. X. Liu, N. A. Gomez, Y. Gao, J. S. Son, S. A. Chae, M. J. Zhu, and M. Du. 2023. Stage-specific nutritional management and developmental programming to optimize meat production. Journal of Animal Science and Technology. 14: 2.   <ol start="2"> <li>Books, non-refereed book chapters, proceedings, instructional media, theses/dissertations</li> </ol> Thesis Title: "CRISPR Cas9 Targeted Single Nucleotide Change at the (+15) Loci of the Alpha-Lactalbumin Gene. Sierra Long, M.S., July 2022. Gutierrez-Castillo, E.  Vitrification of bovine oocytes and embryos. Louisiana State University Dissertation. 2022. Mustapha A Popoola, Suleiman E Bogoro, Jean M Feugang. Biothermoimaging tools for management of climate smart and precision livestock-assisted reproduction. In “Agricultural Biotechnology, Biodiversity and Bioresources conservation and utilization.” (pp. 315-334). 1st Edition, CRC Press, eBook ISBN 9781003178880. https://doi.org/10.1201/9781003178880, 2022.   <ol start="3"> <li>Abstracts</li> </ol> W E Weber, J G Powell, B P Littlejohn, E B Kegley, N N Noga, C R Looney. 2022 Evaluation of Fixed-time Artificial Insemination and Estrus Expression in Angus-cross Heifers with Female Sex-sorted Semen Using 6-day and 7-day CIDR Protocols at Multiple Time Intervals. Journal of Animal Science, Volume 100, Issue Supplement1, April 2022, Page 10, <a href="https://doi.org/10.1093/jas/skac028.018">https://doi.org/10.1093/jas/skac028.018</a> W E Weber, J G Powell, B P Littlejohn, B W Krumpelman, N T Moss, M S Gadberry, C R Looney.  2022. Evaluation of GnRH Administration day in the 7 & 7 Synch Protocol for Fixed-Timed Artificial Insemination in Multiparous Beef Cows. Journal of Animal Science, Volume 100, Issue Supplement3, October 2022, Page 244, <a href="https://doi.org/10.1093/jas/skac247.440">https://doi.org/10.1093/jas/skac247.440</a> <ol start="2022"> <li>E. Weber, J. G. Powell, B. P. Littlejohn, B. W. Krumplman, J. D. Rivera, W. L. Rook, M. S. Gadberry, N. T. Moss, C. R. Looney. Evaluation of the 7 & 7 Synch protocol on GnRH administration day for FTAI in multiparous beef cows. AETA-CETA/ACTE Joint Annual Convention, Louisville, KY in October 2022. Meeting Proceedings pg. 96</li> <li>E. Weber, J. G. Powell, B. P. Littlejohn, B.W. Krumplman, N. T. Moss, M. S. Gadberry, C. R. Looney. Evaluation of GnRH administration day in the 7 & 7 Synch protocol for fixed-timed artificial insemination in multiparous beef cows..ARSBC Lauderdale Scholar Poster Presentation, San Antonio, TX, July 2022.</li> </ol> Guiltinan C, Weldon BM, Soto DA, Ross PJ, Denicol AC. Single-cell transcriptome analysis of fetal and adult bovine ovaries reveals developmental progression in cell population composition and function. Reprod, Fert Dev 35(1-2), 2023. <a href="https://www.publish.csiro.au/RD/pdf/RDv35n2abs">https://www.publish.csiro.au/RD/pdf/RDv35n2abs</a>. Snow A, Donovan A, Winger QA. Investigating the role of thyroid hormone in ovine placenta using trophoblast cell lines. National Veterinary Scholars Symposium, Minneapolis, MN, USA. Donovan A, Anthony RV, Winger QA. Investigating Transthyretin and Thyroid Hormones in CSH Knockdown IUGR Pregnancies. Society for the Study of Reproduction 55th Annual Meeting, Spokane, WA, USA. Tanner A.R., V. C. Kennedy, C. S. Lynch, Q. A. Winger, P. J. Rozance, R. V.  Anthony. Maternal Hyperglycemia Increases Uteroplacental Oxygen Utilization but Fails to Restore Fetal Glucose Concentrations in Chorionic Somatomammotropin RNA Interference Pregnancies. Reprod Sci 2022; Society for Reproductive Investigation 69th Annual Scientific Meeting, Denver, CO. Tanner A.R., V. C. Kennedy, C. S. Lynch, Q. A. Winger, P. J. Rozance, R. V.       Anthony. Varying Degrees of Fetal Growth Restriction in Chorionic Somatomammotropin RNA Interference Pregnancies. Reprod Sci 2022 Society for Reproductive Investigation 69th Annual Scientific Meeting, Denver, CO. Lynch C.S., A. Ali, V. C. Kennedy, A. R. Tanner, Q. A. Winger, P. J. Rozance,  R. V.   Anthony. Assessing the Impact of Placental SCL2A3 RNA Interference at Mid-Gestation. Reprod Sci 2022; Society for Reproductive Investigation 69th Annual Scientific Meeting, Denver, CO. Hord T.K., A. R. Tanner, C.S. Lynch, V. C. Kennedy, Q. A. Winger, P. J.    Rozance, R. V. Anthony. Placental Expression of the IGF Axis Following Chorionic Somatomammotropin RNA Interference. Reprod Sci 2022; Society for Reproductive Investigation 69th Annual Scientific Meeting, Denver, CO. Menjivar N.G., Gad A., Thompson R.E., Meyers M.A., Hollinshead F.K., Tesfaye D. (2022) Bovine Oviductal Organoids: Cellular and Extracellular Response to Heat Stress. Graduate Student Showcase 2022; Page XX. Fort Collins, CO, USA. Tesfaye D., Menjivar N.G., Gad A. Next generation miRNA loading into extracellular vesicles: Potential application for EV-coupled miRNA functional studies. BMS Retreat 2022; Page 29. Estes Park, CO, USA. Menjivar N. G., Gebremedhn S., Gad A., Tesfaye D. (2022) Emerging role of extracellular vesicles as messengers for protection against heat stress in bovine oocytes. Society for the Study of Reproduction 2022 (SSR); 55th Annual Meeting. Spokane, WA, USA. Gad A., Gebremedhn S., Menjivar N., Feugang J., Ishak G., Prochazka R., Tesfaye D., Gastal E. (2022) Dynamics of Extracellular vesicle-coupled microRNAs in equine follicular fluid associated with follicular selection. Society for the Study of Reproduction 2022 (SSR); 55th Annual Meeting. Spokane, WA, USA. Kamryn Joyce, Ahmed Gad, Nico  G Menjivar, Daniella C Heredia, Camila Santos Rojas, Dawit Tesfaye, Angela M Gonella, PSIII-17 Transcriptome Response of Granulosa Cells to Seasonal Heat Stress in Beef Cows, Journal of Animal Science. American Society of Animal Sciences 2022 (ASAS-CSAS-SSASAS) Annual Meeting & Trade Show; Volume 100,Issue Supplement3, October 2022, Pages 242-243, Oklahoma City, OK, USA. <a href="https://doi.org/10.1093/jas/skac247.438">https://doi.org/10.1093/jas/skac247.438</a> Menjivar N. G., Gebremedhn S., Tesfaye D. (2022) Impact of supplementation of granulosa cell-derived extracellular vesicles during bovine oocyte maturation under thermal stress conditions. International Congress on Animal Reproduction 2022 (ICAR); 19th International Conference. Bologna, Italy. Menjivar N. G., Gebremedhn S., Gad A., Tesfaye D. Next generation miRNA loading system into extracellular vesicles: Potential application for EV-coupled miRNA functional studies. Rocky Mountain Reproductive Sciences Symposium 2022 (RMRSS); 15th Annual Conference: Page 25. Fort Collins, CO, USA. Gad A., Joyce K., Menjivar N., Heredia D., Rojas C., Gonella-Diaza A., Tesfaye D. Extracellular vesicle-coupled microRNAs in the follicular fluids of heat-stressed beef cows. Rocky Mountain Reproductive Sciences Symposium 2022 (RMRSS); 15th Annual Conference: Page 14. Fort Collins, CO, USA. Menjivar N. G., Gebremedhn S., Gad A., Krisher R., Yuan Y., Tesfaye D. (2022) Interspecies Translation: Bovine extracellular vesicles impact on human cumulus cells. CSU Demo Day 2022; Annual Showcase. Fort Collins, CO, USA. Ranjitkar S, Shiri M, Sun J, Liu G, Tian XC. 2023 Transcription read-through in in vivo developed bovine oocytes and pre-implantation embryos. Poster at the 49th annual meeting of the International Embryo Technology Society, January 16-19, 2023, Lima, Peru. Salman S, Gungor O, Ranjitkar, S, Zhang D, Oberhaus E, Tian XC. 2023. Hormonal profiles of bovine follicular fluid during the window of in vivo oocyte maturation. Poster at the 49th annual meeting of the International Embryo Technology Society, January 16-19, 2023, Lima, Peru. Losano JDA, Daigneault BW. Environmental modulation of sperm energetics by non-canonical PPARʏ signaling. Society for the Study of Reproduction 55th Annual Meeting, Spokane, WA 2022. P- Losano JDA, Daigneault BW. Detection of novel bioenergetic traits in bovine sperm for improved semen quality analyses. Proceedings of the Eighth UF/IFAS Animal Sciences Symposium, St. Augustine, FL 2022. G-McGraw M, Daigneault BW. Functional characterization of PPAR

Impact Statements

Impacts (Objective 1) Continued research has identified an improved method of estrous synchronization (ES) and Fixed-Timed Artificial Insemination (FTAI) using Controlled Internal Drug Releasing (CIDR) for 14 days (injections 7 &7 days) of Gonadotropin Releasing Hormone (GnRh) and Prostaglandin (PGF). This new protocol was developed after combining our data on using CIDR + PGF for pre-synchronization which improved conception rates but required more labor for cattle handling. We also utilized our experience comparing 6 vs. 7-day CIDR exposure to improve the 14 day, 7 & 7 injection scheme to use a modified 8 & 6 protocol. In more than 500 inseminations of conventional and sexed-sorted frozen semen, performed in 4 different facilities, the new 14-day program has legitimate conception rates above 70-80%, which is 20-30% above the national average. This research was a collimation of research over 3 years and has been tested in commercial herds throughout Arkansas during this year. Single-cell RNA sequencing is a powerful technique to help us unravel the complex nature of the ovary and how this organ undergoes the dramatic remodeling necessary throughout a female’s lifespan to ensure fertility. The new follicle isolation protocol opens up opportunities to study preantral folliculogenesis in the cow, an area of reproductive biology that has been largely unexplored due to the difficult access to these follicles. The potential involvement of macrophages in increasing Sertoli cell population and absence of involvement of CSF1, AR, and SRD5A2 advances our understanding of this process. Investigating the impact of genetic and pharmacological approaches to modulate NRF2-mediated oxidative stress response in bovine preimplantation embryos will provide a unique opportunity to enhance survival and mitigate the long-term impact of preimplantation period oxidative damage on fetal development and offspring health. Moreover, the study will identify genes and genome regions which can be targets for future interventions to enhance viability of embryos from assisted reproductive technologies. Moreover, the research on extracellular vesicles-mediated molecular signaling in ovarian follicle and oocytes in Dr. Tesfaye lab will facilitate the development of diagnostic markers associated with maternal physiology and embryo developmental competence and future potential therapeutic application of EVs in assisted reproductive technologies. The introduction of studies of intergenic transcription in the field of reproduction will lead to a wave of new studies in this subject area. These studies will enhance our understanding of transcription regulation during embryogenesis and animal reproduction in general. We have determined that current industry assessment of frozen-thawed bovine sperm is not sufficient to identify differences in the bioenergetic capacity of sperm when maintained over time. In addition, we have characterized the temporal bioenergetic plasticity of bovine sperm maintained for 24 hr and have revealed a shift in the bioenergetic status for quiescent to glycolytic. Additionally, we have determined that incubation in heterospermic conditions seems to elicit a compensatory response that remains undefined. We have developed a media that appears suitable for prolonged storage of frozen-thawed and fresh bovine sperm conducive for in vitro fertilization and artificial insemination trials. Positive results may impact the cattle industry by providing flexibility between sperm handling and fertilization to increase reproductive efficiency, while lowering dependance on cryopreservation of sperm for embryo production. We have determined that PPARG is an early developmental control gene that influences the blastocyst transcriptome towards early placental formation. Our work filled a significant knowledge gap in the study of translational regulation over a period of rapid developmental change and provided an extensive database that can be mined for more detailed insights into bovine oocyte and preimplantation development. The development of training deep neural networks approach and the application of transfer learning approach enable imputing single-cell DNA methylome profiles to predict methylation in scarce material samples, such as oocytes, and embryos. Understanding the molecular characteristics of oocyte competence will provide insight into improving the developmental competence of in vitro produced embryos. Oocyte maturation: Dominant follicle removal (DFR) and a shorter interval of OPU following DFR in Bos taurus X Bos indicus cattle increases the number of higher quality cumulus-oocyte-complexes (COCs). This allows for the optimization of in vitro embryo production (IVP) from Bos indicus crossbred cattle. Embryogenesis: Cows fed supplemental rumen-protected methionine may have higher quality embryos that may lead to higher pregnancy rates in dairy cattle. Differential localization of key proteins involved in meiotic spindle formation will provide an avenue to study this process in in vitro matured bovine oocytes and potentially improve the effectiveness of this process. The methods and advanced mathematical models developed in this project will help extend the application of MFA framework to complex mammalian systems. Unraveling the metabolic basis of normal early embryo development will provide significant benefits to human and animal reproductive health. Improved embryonic competency following in vitro production and cryopreservation would stimulate the industry by lowering costs, especially those related to recipient management. Seasonal and developmental changes affecting ovarian follicular fluid composition are demonstrated in the mare. Subsets of miRNA affecting mRNA expression are identified as potential candidate to improve female fertility through in vitro oocyte maturation or in vitro follicle culture. Near-Infra-Red Spectroscopy (NIRS) combined with aquaphotomics are revealed as possible tools to assist in the rapid screening of Good vs. Poor semen quality prior to breeding. Various technical approaches enabled potential biomarkers of boar semen quality, reinforcing the potential of the NIRS for a rapid screening tool for boar semen quality. Investigating spermatozoa and corresponding seminal plasma permits a better understanding of molecular changes during aging, providing additional knowledge for possible interventions to slow or minimize the aging impact on male fertility. Phenotype of GDF9 knockout pigs will expand our knowledge on oogenesis and folliculogenesis. E-64 supplementation during IVM is a promising tool to improve the efficiency of OPU-IVF program. Regulating CTSL level in oocytes and early embryos is a promising approach to improve the efficiency of IVP technology. As a result of these studies, we acquired a change in fundamental knowledge regarding how the GnRH-II/GnRHR-II system regulates 17ß-estradiol levels and follicular dynamics in porcine females, representing a potential avenue for future reproductive therapies. Together, these data strongly suggest that GnRH-II and its receptor play a crucial role in regulating follicular recruitment and development during the estrous cycle, impacting overall fertility outcomes of gilts. As a result of these experiments, we expect to better understand how the GnRH-II receptor is regulated within extra-pituitary tissues related to reproduction. New pharmacological agents may be developed to manipulate the reproductive axis, leading to enhanced fertility rates and reduced incidences of endocrine disorders impacting reproduction. Data from these experiments could lead to novel swine-specific contraceptive methods to manage feral pig populations. As a result of this project, we expect to see a change in knowledge regarding the mechanisms underlying regulation of ovarian steroidogenesis by GnRH-II and its receptor and how this hormone-receptor complex is involved in determining ovulation rate and subsequently, litter size, in pigs. A genetic test to identify fertile and/or subfertile female gametes could enhance traditional selection procedures and novel pharmacological agents could improve swine production practices. Ultimately, this will lead to a change in condition, increasing profitability and therefore, sustainability for pork producers. Conceptus elongation is a critical period for maternal recognition of pregnancy in cattle. Understanding the normal mechanisms regulating embryonic and placental development is necessary to acquire basic knowledge that can serve as a foundation to diagnose abnormal embryogenesis. The long-term goal of this research is to improve cattle production systems and reduce the impact of reproductive problems in cattle operations. Collectively, results from our studies in the bovine uterus help elucidate the long-term impact of postpartum metritis on reproductive performance. Further understanding of the biological processes regulating uterine inflammation, remodeling and repair are necessary to develop alternative strategies for the treatment of uterine diseases to reduce its impact on fertility. A simple but efficient technique that can increase ATP concentrations in oocytes could be adapted to enhance oocyte cytoplasmic maturation, embryo production and embryo quality. Improvement in sheep oocyte maturation and embryo development using L-OPU derived oocytes could have a significant impact on improving embryo development and lambing rates following SCNT. Maternal nutrition not only affects fetal development, but also embryonic development, which generates long-term impacts on offspring performance. (Objective 2) Examination of multiple genes and most likely off-targets will be needed to reach a conclusion whether such off targets following editing of zygotes are an occasional or an extremely rare event. Understanding the role of LIN28A and LIN28B in sheep placenta will improve our understanding of the genetic regulation of placenta development and result in methods to improve somatic cell nuclear transfer in ruminants. Dr. Winger’s lab has initiated studies into how thyroid hormones are regulated by the placenta. Transthyretin (TTR) is a tetrameric serum protein composed of four identical subunits (55kDa) mainly synthesized and secreted by the liver for the purpose of transport of thyroxin (T4). TTR is detected in both fetal and maternal serum of sheep during pregnancy. TTR from the trophoblast cells is secreted on the maternal side of the placenta and is believed to transport maternal T4 to the fetus. This proposal will investigate if TTR from the placenta is critical for T4 uptake by the placenta and/or transport to the fetus. Naïve pluripotency in the bovine has not been achieved after decades of efforts. It establishment will significantly improve our ability to modify the bovine genome through genetic engineering including gene-editing. We have streamlined the ability to produce gene edited bovine embryos with high efficiency and throughput towards the goal of producing gene edited cows conducive to microbiome and metabolomic studies for increasing both reproductive and feed efficiency. Bovine TSCs fill a gap and add invaluable information of placenta development of an ungulate species. The bovine TSCs not only serves a model to study the unique placentation process in the ruminants and early pregnancy failure, but also enables the first generation of blastocyst-like structures (blastoids) from a large livestock species. The bovine blastoids represent a valuable model to study early embryo development and understand the causes of early embryonic loss. Upon further optimization, bovine blastoid technology could lead to the development of new artificial reproductive technologies for cattle breeding, which may enable a paradigm shift in livestock reproduction. Development of Human Biomedical Models: Topology-optimized PCL cages with surface adsorbed BMP-2 may allow for patient-specific designed spinal fusion prostheses resulting in more complete intervertebral fusion. Improvements in the outcomes of bovine oocyte cryopreservation will provide an additional tool for breeding of elite cattle including those resulting from genetic modification. Improved outcomes from bovine intra cytoplasmic sperm injection will provide an additional method for incorporation of gene editing techniques and fertilization with spermatozoa for which there are limited numbers such as sex sorted semen. Ability to effectively assess and reduce off-targeting events, caused by the genome editing systems, will expand application of genome editing systems in livestock. From a redox metabolism standpoint, these studies are uncovering previously unrecognized systems that support survival and function of liver cells, which, in turn, is providing insights into better therapies for treating some liver diseases or toxicities. From more of a bioengineering standpoint, these studies are demonstrating the power of being able to genetically manipulate hepatic metabolism in adult animals. This promises to provide interesting new ways to genetically modify nutrient use or intermediary metabolism of animals that could improve food-production characteristics of livestock without generating genetically modified food products. Introduction of the SCD mutation in the sheep β-globin gene results in a similar phenotype at the blood level, thereby potentially producing a similar disease phenotype and progression in the sheep model. These findings support the translational relevance of the SCD sheep model and highlight the potential for its future use in the development of novel therapies for SCD that could be highly impactful. The establishment of pregnancy in two IFNAR2–/– ewes demonstrates that the accepted mechanism of pregnancy recognition in ruminants, which involves IFNT interacting with IFNAR, is incorrect or is not required for establishment of pregnancy in ruminants. Unless the effects of IFNT are mediated by an alternative receptor, it appears that IFNT is not required for pregnancy recognition or the development of a healthy ruminant fetus. Cas-CLOVER gene editing tool could reduce potential off-target effect when use for animal model development.

Date of Annual Report: 01/17/2024

Report Information

Annual Meeting Dates: 01/07/2024 - 01/07/2024
Period the Report Covers: 01/01/2023 - 12/31/2023

Participants

Participants
In attendance were Anna Denicol (California), Quinton Winger (Colorado), X. Cindy Tian (Connecticut), Brad Daigneault (Florida), Zongliang (Carl) Jiang (Florida), Curt Youngs (Iowa), Kenneth Bondioli (Louisiana), Carol L. Keefer (Maryland), Jean Feugang (Mississippi), Kiho Lee (Missouri), Brett White (Nebraska), Jingyue (Ellie) Duan (New York), Joao Gabriel Nascimento Moraes (Oklahoma), Celina Checura (South Carolina), Irina Polejaeva (Utah), Ying Li (Utah).

In person attendance (14)
Dr. Kenneth Bondioli, Dr. Brad Daigneault, Dr. Anna Denicol, Dr. Quinton Winger, Dr. Curt Youngs, Dr. Jean Feugang, Dr. Jaoa Moraes, Dr. Brett White, Dr. Irina Polejaeva, Dr. Ying Liu, Dr. Curt Youngs, Dr. Ellie Duan, Dr. Min Du, Dr. Lacey Luense

Zoom attendance (7)
Dr. Cindy Tian, Dr. Carol Keefer, Dr. Trish Berger, Dr. Ken White, Dr. Carl Jiang, Dr. Kiho Lee, Dr. Celina Checura

Brief Summary of Minutes

Brief Summary of Minutes of 2023 Annual Meeting

Jan 7th, 2024

Hyatt Regency Denver at Colorado Convention Center, Denver, Colorado

Conference room: Agate C

Zoom link: https://okstate-edu.zoom.us/j/92286999220

9:00 AM – 9:45 AM (45 min)

9:00: Dr. Joao Moraes started the meeting and welcomed the participants.

9:02: All participants introduced themselves in the room and over the Zoom.

9:04: Dr. Joao Moraes presented the agenda for the meeting.

9:05: Dr. Joao Moraes proposed a Motion to approve minutes from 2023 meeting, Dr. Irina Polejaeva seconded.

09:06-09:40: Dr. Brett White lead the discussion regarding the W4171 Project Renewal, everyone in the room and over the Zoom all contributed to the discussion:

Object 1&2 title change. Genetic engineering includes everything. Advancing the fields of reproductive physiology, not just refining the method. Add the aspect of Molecular, cellular, omics technology and data analysis. Objective 2: Add intentional genomic alternation

Working groups set up for each station to sign up for either objective(s).

Counting the number of transgenic animal models on the ground. Several stations mentioned one or more of transgenic animal models that on the ground made by their group.

9:40: Dr. Ken White has discussed the process of submission of annual final report and the renewed W4171 multistate project.

Secretary Dr. Ellie Duan need to 30 days submission this annual reports summary to Ken White.

The renewed W4171 project has to be submitted on 1/15/2024

The W4171 will be end on 9.30, after that the stational direction can not use the station funds. The late submission of renewal project may have 6 month to 1 year delay

Each station: take 1 station lead to fill out appendix E for each station for the new W5171, this can happen after 1/15/2024

Each station must select the object(s) that each station chose to work on in W5171. This need to be done by the first of March 2024.

Need to identify 5 reviewers that agree to read and review the proposal prior to the project proposal submission.

9:45 AM – 12: 30 PM

Station Reports (10-15 min per station) –Station Reports were discussed in the reverse order that they appear in the merged Station Report draft (descending alphabetical order by Station)

10 stations:

Washington- Dr. Min Du

Early embryo development, embryonic muscle, and adipose development, how are same MSC cells have their final fate decided during development. Understand this process will have potential implication in the livestock growth improvement.

Imprinting genes, impact of oocyte/sperm genome on the early embryos. Maternal obesity impact on the offspring development.

Utah - Dr.Ying Liu

Oocyte maturation with cytokine-supplemented medium

Effect of serum-free maturation medium on bovine oocyte maturation

- Dr. Irina Polejaeve

Genetic engineering livestock

SCNT method for genetic engineering
- Anti-GAL GALKO sheep produce anti-gal antibodies similar to humans.

South Carolina - Dr. Celina Checura

Photobiomodulation, activate the mitochondrial could potentially change the embryo cleavage and metabolism

Overall increase in embryonic development rates and high cell numbers

Mitochondrial number will not increase in early embryonic development, only increase after blastocyst stage

Oklahoma - Dr. Joao Moraes

Uterine disease in long-term fertility
- Endometrial 16S in healthy vs. metritis D7

Baseline uterine biology, what heathy cells, Uterine biology sncRNA-seq
- Spatial RNA-seq in D0, D7, D30 healthy vs. metritis

Aim to identify gene drives the conceptus elongation
- Bulk RNA-seq, micro RNA-seq of D45, D50 embryos

Nebraska – Brett White

GnRHR-II KD pig
- GnRH-II function in regulating steroidogenesis

New York – Dr. Ellie Duan

Maternal factor impact the early zygotic genome activation
- Profiled the general expression and protein changes
- New method developed to do CUT&TAG in limited cell numbers

Sex-specific development difference in early development
- Whether the cultural medium will change the male develop faster
- Whether XCI process slow the female embryos down

Transposon element expression during pre-implantation development
- Intergenic region

Montana - Skip

Missouri - Dr. Kiho Lee

New ways to activate oocytes

The identification of off-targeted events introduced by the CRISPR-Cas9 system in gene-edited pigs. Off target event happens in the intronic region.

Compare the off-target to the non-reference genome but to the sequencing of the donor cell line.

Mississippi - Dr. Jean Feugang

The gradual decline of semen quality.

Predict the poor quality of bull semen.

Characterization of seminal extracellular vesicles of divergent sperm quality.

Maryland - Dr. Carol Keefer

Reported on her work with energy metabolism in embryos.
- Discussion: do embryos store energy in the form of glycogen?
- Discussion: do they divide fast as cancer cells?

12:20 - 1:15 PM – Lunch Break

1:15 PM Afternoon session start

1:15 PM – 2: 45 PM (90 min) Continue Station Reports

8 stations:

Louisiana - Dr. Kenneth Bondioli

Localization of key proteins involved in meiotic spindle formation. Occyte cryopreservation.
- Metabolic profiling from cytoplasm of single oocyte
- Spatial heatmap of individual oocyte
- Characterize different proteins from MS

In vitro produced embryos has different ZP

Improved methods developed in his lab for ICSI.

Iowa – Dr. Curt Youngs

Presented some of his work overseas including word done with alpacas in Peru and dairy cattle in Ethiopia.

Embryo cryopreservation alpaca
- Sexed-semen, community based

Microbiota work in sheep, virginal microbiome. Shift in microbiota changes throughout gestation and pregnancy
- 95% pregnancy happen in the left virginal horn, with ovulation happen in both ovaries.

Illinois

Skip

Florida - Dr. Brad Daigneault

Optimizing cool condition for prolonged maintenance of equine

How environmental factor impact the sperm function (post ejaculation)

Role of PPARG in bovine embryos: Activation and inhibition, changes some genes. KO PPARG no change of blastocysts.

Separation and passage embryo cells from every cleavage

Zongliang Jiang

Molecular mechanism of early embryonic development, particularly during pre-implantation development.

Profile the bovine pre-implantation embryo using single cell sequencing.

Bovine blastoids derived from trophoblast stem cells.

Connecticut – Dr. X. Cindy Tian

Oocyte maturation follicular fluid to improve maturation conditions

Intergenic transcription forms read-through and read-ins during pre-implantation embryos. Potential changes when cell under stress or ART, IVF, Cloning.

Colorado - Dr. Quinton Winger

How the placenta and embryo use the T4 hormone from mom-placenta to fetus.

California - Dr. Trish Berger

Nonfunctional AR in pigs on X chromosome, male vs. female

Anna Denicol

Ovary biology, early-stage follicle, optimize cultural condition

FSH in primary follicles. Follicle growth, Stimulate cows with FSH

Single-cell transcriptomics, ovarian biology

Embryonic stem cells from early vs late stage blastocysts

Texas – Dr. Lacey Luense

Epigenetics reprogramming of sperm retained into embryo

10% of histone retention in cattle sperm, 5% in human and mice sperms

Abnormal sperm chromatin-associated with pregnancy loss

Gcn5 mouse model

2: 45 PM- 3:00 (15 min)

Nomination for Chair and Secretary of the W4171 2024-2025 Annual Meeting

Joao Moraes nominated Dr. Ellie Duan for Chair of the 2025 meeting. Dr. Polejaeva seconded.

Joao Moraes nominated Dr. Lacey Luense for secretary of the 2025 meeting. Dr. Daigneault seconded.

2025 Meeting Venue, Date and Time – Member discussion

Possible dates for the 2025 annual meeting will be near IETS time, which is 2025 1.18-1.21 in Texas

Once the schedule of activities for IETS has been released, there will be a Doodle Pool to decide the time for the 2024 annual meeting.

We could hold meet with the pre-conference time, so there will not be a 2-day gap like this year.

3:00 Work on Project Renewal proposal.

Dr. White lead the draft for the new project on two google docs

In this document, each station to add their information (1-2 sentences) under each Working Group and Objective:

This document has the new project draft for everyone to sign up a session to update the literature review, everyone has signed up for a section to work on re-writing and updating the literature list.

Oocyte Maturation - Cindy Tian and Ken Bondioli

Fertilization and Sperm Biology - Brad Daigneault and Jean Feugang

Embryo Development - Carl and Carol

Epigenetics- Ellie Duan, Lacey Luense

Nuclear Transfer - Irina Polejaeva

Cryopreservation - Curt Youngs

Stem Cell Biology- Anna Denicol, Joao Moraes, Zongliang Jiang

Genome Editing - Kiho Lee

6:00 Dr. Youngs - motion to end the meeting. Dr. Polejaeva seconded. End minutes.

Accomplishments

ACCOMPLISHMENTS Objective 1- Understand the biology of gamete development, fertilization and embryogenesis including the underlying cellular and molecular mechanisms.   <ol> <li>Detailed characterization of the cell populations that make up the adult bovine ovary based on single cell transcriptional profiling.</li> </ol>   <ol start="2"> <li>Validation of a RNA interference strategy to study the roles of FSH in preantral follicles.</li> </ol>   <ol start="3"> <li>Optimization of the ovarian biopsy technique in cattle.</li> </ol>   <ol start="4"> <li>Established the detailed profiles of proteomics and metabolomics events in follicular fluid in pre-ovulatory follicles.</li> </ol>   <ol start="5"> <li>Continued to refine the culture condition of putative naïve bovine embryonic stem cells.</li> </ol>   <ol start="6"> <li>Conducted studies for the separation of bovine sperm for sex selection.</li> </ol>   <ol start="7"> <li>Using the commercial protocol, high cleavage rate and a blastocyst rates were achieved. Large numbers of embryos will be needed to improve the dairy cattle genetics in The efficiency of the present IVF system will enable that large-scale embryo production.</li> </ol>   <ol start="8"> <li>A White-tailed deer (WTD) IVM and IVF protocol was tested and produced high cleavage and a blastocyst rate s. Furthermore, embryos were produced using a commercial bovine IVF protocol adapted to have 24 h of fertilization (as opposed to 18–22 h of fertilization). Overall, the adapted protocol produced blastocyst and cleavage rates for WTD similar to those achieved when using the protocol on cattle. With continued adjustments, the protocol could be optimized for IVF embryo production in WTD.</li> </ol>   <ol start="9"> <li>Many research tools can accurately determine embryo quality/viability; however, most are invasive, expensive, laborious, technically sophisticated, and/or time-consuming, making them futile in the context of in-field embryo evaluation. Time-lapse monitoring and artificial-intelligence-based automated image analysis also have the potential for accurate embryo evaluation; however, further research is warranted to innovate economically feasible options for in-field applications.</li> </ol>   <ol start="10"> <li>Raman spectroscopy was used for lipid profiling of single in vivo and in vitro matured oocytes. Spatial mapping of lipid droplets within oocytes indicated higher levels of fatty acids in in vitro matured oocytes compared to in vivo oocytes suggesting incomplete metabolism of fatty acids during in vitro maturation.</li> </ol>   <ol start="11"> <li>Raman spectroscopy was used to characterize the secondary structure of zona pellucida proteins of in vivo and in vitro derived bovine embryos. This analysis revealed no difference in the α-helix and β-sheet ration between in vitro and in vivo derived embryos.</li> </ol>   <ol start="12"> <li>A modified procedure for intracytoplasmic sperm injection (ICSI) was used to fertilize vitrified and non-vitrified bovine oocytes. This procedure yielded a higher proportion of blastocyst compared to a conventional procedure and it was demonstrated that these ICSI derived embryos capable of producing pregnancies upon transfer.</li> </ol>   <ol start="13"> <li>Utilization of Glucose and Fructose in Bovine Embryos Assessed by Metabolic Flux Analysis.</li> </ol>   <ol start="14"> <li>Raman and Near infra-red spectroscopies have been used effectively used on extended boar semen to assess quality decline during storage, providing semen-specific spectral profiles of long-term (Good) and short-term (Poor) survival during chilled storage.</li> </ol>   <ol start="15"> <li>Using single-cell RNA-sequencing (scRNA-seq), the Washington Station is studying the impacts of maternal obesity on embryonic development and found that maternal obesity impairs embryonic myogenesis but enhances fibro-adipogenesis, which correlates with the increased fibrosis in offspring.</li> </ol>   <ol start="16"> <li>In addition, the Washington Station also analyzed the impacts of cold exposure of bulls on DNA methylation of sperm cells, which could be passed on to the resulting embryos to affect offspring development. DNA methylation changes were identified in a group of genes including imprinted genes, which are known to affect offspring development.</li> </ol>   <ol start="17"> <li>FLI-supplemented IVM aids in proper meiotic spindle configuration at MII in bovine oocytes and promotes nuclear progression through meiosis I in unmatured oocytes.</li> </ol>   <ol start="18"> <li>The low oxygen level during IVM could improve oocyte quality but does not increase oocyte maturation and subsequent blastocyst development rates.</li> </ol>   <ol start="19"> <li>Commercial serum-free medium improves the quality of oocytes and PA blastocysts, but does not increase the maturation and embryo development. Additional supplementation of resveratrol does not improve the oocyte quality and embryos development.</li> </ol>   <ol start="20"> <li>Supplementing iSCNT embryos with donor cell mitochondria and/or demethylase mRNA that promotes cellular reprogramming and post-EGA progression may increase in vitro developmental abilities.</li> </ol>   <ol start="21"> <li>Embryogenesis- Determine if abnormal paternal histones alter cell differentiation in preimplantation embryo development.</li> </ol>   <ol start="22"> <li>Determine if post-translational histone modifications are altered in embryos generated from sperm with abnormal paternal histones.</li> </ol>   <ol start="23"> <li>Bioinformatic analysis of H3K27ac Cut&Run from sperm with abnormal paternal histones suggests placement at genes critical for preimplantation embryo development.</li> </ol>   <ol start="24"> <li>Reduced H3K27ac in sperm at genes involved in multiple chromatin pathways may alter histone modifications in preimplantation embryos.</li> </ol>   <ol start="25"> <li>A defined photobiomodulation system (time, wavelength, and intensity) that affects oocyte competence is being developed. The treatment improves ATP concentrations, embryonic development, and embryo quality. The increase in ATP production is not permanent, and the treatments do not alter the proportion of oocytes reaching MII by the end of maturation.</li> </ol>   <ol start="26"> <li>Generated RNAseq and microRNA sequencing data from spherical (0.05 - 0.2 mm), ovoid (0.2 - 5mm), tubular (5 - 15mm), and filamentous (16 - 35 mm) bovine conceptuses.</li> </ol>   <ol start="27"> <li>Generated novel data contributing to the current understanding of how postpartum uterine disease programs long-term infertility in dairy cattle.</li> </ol>   <ol start="28"> <li>Characterize the dynamic of MOF/H4K16ac during preimplantation embryogenesis via qPCR and immunofluorescence staining</li> </ol>   <ol start="29"> <li>Established the CUT&Tag-T7 protocol to profile MOF/H4K16ac genomic distribution in low cell number embryo samples</li> </ol>   <ol start="30"> <li>Sexed embryos using PCR method & Generated full-length transcriptomes of male and female blastocysts</li> </ol>   <ol start="31"> <li>Profiled the transposon elements in the bovine newest genome. Characterized the transcription level of TEs during bovine early preimplantation development</li> </ol>   <ol start="32"> <li>During the follicular phase of the estrous cycle, concentrations of GnRH-II in follicular fluid did not differ between GnRHR-II KD and littermate control gilts.</li> </ol>   <ol start="33"> <li>Prior to the onset of estrus (24 h), circulating GnRH-II levels were diminished in GnRHR-II KD compared to littermate control females.</li> </ol>   <ol start="34"> <li>There was a strong positive correlation between circulating concentrations of 17ß-estradiol and GnRH-II.</li> </ol>   <ol start="35"> <li>Theca cells from GnRHR-II KD females expressed 40% less GnRHR-II protein than control theca cells or transgenic granulosa cells.</li> </ol>   <ol start="36"> <li>Levels of GnRHR-II protein in granulosa cells were similar between lines.</li> </ol>     <ol start="37"> <li>Development and validation of effective protocols for in-house synthesis of heavy sulfur ([34S])-labeled methionine and cystine.</li> </ol>   <ol start="38"> <li>Train and develop expertise in microsurgical insertion of jugular catheters in mice for whole-mouse metabolic labeling studies.</li> </ol>   <ol start="39"> <li>Develop and validate protocols for measuring most biologically important sulfur amino acid metabolites by liquid chromatography-mass spectrometry.</li> </ol>   <ol start="40"> <li>Quantitation of the flux of the sulfur and all carbon atoms from methionine into downstream metabolites in the methionine cycle, methionine salvage pathway, transsulfuration, and glutathione biosynthesis pathways, as well as several other key pathways.</li> </ol>   <ol start="41"> <li>Quantitation of the flux of the sulfur and all carbon atoms from cystine into downstream metabolites in transsulfuration, per / poly-sulfide production, and glutathione biosynthesis pathways, as well as several other key pathways.</li> </ol>   <ol start="42"> <li>Quantitative verification of the role of the methionine catabolism pathway in redox homeostasis.</li> </ol>   <ol start="43"> <li>Identification of a novel non-canonical non-reductive cystine catabolism pathway that also supports redox homeostasis.</li> </ol>   <ol start="44"> <li>Supplementation of the in vitro maturation and embryo culture medium with recombinant cathepsin L significantly improved the developmental competence of bovine oocytes and embryos.</li> </ol>   <ol start="45"> <li>Developed a new oocyte activation approach that can be used for SCNT.</li> </ol>   <ol start="46"> <li>Optimization of stallion sperm capacitating conditions for equine in vitro fertilization</li> </ol>   <ol start="47"> <li>Optimization of cooling conditions for prolonged maintenance of stallion sperm</li> </ol>   <ol start="48"> <li>Reproductive parameters of Florida Native Sheep</li> </ol>   <ol start="49"> <li>Environmental influences on post-ejaculatory sperm function</li> </ol>   <ol start="50"> <li>PPAR-gamma influences developmental competence and trophectoderm lineage specification in bovine embryos</li> </ol>   <ol start="51"> <li>Molecular and cellular programs underlying the development of bovine pre-implantation embryos</li> </ol>   <ol start="52"> <li>High-resolution ribosome fractionation and low-input ribosome profiling of oocytes and preimplantation embryos in both bovine and mouse has enabled us to define the translational landscapes of early embryogenesis.</li> </ol>   <ol start="53"> <li>Single-cell transcriptomic analysis of bovine pre- and peri-implantation embryo development at day 8, 12, 14, 16 and 18, when most pregnancy failure occurs in cattle.</li> </ol>   <ol start="54"> <li>Genetic and pharmacological approaches to modulate NRF2-mediated oxidative stress response in bovine preimplantation embryos</li> </ol>   <ol start="55"> <li>Application of granulosa cells and oviductal organoid derived extracellular vesicles mediated molecular signaling in modulating stress response in bovine granulosa cells and preimplantation embryos</li> </ol>     Objective 2 - Refine methods to produce animals by genetic engineering or genome editing for the improvement of livestock production efficiency and development of human biomedical models.   <ol> <li>CRISPR editing of porcine embryos.</li> </ol>   <ol start="2"> <li>Creation of monoallelic fetuses (at least through day 18) is possible with electroporation of oocytes prior to fertilization.</li> </ol>   <ol start="3"> <li>Identifying the role of thyroid hormone in the regulation and function of the placenta and to learn how maternal thyroid hormone is transported to the fetus.</li> </ol>   <ol start="4"> <li>Identified alternatives methods for producing genetically identical bovine embryos through multiple passaging techniques.</li> </ol>   <ol start="5"> <li>Optimized conditions for knocking our receptors of immune function.</li> </ol>   <ol start="6"> <li>Establishment of bovine trophoblast stem cells (TSCs).</li> </ol>   <ol start="7"> <li>Development of bovine stem cell derived embryos (blastoids).</li> </ol>   <ol start="8"> <li>The laboratory has recently edited the bovine genome to knockout immune cell receptors with a goal of understanding how loss of this receptor impacts colostrum production and immune deficiency in neonates. These strategies will also advance a human biomedical model to better understand roles of this receptor in acquired immunity.</li> </ol>   <ol start="9"> <li>The genetic modification of cattle is a powerful tool for biotechnology. Transgenic cattle that produce human proinsilin and insulin in their milk were produced. Mass spectrometry showed the presence of human insulin more than proinsulin, proteases that could convert proinsulin into insulin. Results suggest that endogenous milk proteases convert recombinant human proinsulin to insulin in milk because mass spectrometry detected human C- peptide. Endogenous milk proteases appear to convert recombinant proinsulin to functional insulin. For the first time, it was demonstrated that mature bioactive human insulin can be produced in cows milk, which could potentially revolutionize how insulin is administered to diabetic patients.</li> </ol>   <ol start="10"> <li>Findings suggest that 0.1–1 mM Sr enhances in vitro osteogenic differentiation of pASCs. Future studies are warranted to determine effects of Sr on in vivo osteogenic differentiation of pASCs.</li> </ol>   <ol start="11"> <li>Platlet rich-plasma (PRP) was determined to be a media supplement with similar effects as FBS, potentially making it a suitable substitute for in vitro expansion of ASC populations. These results are likely partly explained by similarities in growth factor concentrations and their effects. Further characterization of PRP will be necessary to tease out the specific growth factor/s responsible for the increase in swine ASC migration.</li> </ol>   <ol start="12"> <li>Using calcium phosphate resulted in a fibrin scaffold that coagulated faster (p = 0.022), had a rougher surface, higher stiffness, and desirable properties for practical use during surgical operations and scaffolds used in bone tissue engineering. Further, osteogenic differentiation was enhanced on scaffolds treated with calcium phosphate. In addition, fibrin scaffolds treated with RBC lysis buffer were also stiffer than untreated controls. These results are partly explained by ASC attachment and fibrin polymerization during</li> </ol>   <ol start="13"> <li>Preliminary research indicates that patch repair of intestinal perforations is likely inferior to sutures alone, although repair with the patch outside the bowel wall cannot be completely excluded because of the high variability of the Further research may try alternate patch types or different variations to find a non-inferior closure method.</li> </ol>   <ol start="14"> <li>A topology-optimized PCL cage with BMP-2 is capable of resulting in an intervertebral fusion, similar to a conventional ring-based design of the same bioresorbable material.</li> </ol>   <ol start="15"> <li>The RapidVent Emergency Ventilator withstands continuous use over an extended period and allows for the control of physiological parameters of the pig. Weight added to the ribs of the animal may be a viable model for a viable model for labored breathing with more evidence.</li> </ol>   <ol start="16"> <li>Successfully produce a novel Gal KO sheep model lacking the Gal antigen and expressing cytotoxic anti-Gal antibody by 2 - 3 months of age increasing to clinically relevant levels by 6 months.</li> </ol>   <ol start="17"> <li>TAMU broke ground on new Animal Reproductive Biotechnology Center in November. This center is focused on gene editing in ruminants and will be used to improve reproductive efficiency in livestock species and their development as biomedical models for human research.</li> </ol>   <ol start="18"> <li>Development of mouse models with liver-specific Cas9 expression combined with liver-specific mutations in redox system genes.</li> </ol>   <ol start="19"> <li>Development of AAV8-sgRNA vectors that target key genes encoding redox system genes, metabolic genes, cancer genes, transporters, and others.</li> </ol>   <ol start="20"> <li>Validation of AAV8-sgRNA-mediated CRISPR / Cas9 gene disruptions in mouse livers by endonuclease-7 assay.</li> </ol>   <ol start="21"> <li>Detection of off-targeting events in genome edited pigs.</li> </ol>

Publications

Publications <ol> <li>Candelaria JI, Denicol AC. Assessment of ovarian tissue and follicular integrity after cryopreservation via slow freezing or vitrification followed by in-vitro culture. Fert & Steril Science. 2023 (in press).</li> <li>Castro B, Candelaria JI, Austin MM, Shuster CB, Gifford CA, Denicol AC, Hernandez Gifford JA. Low-dose lipopolysaccharide exposure during oocyte maturation disrupts early bovine embryonic development. Theriogenology. 2024. 15;214:57-65.</li> <li>Jara TC, Park K, Vahmani P, Van Eenennaam AL, Smith LR, Denicol AC. Stem cell-based strategies and challenges for production of cultivated meat. Nat Food. 2023 Oct;4(10):841-853.</li> <li>Denicol AC, Siqueira LGB. Maternal contributions to pregnancy success: from gamete quality to uterine environment. Anim Reprod. 2023 Sep 8;20(2):e20230085.</li> <li>Botigelli RC, Guiltinan C, Arcanjo RB, Denicol AC. In vitro gametogenesis from embryonic stem cells in livestock species: recent advances, opportunities and challenges to overcome. J of Animal Sci 2023. 3:101:skad137.</li> <li>Morton AJ, Candelaria JI, McDonnell SP, Denicol AC. Review: Roles of follicle-stimulating hormone in preantral folliculogenesis of domestic animals: what can we learn from model species and where do we go from here? Animal 2023. Suppl 1:100743.</li> <li>Camargo LSA, Saraiva NZ, Oliveira CS, Carmickle A, Lemos DR, Siqueira LGB, Denicol AC. Perspectives of gene editing for cattle farming in tropical and subtropical regions. Anim Reprod. 2023 19(4):e20220108.</li> <li>Johnson, A. K., F. K. Hollinshead, T. Berger, R. F. Cotterman, C. J. Caruso, and A. J. Conley. 2023. Anti-Mullerian hormone and inhibin-B concentrations vary cyclically in nonovulating queens within reference ranges established for determining gonadal status in cats. J Am Vet Med Assoc 261(12):1796-1803. doi: 10.2460/javma.23.06.0320</li> <li>Kang, S., and T. Berger. 2023. Macrophages in juvenile pig testis: Link with increase in Sertoli cells induced by oestradiol suppression. Reprod Domest Anim 58(4):564-568. doi: 10.1111/rda.14322</li> <li>Zacanti, K., I. Park, B. R. McNabb, T. M. Urbano, E. A. Maga, B. J. Nitta-Oda, J. D. Rowe, S. L. Hennig, P. Ross, and T. Berger. 2023. Gender disparity in survival of early porcine fetuses due to altered androgen receptor or associated U2 spliceosome component. Sci Rep 13(1):15072. doi: 10.1038/s41598-023-41665-6</li> <li>Omar A, Puma LL, Whitcomb L, Risk B, Witt A, Bruemmer J, Winger Q, Bouma G, Chicco A. High-fat diet during pregnancy promotes fetal skeletal muscle fatty acid oxidation and insulin resistance in an ovine model. Accepted. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology. Am J Physiol Regul Integr Comp Physiol. 2023 Nov 1;325(5):R523-R533.</li> <li>Hord TK, Tanner AR, Kennedy VC, Lynch CS, Winger QA, Rozance PJ, Anthony RV. Impact of Chorionic Somatomammotropin In Vivo RNA Interference Phenotype on Uteroplacental Expression of the IGF Axis. Life (Basel). 2023 May 26;13(6):1261.</li> <li>Menjivar NG, Gad A, Thompson RE, Meyers MA, Hollinshead FK, Tesfaye D. Bovine oviductal organoids: a multi-omics approach to capture the cellular and extracellular molecular response of the oviduct to heat stress. BMC Genomics 2023; 24(1):646.</li> <li>Gad A, Joyce K, Menjivar NG, Heredia D, Rojas CS, Tesfaye D, Gonella-Diaza A. Extracellular vesicle-microRNAs mediated response of bovine ovaries to seasonal environmental changes. J Ovarian Res. 2023 May 23;16(1):101.</li> <li>Menjivar NG, Gad A, Gebremedhn S, Ghosh S, Tesfaye D. Granulosa cell-derived extracellular vesicles mitigate the detrimental impact of thermal stress on bovine oocytes and embryos. Frontiers in Cell and Developmental Biology; 11.</li> <li>Gebremedhn S, Gad A, Ishak GM, Menjivar NG, Gastal MO, Feugang JM, Prochazka R, Tesfaye D, Gastal EL. Dynamics of extracellular vesicle-coupled microRNAs in equine follicular fluid associated with follicle selection and ovulation. Mol. Hum. Reprod. 2023.</li> <li>Jessica Kurzella, Dennis Miskel, Franca Rings, Ernst Tholen, Dawit Tesfaye, Karl Schellander, Dessie Salilew-Wondim, Eva Held-Hoelker, Christine Große-Brinkhaus, Michael Hoelker: (2023): Mitochondrial bioenergetic profiles of warmed bovine blastocysts are typically altered after cryopreservation by slow freezing and vitrification. Theriogenology 214, 21-32.</li> <li>Dlamini NH, Nguyen T, Gad A, Tesfaye D, Liao SF, Willard ST, Ryan PL, Feugang JM. 2023. Characterization of Extracellular Vesicle-Coupled miRNA Profiles in Seminal Plasma of Boars with Divergent Semen Quality Status. Int J Mol Sci. 24(4): 3194. (IF:6.208 Cit: 0)</li> <li>Rabaglino MB, Salilew-Wondim D, Zolini A, Tesfaye D, Hoelker M, Lonergan P, Hansen PJ. 2023. Machine-learning methods applied to integrated transcriptomic data from bovine blastocysts and elongating conceptuses to identify genes predictive of embryonic competence. FASEB J. 37(3)</li> <li>Acevedo C, Barfield JP. 2023. Reproductive physiology of bison and application of assisted reproductive technologies to their conservation. Animal, 17:100842.</li> <li>Pasquariello R, Zhang M, Herrick JR, Ermisch A, Becker J, Schoolcraft WB, Barfield JP, Yuan Y, Krisher RL. 2023. Lipid Enriched Reduced Nutrient Culture Medium Improves Bovine Blastocyst Formation. Reprod Fertil. 2023 Nov 1:RAF-23-0057. doi: 10.1530/RAF-23-0057. Epub ahead of print. PMID: 37971749.</li> <li>Zhu J, He X, Bernard, D, Shen J, Su Y, Wolek, A, Issacs B, Mishra N, *Tian, X, *Garmendia A, *Tang, Y. 2023. Identification of New Compounds against PRRSV Infection by Directly Targeting CD163. Journal of Virology eooo5423, published on May 3, 2023, doi: 10.1128/jvi.00054-23</li> <li>Losano JDA, Souders C, Martyniuk C, Daigneault BW. Characterization of bioenergetic and kinematic plasticity of bovine sperm reveals novel and dynamic temporal traits. Reproduction. 2023 Jul 3;166(2):135-147. doi: 10.1530/REP-23-0095. Print 2023 Aug 1.</li> <li>Losano JDA, Daigneault BW. Pharmacological perturbation of peroxisome-proliferator-activated receptor gamma alters motility and mitochondrial function of bovine sperm. Andrology. 2023 Jan;11(1):155-166. doi: 10.1111/andr.13308. Epub 2022 Oct 20.</li> <li>Molecular and cellular programs underlying the development of bovine pre-implantation embryos. Jiang Z#. Reproduction, Fertility and Development. 2023 September 21. PMID:38064195.</li> <li>Mouse trophoblast cells have attenuated responses to TNFα and IFNγ and can avoid synergic cytotoxicity of the two cytokines. Fendereski M, Ming H, Jiang Z, Guo Y. Journal of Immunology. 2023 Dec 8. PMID: 38054905</li> <li>Iyyappan R, Aleshkina D, Ming H, Dvoran M, Kakavand K, Jansova D, Del Llano E, Gahurova L, Bruce AW, Masek T, Pospisek M, Horvat F, Kubelka M, Jiang Z, Susor A. The translational oscillation in oocyte and early embryo development. Nucleic Acids Research. 2023 Nov 10. PMID: 37950888.</li> <li>Hoorn QA, Rabaglino MB, Maia TS, Sagheer M, Fuego D, Jiang Z, Hansen PJ. Transcriptomic profiling of the bovine endosalpinx and endometrium to identify putative embryokines. Phyiol Genomics. 2023 Nov 1. PMID: 37720990.</li> <li>Zhu L, Tillquist N, Scatolin G, Gately R, Kawaida M, Reiter A, Reed S, Zinn S, Govoni KE, Jiang Z#. Maternal restricted- and over-feeding during gestation perturbs offspring sperm epigenome in sheep. Reproduction. 2023 Aug 1. PMID: 37647207.</li> <li>Stanton DL, Graf A, Maia TS, Blum H, Jiang Z, Hansen PJ. Absence of a molecular circadian clock in the preimplantation embryo is a conserved characteristic in the mammal. Reproduction. 2023 Jul 31;166(3):199-207. PMID: 37387479.</li> <li>Scatolin GN, Ming H, Wang Y, Zhu L, Castillo EG, Bondioli K, Jiang Z#. Single-cell transcriptional landscapes of bovine peri-implantation development. bioRxiv. 2023 Jun 14:2023.06.13.544813. PMID: 37398069</li> <li>Ming H, Zhang M, Rajput S, Logsdon D, Zhu L, Schoolcraft WB, Krisher R, Jiang Z#, Yuan Y#. In vitro culture alters cell lineage composition and cellular metabolism of bovine blastocyst. bioRxiv. 2023 Jun 11:2023.06.09.544379. PMID: 37333292.</li> <li>Wang Y, Yu L, Li J, Zhu L, Ming H, Pinzon-Arteaga C, Sun H, Wu J, Jiang Z#. Establishment of bovine trophoblast stem cells. Cell Reports. 2023 May 30. PMID: 37146606.</li> <li>Pinzon-Arteaga C, Wang Y, Wei Y, Li L, Orsi A, Scatolin G, Liu L, Sakurai M, Ye J, Yu L, Li B, Jiang Z#, Wu J#. Bovine blastocyst like structures derived from stem cell cultures. Cell Stem Cell. 2023 May 4. PMID: 37146582.</li> <li>Ho, N.C., Hollister, S., Agrawal, V., Flanagan, C. Lee, C., Wheeler, M.B., Wang,</li> <li>, Ebramzadeh, E., Sangiorgio, S. (2023) Evaluation of topology optimization using 3D printing for bioresorbable fusion cages: A biomechanical study in a porcine model.</li> <li>Spine, online September 20, 2022 - Volume - Issue - 10.1097/BRS.0000000000004491, doi: 10.1097/BRS.0000000000004491</li> <li>Hojati, A., Policarpio, J., Wheeler, M.B., Rowitz, B. 2023. Repair of Bowel</li> <li>Perforation: Current State of the Art. J Regenerative Medicine & Biology Res. 2023;4(1):1-6. https://doi.org/10.46889/JRMBR.2023.</li> <li>Maki, A.J., Rabel, R.A.C., and Wheeler, M.B. (2023). In Vitro Migration of Porcine Adipose- Derived Stem Cells in Platelet-Rich Plasma. International Journal of Regenerative Medicine Volume 6(1): 2-9 doi: 10.31487/j.RGM.2023.01.01</li> <li>Hojati A., Policarpio, J., Yerrabelli, R.S., Wheeler, M.B., Rowitz, B. (2023). Patch Repair for Intestinal Perforation Closure in an Ex-Vivo Porcine Model with BurstPressure Characterization . J Reg Med Biol Res. 2023;4(1):1-5. DOI:10.46889/JRMBR.2023.4106</li> <li>Marchioretto, P., Allen, C.A., Rabel, R.A.C., Ole-Neselle, M.M.B., Wheeler, M.B. (2023). Development of Genetically Improved Tropical-Adapted Dairy</li> <li>Cattle" Animal Frontiers. Volume 13, Issue 5, October 2023, Pages 7–15, https://doi.org/10.1093/af/vfad050</li> <li>Maki, A.J., Rabel, R.A.C., and Wheeler, M.B. (2023). Calcium Phosphate</li> <li>Treatment Enhances Osteogenic Differentiation of Porcine Adipose-Derived Stem Cells on Fibrin Scaffolds. International Journal of Regenerative Medicine 6:2, 2-10. http://dx.doi.org/10.31487/j.RGM.2023.02.01</li> <li>Rabel, R.A.C.; Marchioretto, P.V.; Bangert, E.A.; Wilson, K.; Milner, D.J.; Wheeler, M.B. Pre-Implantation Bovine Embryo Evaluation—From Optics to Omics and Beyond. Animals 2023, 13, 2102. https://doi.org/10.3390/ani13132102</li> <li>Baloch AR, Feugang JM, Rodríguez-Osorio N. Genomic and epigenomic applications in animal and veterinary sciences. Frontiers in Veterinary Science. 2023 Mar 20;10:1167079. https://doi.org/10.3389/fvets.2023.1167079.</li> <li>Dlamini NH, Nguyen T, Sheng Q, Vance C, Willard ST, Ryan PL, Gad A, Tesfaye D, Liao ST, Feugang JM. 60 Molecular Characterization of Boar Semen in Relation to Sperm Quality. J Anim Sci. 2023 Nov 1;101(Supplement_2):31-2. https://doi.org/10.1093/jas/skad341.035</li> <li>Hasan MS, Wang Y, Feugang JM, Zhou H, Liao SF. RNA sequencing analysis revealed differentially expressed genes and their functional annotation in porcine longissimus dorsi muscle affected by dietary lysine restriction. Frontiers in Veterinary Science. 2023;10. https://doi.org/10.3389/fvets.2023.1233292.</li> <li>Xu, D., B. Wan, K. Qiu, Y. Wang, X. Zhang, N. Jiao, E. Yan, J. Wu, R. Yu, S. Gao, M. Du, C. Liu, M. Li, G. Fan, J. Yin. (2023). Single-Cell RNA-Sequencing Provides Insight into Skeletal Muscle Evolution during the Selection of Muscle Characteristics. Advanced Science, e2305080.</li> <li>Santo, M.M., T.C. Costa, W. Silva, L.Z. Pistillo, D. V. Teixeira Junior, L.L. Verardo, P.V.R. Paulino, C.B.Sampaio, M.P. Gionbelli, M. Du, and M.S. Duarte. (2023). Nutrient supplementation of beef female calves at pre-weaning enhances the commitment of fibro-adipogenic progenitor cells to preadipocytes. Meat Science. 204: 109286.</li> <li>Du, M. (2023). Prenatal development of muscle, adipose and connective tissues, and its impact on meat quality. Meat and Muscle Biology. 7: 16230.</li> <li>Liu, X., L. Zhao, Y. Chen, J.S. Son, S. A. Chae, Q. Tian, Y. Gao, J. M. de Avila, M. J. Zhu, and M. Du.* (2023). AMP-activated protein kinase inhibition in fibro-adipogenic progenitors impairs muscle regeneration and increases fibrosis. Journal of Cachexia, Sarcopenia and Muscle. 14: 479-492.</li> <li>Zhao, L. X. Liu, N. A. Gomez, Y. Gao, J. S. Son, S. A. Chae, M. J. Zhu, and M. Du.* (2023). Stage-specific nutritional management and developmental programming to optimize meat production. Journal of Animal Science and Technology. 14: 2.</li> <li>Keim, J., Liu, Y., Regouski, M., Stott, R. D., Singina, G. N., White, K. L., & Polejaeva, I.* (2023) Cytokine supplemented maturation medium improved development to term following somatic cell nuclear transfer (SCNT) in cattle. Reproduction, Fertility and Development, 35, 575-588.</li> <li>Kerschner J.L., A. Paranjapye, M. Schacht, F. Meckler, F. Huang, G. Bebek, A. Van Wettere, M. Regouski, I. Viotti Perisse, K.L. White, I.A. Polejaeva, S-H Leir, A. Harris* (2023). Transcriptomic analysis of lung development in wildtype and CFTR-/- sheep suggests an early inflammatory signature in the CF distal lung. Functional & Integrative Genomics 23(2), 135.</li> <li>McGregor C.G.A.*, G.W. Byrne, Z. Fan, C.J. Davies and I.A. Polejaeva* (2023) Genetically Engineered Sheep: a New Paradigm for Future Preclinical Testing of Biological Heart Valves. The Journal of Thoracic and Cardiovascular Surgery. https://doi.org/10.1016/j.jtcvs.2023.02.007</li> <li>Van Wettere A.J., S-H Leir, C.U. Cotton, M. Regouski, I. Viotti Perisse, J.L. Kerschner, A. Paranjapye, Z. Fan, Y. Liu, M. Schacht, K.L. White, I.A. Polejaeva*, Ann Harris* (2023) Early developmental phenotypes in the cystic fibrosis sheep model. FASEB Bioadv 2023, 5: 13-26.</li> <li>Sellmer Ramos, J.G.N. Moraes, M. O. Caldeira, S. E. Poock, T. E. Spencer, and M. C. Lucy. 2023. Impact of postpartum metritis on the reestablishment of endometrial glands in dairy cows. Dairy Sci. Communications. doi: 10.3168/jdsc.2022-0338.</li> <li>White, B.R., R.A. Cederberg, D.H. Elsken, C.E. Ross, C.A. Lents and A.T. Desaulniers. 2023. Role of gonadotropin-releasing hormone-II and its receptor in swine reproduction. Mol. Reprod. Dev. 90:469–479.</li> <li>Desaulniers, A.T., and B.R. White. 2023. Role of gonadotropin-releasing hormone 2 (GnRH2) and its receptor in human reproductive cancers. Front. Endocrinol. (Accepted).</li> <li>DeBlasi, J. M., A. Falzone, S. Caldwell, N. Prieto-Farigua, J. R. Prigge, E. E. Schmidt, I. I. C. Chio, F. A. Karreth, and G. M. DeNicola. 2023. Distinct Nrf2 signaling thresholds mediate lung tumor initiation and progression. Cancer Research 83(12):1953-1967.</li> <li>Karpova, Y., D. J. Orlicky, E. E. Schmidt, and A. V. Tulin. 2023a. Disrupting Poly (ADP-ribosyl) ating Pathway Creates Premalignant Conditions in Mammalian Liver. International journal of molecular sciences 24(24):17205.</li> <li>Karpova, Y., D. J. Orlicky, E. E. Schmidt, and A. V. Tulin. 2023b. Disrupting Poly(ADP-ribosyl)ating Pathway Creates Premalignant Conditions in Mammalian Liver. Int J Mol Sci 24(24)doi: 10.3390/ijms242417205</li> <li>Martí-Andrés, P., S. Pérez, S. Rius-Pérez, I. Torres-Cuevas, H. Colino-Lage, D. Guerrero-Gómez, E. Morato, A. Marina, P. Michalska, and R. León. 2023. TRP14 is the cellular cystine reductase and also reduces cysteinylated proteins. Free Radical Biology and Medicine 201:15.</li> <li>Schmidt, E., C. Miller, S. Austad, Z. Seaford, and R. Noyd. 2023. Rewired Sulfur Amino Acid Metabolism Provides Thiols Supporting Cell Survival in the Absence of Disulfide Reducing Power. Free Radical Biology and Medicine 208:S31-S32.</li> <li>Shearn, C. T., A. L. Anderson, M. W. Devereux, D. J. Orlicky, C. Michel, D. R. Petersen, C. G. Miller, S. Harpavat, E. E. Schmidt, and R. J. Sokol. 2022. The autophagic protein p62 is a target of reactive aldehydes in human and murine cholestatic liver disease. Plos one 17(11):e0276879.</li> <li>Shearn, C. T., A. L. Anderson, C. G. Miller, R. C. Noyd, M. W. Devereaux, N. Balasubramaniyan, D. J. Orlicky, E. E. Schmidt, and R. J. Sokol. 2023. Thioredoxin reductase 1 regulates hepatic inflammation and macrophage activation during acute cholestatic liver injury. Hepatology Communications 7(1)</li> <li>Stancill, J., P. Hansen, A. Mathison, E. Schmidt, and J. Corbett. 2022a. Thioredoxin reductase 1 is required for maintenance of pancreatic beta-cell function and identity. Free Radical Biology and Medicine 192:136-137.</li> <li>Stancill, J., P. Hansen, E. Schmidt, and J. Corbett. 2022b. Chronic thioredoxin reductase deficiency disrupts redox homeostasis and impairs beta-cell function. In: FREE RADICAL BIOLOGY AND MEDICINE. p 67-68.</li> <li>Stancill, J. S., P. A. Hansen, A. J. Mathison, E. E. Schmidt, and J. A. Corbett. 2022c. Deletion of thioredoxin reductase disrupts redox homeostasis and impairs β-cell function. Function 3(4):zqac034.</li> <li>A. Ezz, M. Takahashi, R.M. Rivera, A.Z. Balboula. (2023). Cathepsin L regulates oocyte meiosis and early embryonic development, Cell Proliferation, e13526.</li> <li>Jung GI, Londoño-Vásquez D, Park S, Skop AR, Balboula AZ, Schindler K. (2023). An oocyte meiotic midbody cap is required for developmental competence in mice. Nature Communications. 14(1): 7419.</li> <li>Lockhart KN, Drum JN,  Balboula AZ, Spinka CM, Spencer TE, Ortega MS. (2023). Sire modulates developmental kinetics and transcriptome of the bovine embryo. Reproduction. 166(5): 337-348.</li> <li>Ho KT, Balboula AZ, Homma K, Takanari J, Bai H, Kawahara M, Thi Kim Nguyen K, Takahashi M. (2023). Synergistic effect of standardized extract of Asparagus officinalis stem and heat shock on progesterone synthesis with lipid droplets and mitochondrial function in bovine granulosa cells, J Steroid Biochem Mol Biol, 225:106181.</li> </ol>   Books, non-refereed book chapters, proceedings, instructional media, theses/dissertations <ol> <li>Sarah A. Womack, M.S. February 2023, Thesis Title: " Development of a Porcine Model for the Testing of The RapidVent Emergency Ventilator for the Treatment of Covid-19"</li> <li>Paula Marchioretto Viero, M.S. July 2023. Thesis Title: " Development of Tropical-Adapted Herds Facilitated by the Use of Dominant Follicle Removal Technique"</li> <li>Ahern, D.F. 2023. The hypogonadotropic hypogonadism phenotype of KISS1-edited pigs is unchanged after treatment with hormones targeting the HPG axis. M.S. University of Nebraska-Lincoln.</li> <li>Ranjitkar S. 2023. Improving farming efficiency through understanding of embryo development, mycoplasma control and gene PhD thesis. University of Connecticut.</li> <li>Barfield, JP. 2023. How can a baby have 3 parents? In: The Conversation on Biotechnology (Ed Marc Zimmer), Johns Hopkins Press, Baltimore, pp 182-186.</li> </ol> Abstracts <ol> <li>Guertin J, Losano JDA, Callaham J, Daigneault BW. Optimization of cooling conditions for prolonged maintenance of stallion sperm for assisted reproductive technologies. University of Florida, Animal Sciences 9th Annual Graduate Student Research Symposium, 2023.</li> <li>Bishman JA, McGraw MM, Daigneault BW. Embryo complementation as an alternative cloning strategy drives trophoblast stem cell development through multiple passaging. Undergraduate Scholars Symposium, University of Florida, 2023</li> <li>July 19, 2023. Investigating placenta insufficiency, fetal growth and thyroid hormone in sheep using RNA interference. 7th Food Science and Human Nutrition (IFHN-2024) Meeting. Frankfurt, Germany.</li> <li>July 10, 2023. Regulation of Trophoblast Function by Histone Lysine Demethylase and Nuclear Hormone Receptors, Dual Purpose with Dual Benefit. Agriculture and Food Research Initiative, 2023 Annual Animal Reproduction Program Project Director Meeting, Ottawa, ON. Canada.</li> <li>March 2, 2023. Investigating placental insufficiency and fetal growth restriction in sheep using RNAi. University of Florida, Department of Animal Sciences, Gainesville, Florida.</li> <li>“From stress to success: Exploring the role of extracellular vesicle in stress adaptation”. IETS Morula virtual webinar, November 14, 2023.</li> <li>“The impact of heat stress on reproductive performance and potential mitigation strategies” A scientific workshop on Designing innovative livestock breeding strategies in Ethiopia. May 30 – June 2, 2023, in Bahir Dar, Ethiopia.</li> <li>“The negative impact of environmental thermal stress on animal’s reproductive performance and potential mitigation strategies” Guest seminar at Bio- and Emerging Technology Institute, Ministry of Science and Technology, June 12, 2023. Addis Ababa, Ethiopia.</li> <li>2023, Reproduction and ART in bison, International Ruminant Reproduction Symposium, Galway, Ireland.</li> <li>2023, Sanitary controls during collection of oocytes and processing of IVP embryos for international trade: The researcher’s perspective, Health and Safety Advisory Committee, International Embryo Technology Society, Lima, Peru.</li> <li>2023, Updates to IVF embryo staging and grading, Canadian Embryo Transfer Association, Orlando, Florida.</li> <li>Kennedy VC, Lynch CS, Tanner AR, Winger QA, Rozance PJ, Anthony RV.</li> <li>Fetal hypoglycemia induced by placental SLC2A3 RNA interference alters fetal pancreas development and function at mid-gestation. Rocky Mountain Reproductive Sciences Symposium 2023; 16th Annual Conference.</li> <li>Dunn B, Menjivar NG, Gad A, Thompson RE, Meyers MA, Hollinshead FK, Tesfaye D. Generation of physiologically relevant extracellular vesicles from bovine oviductal organoids. Graduate Student Showcase 2023. Fort Collins, CO, USA. (Poster Presentation)</li> <li>Ramírez GG, Gad A, Menjivar NG, Tesfaye D. Go Greens: Phytochemicals against heat stress-associated infertility. Graduate Student Showcase 2023. Fort Collins, CO, USA. (Poster Presentation)</li> <li>Wittenstein J, Gad A, Menjivar N, Tesfaye D. Modulation of embryonic oxidative stress response to mitigate oxidative damage during preimplantation period. Graduate Student Showcase 2023. Fort Collins, CO, USA. (Poster Presentation)</li> <li>Menjivar NG, Gad A, Hessock EA, Tesfaye D. Extracellular vesicle-mediated delivery of stress-associated miRNAs into bovine granulosa cells. Society for the Study of Reproduction 2023; 56th Annual Meeting. Ottawa, Canada. (Poster Presentation)</li> <li>Gad A, Menjivar NG, Hessock E, Tesfaye D. Regulation of miRNA packaging and release via extracellular vesicles in ovarian and oviductal cells in response to heat stress: Interplay between sequence motifs and RNA binding proteins Society for the Study of Reproduction 2023; 56th Annual Meeting. Ottawa, Canada. (Poster Presentation)</li> <li>Thompson RE, Gad A, Meyers MA, Menjivar NG, Tesfaye D, Hollinshead FK. Characterization and loading of extracellular vesicles secreted by bovine oviductal organoids as a first step in developing a novel contraceptive. Theriogenology Annual Conference. Birmingham, AL, USA.</li> <li>Menjivar NG, Gad A, Thompson RE, Meyers MA, Hollinshead FK, Tesfaye D. 068 Bovine Oviductal Organoids: 3-D biomimetic culture system to study the maternal cellular and extracellular response to thermal stress. Animal-science proceedings 2023; 14(3):473. (Poster Presentation)</li> <li>Gad A, Joyce K, Menjivar NG, Heredia D, Rojas CS, Gonella-Diaza A, Tesfaye D. 093 Ovarian follicle extracellular vesicle-microRNAs mediated response of beef cows to environmental heat stress. Animal-science proceedings 2023; 14(3):489. (Poster Presentation)</li> <li>Joyce K, Gad A, Menjivar N, Heredia D, Santos-Rojas C, López-Duarte MC, Venturini M, Tesfaye D, Gonella-Diaza A. 032 Transcriptome response of oocytes to seasonal heat stress in beef cows. Animal-science proceedings 2023; 14(3):450. (Poster Presentation)</li> <li>Menjivar NG, Gad A, Thompson RE, Meyers MA, Hollinshead FK, Tesfaye D. Sorting it out: EV-microRNA-guided response of bovine reproductive tissues to heat stress in vivo versus in vitro. Rocky Mountain Reproductive Sciences Symposium 2023; 16th Annual Conference: Pages 56-57. Fort Collins, CO, USA. (Poster Presentation)</li> <li>Hessock EA, Menjivar NG, Gad A, Tesfaye D. Improving the antioxidant capacity of bovine embryos to enhance viability under oxidative stress condition. Rocky Mountain Reproductive Sciences Symposium 2023; 16th Annual Conference: Pages 50-51. FortCollins, CO, USA. (Poster Presentation)</li> <li>Menjivar N, Gad A, Tesfaye D. Granulosa cell-derived extracellular vesicles alter oocyte function and promote survival under heat stress conditions. CVMBS Research Day 2023: Page 36. Fort Collins, CO, USA. (Oral Presentation)</li> <li>Souza LA, Menjivar NG, Gad A, Silveira da JC, Tesfaye D. Investigating the levels of LHCGR in extracellular vesicles and its relationship with ovarian follicle development in bovine. CVMBS Research Day 2023: Page 18. Fort Collins, CO, USA. (Oral Presentation)</li> <li>Thompson RE, Meyers MA, Menjivar NG, Gad A, Tesfaye D, Hollinshead FK. Bovine oviductal organoids as a biomimetic system to evaluate extracellular vesicles. CVMBS Research Day 2023: Page 23. Fort Collins, CO, USA. (Oral Presentation)</li> <li>Hessock E, Gad A, Menjivar NG, Tesfaye D. Modulation of embryonic oxidative stress response to mitigate oxidative damage during the preimplantation period and beyond. CVMBS Research Day 2023: Page 52. Fort Collins, CO, USA. (Poster Presentation)</li> <li>Xiao-Kim E, Schountz T, Graham JK, Barfield JP. Improving Cryopreservation of Artibeus jamaicensis Bat Spermatozoa, 49th Annual International Embryo Technology Society Conference, Lima, Peru. (Poster Presentation)</li> <li>Acevedo C, Rajput S, Yuan Y, Krisher R, Barfield JP. Growth factor receptors in bison blastocysts and expanded blastocysts, 49th Annual International Embryo Technology Society Conference, Lima, Peru. (Poster Presentation)</li> <li>Acevedo C, Rajput S, Yuan Y, Krisher R, Menjivar N, Barfield JP. Development of a bison-specific embryo culture system through targeted supplementation of media with stage-specific growth factors. Rocky Mountain Reproductive Sciences Symposium 2023; 16th Annual Conference: Page 18. Fort Collins, CO, USA. (Poster Presentation)</li> <li>Rabel R.A.C., Marchioretto P.V., Long S.A., Rodriguez-Zas S., Lindsey B.R., Mathews, G., Milner, D.J., Wheeler M.B. (2023) Dominant follicle removal in Gyr × Holstein crossbreds accelerates development of ovarian follicles. Reproduction, Fertility and Development 35, 226-226. https://doi.org/10.1071/RDv35n2Ab196</li> <li>Monzani, P.S., Sangalli, J.R., Sampaio, R.V., Guemra, S., Zanin, R., Adona, P.R., Berlingieri, M.A., Cunha Filho, L F.C., Mora-Ocampo, I.Y., Pirovani, C P., Meirelles, F.V., Ohashi, O. and Wheeler M.B. (2023). Human proinsulin and insulin production in the milk of transgenic cattle. Reproduction, Fertility and Development 36(2) 231 https://doi.org/10.1071/RDv36n2Ab155</li> <li>Marchioretto, P.V., Rodriguez-Zas, S L., Womack, S.A., Lindsey, B.R., Milner, D.J., Rubessa, M., Wilson, K.C. and Wheeler, M.B. (2023). Effect of dominant follicle removal before ovum pickup in Girolando cattle. Reproduction, Fertility and Development 36(2) 251- 252 https://doi.org/10.1071/RDv36n2Ab193</li> <li>Glassey, J.R., Rabel, R.A.C., Milner, D.J. and Wheeler, M.B. (2023) Strontium enhances in vitro osteogenic differentiation of porcine adipocyte–derived stem cells. Reproduction, Fertility and Development 36(2) 265-266 https://doi.org/10.1071/RDv36n2Ab220</li> <li>Bangert, E., Shipley, C., Rabel, R.A.C., Garrett, E., Milner, D. J., Marchioretto, P.V., Spencer, K., Allen, C., and Wheeler, M.B. (2023). The efficiency of an adapted bovine IVF protocol to produce in vitro-derived embryos from oocytes collected via surgical ovum pickup from live white-tailed deer (Odocoileus virginianus) donors under captivity in central Illinois. Reproduction, Fertility and Development 36(2) 165-166 https://doi.org/10.1071/RDv36n2Ab32</li> <li>Zimmerman, L.A., Bangert, E. A., Rabel, R. A C., Milner, D. J., Marchioretto, P.V., Allen, C and Wheeler, M.B. (2023). The in vitro production of Gyr X Jersey bovine embryos from oocytes collected via ovum pickup for use in the tropics. Reproduction, Fertility and Development 36(2) 155 https://doi.org/10.1071/RDv36n2Ab12</li> <li>Womack, S.A., Bethke, E.B., King, W P., Milner, D.J., Rubessa, M., Marchioretto, P.V., and Wheeler, M.B. (2023). Development of a porcine model for the testing of the RapidVent emergency ventilator for the treatment of COVID-19 infection. Reproduction, Fertility and Development 36(2) 219 https://doi.org/10.1071/RDv36n2Ab132</li> <li>Girka, E., Brewer, A., Sheikh, E., Gartia, M.R. Bondioli, K.R.   Lipid profiling of bovine oocytes matured in vivo and in vitro. Reprod. Fertil. and Dev. 36 (1-2):257.</li> <li>Brewer, A., Girka, E., Pryor, J.H., Bondioli, K.R., Looney, C.R.   Raman micro spectroscopy to characterize secondary structure of zona pellucida proteins of in vitro and in vitro derived bovine embryos.  Reprod. Fertil. and Dev. 36 (1-2):200.</li> <li>Gatenby, L., Brewer, A., Looney, C. Bondioli, K.R.   Embryo transfer of ICSI generated embryos.  Reprod. Fertil. and Dev. 36 (1-2): 224.</li> <li>Dalton, A., Girka, E., Brewer, A. Bondioli, K.   The influence of different cryoprotectants on mitochondrial function in vitrified bovine oocytes.  Reprod. Fertil. and Dev.  36 (1-2): 272.</li> <li>M Thoresen, EH King, S Cornejo, CD Barber, D Vanover, HE Peck, JY Joo, JM Feugang, PJ Santangelo, AR Woolums. Bovine preputial epithelium produces antibodies against Tritrichomonas foetus following aerosol application of synthetic mRNA. World Buiatrics Congress, Cancun Mexico, Accepted 2023.</li> <li>Marhant N, Dlamini N, Kameni S, Feugang J, Rios L. A preliminary study of the effect of dietary cottonseed on the follicular reserve of does artificially infected with Haemonchus contortus. American Society of Animal Sciences, Southern section, Louisville, KY, USA. Accepted, 2023.</li> <li>KM Johnson, SL Kameni, NH Dlamini, SF Liao, JM Feugang. Discriminating highly resilient spermatozoa during long-term chilled storage. American Society of Animal Sciences, Southern section, Louisville, KY, USA. Accepted, 2023.</li> <li>SL Kameni, B Semon, GO Ariumbold, JM Feugang. Exploring the potential of Raman spectroscopy to discriminate boar semen samples during storage. International Embryo Technology Society, Denver, CO, USA. Accepted 2023.</li> <li>N Dlamini, T Nguyen, O Pechanova, T Pechan, J Feugang. P41. Proteomic profiling of Extracellular vesicles in Seminal Plasma with Divergent Quality status. Proceeding of the American Society of Andrology (ASA), Boston, USA, 2023.</li> <li>T Nguyen, NH Dlamini, Q Sheng, LD Chen, P Asmita, CK Vance, ST Willard, PL Ryan, JM Feugang. Q2.09. Semen quality evaluation using Near-Infrared Spectroscopy. J Mississippi Academy of Sciences, 68(1):66-67, 2023.</li> <li>N Dlamini, T Nugyen, S Kundu, ST Willard, PL Ryan, J Feugang. Q2.10. Exploring Seminal Plasma Extracellular Vesicle miRNA in relation to Sperm Quality. J Mississippi Academy of Sciences, 68(1):67, 2023.</li> <li>Notsile H. Dlamini, T. Nguyen, Qinyu Sheng, Carrie K Vance, Scott T Willard, Peter L Ryan, Ahmed Gad, Dawit Tesfaye, Shengfa F Liao, Jean M. Feugang. Molecular characterization of boar semen in relation to sperm quality. American Society for Animal Sciences-Midwest meeting, Madison, Wisconsin, 2023.</li> <li>Perisse, I. V., Adams, M. E., White, K. L., Polejaeva, I. (2023). Precise Plasmid Vector Integration into the Rosa26 Gene of Sheep Fetal Fibroblasts using Integrase Editing. (vol. 32, pp. 1-22). Transgenic Research. https://link.springer.com/article/10.1007/s11248-023-00372-8</li> <li>Davies, C., Peterson, E. K., Brothers, M. J., Thomas, A. J., Rutigliano, H. M., Lee, Y.-M., Polejaeva, I. A. (2023). Rethinking the genetic basis of pregnancy recognition in ruminants: pregnancy in type I interferon receptor (IFNAR2) knockout sheep ISAG 2023 Abstract Book. (pp. 39). International Society for Animal Genetics.</li> <li>Thornton, A. E., Peterson, E., Thomas, A., Regouski, M., Liu, Y., White, K. L., Davies, C. J., Polejaeva, I., Rutigliano, H.* (2023). The role of extracellular vesicles in immunomodulation during bovine pregnancy. Proceedings of the International Embryo Transfer Society Meeting 2023.</li> <li>Adams, L., Liu, Y., B. Durrant, B., Young, C., E. Ruggeri, E., R. Krisher, R., Patrick, T., Polejaeva, I.* (2023). Quantification of mitochondrial DNA copy number in interspecies somatic cell nuclear transfer embryos. (2nd ed., vol. 35, pp. 136-136). Reproduction, Fertility and Development.</li> <li>Blocher, R., Liu, Y., Adams, L., Polejaeva, I.* (2023). Evaluation of mitochondrial quantity and distribution in bovine oocytes matured in cytokine-supplemented medium. (2nd ed., vol. 35, pp. 238-239). Reproduction, Fertility and Development.</li> <li>Culler H, Richey KD, Checura CM. Photobiomodulation during oocyte maturation enhances blastocyst rates but not blastocyst ATP content. Prisma Health Education and Research Institute Showcase 2023; Columbia, SC, USA: 2023: 13.</li> <li>Richey K, Hersom M, Bridges W, Checura CM. Effects of Photobiomodulation on Bovine Oocyte ATP Levels and Nuclear Stage Progression. Prisma Health Education and Research Institute Showcase 2023; Columbia, SC, USA: 2023: 18</li> <li>Richey K, Culler H, Hersom M, Bridges W, Checura CM. Effect of photobiomodulation treatment on ATP concentrations in bovine oocytes during maturation. American Society of Animal Sciences Annual Meeting, Albuquerque, NM, USA: 2023: PSIII-4. https://doi.org/10.1093/jas/skad281.461</li> <li>Kirkman K, Mandel A, Thomason L, Wilhelm C, Richey K, Checura CM. Comparison of Two Commercial Bovine IVF Media. Clemson University 18th Annual Focus on Creative Inquiry Forum, Clemson, SC. 2023: 92.</li> <li>Richey K, Hersom M, Bridges W, Checura CM. Using Photobiomodulation to Improve Bovine Oocyte Maturation. American Society of Animal Sciences Southern Section, Raleigh, NC, USA: 2023: 75. https://doi.org/10.1093/jas/skad068.027</li> <li>Buckmaster, K. L., Melton, C., Lopes, M.J.A., Lamberti G.S.E., Briggs E. A., Lalman D.L., Moraes J.G.N. The Role of Interferon-Stimulated Genes in Early Recognition of Pregnancy in Cattle. 2024 Research Day at the Capitol, Oklahoma State University, March 18-19, 2024, Oklahoma City, Oklahoma, USA. (Abstract submitted).</li> <li>H. McDonald, C. P. Johnson, T. P. Vining, M. D. Major, G.S.E. Lamberti, M.I.A. Lopes, N.F.F. Bonmann, J.G.N. Moraes, and P. A. Beck. Effects of sire selection on puberty and reproduction of offspring selected as replacement heifers. 2024 American Society of Animal Sciences (ASAS) Southern Section Meeting, January 27-30, 2024, Louisville, Kenticky, USA (Abstract submitted).</li> <li>Lamberti G.S., Goldkamp A.K., Lopes, M.J.A., Bonmann, N.F. F., Van Rhijn S., Hagen, D.E., Moraes, JGN. Unraveling the biology of conceptus elongation in cattle. 2024 American Society of Animal Sciences (ASAS) Southern Section Meeting, January 27-30, 2024, Louisville, Kenticky, USA (Abstract submitted).</li> <li>Sellmer Ramos, J. G. N. Moraes, M. O. Caldeira, S. E. Poock, A.L. Patterson, T. E. Spencer, and M. C. Lucy. Metritis is associated with abnormal invasion of uterine glands into the myometrium (adenomyosis). 2023 Dairy Cattle Reproduction Council Annual Meeting, November 14-16, at the Sheraton Salt Lake City Hotel in Salt Lake City, UT.</li> <li>Buckmaster, K. L., Melton, C., Lopes, M.J.A., Lamberti G.S.E., Briggs E. A., Lalman D.L., Moraes J.G.N. The Role of Interferon-Stimulated Genes in Early Recognition of Pregnancy in Cattle. 2023 Undergraduate Research Symposium, Oklahoma State University, April 18, 2023, Stillwater, USA.</li> <li>Allen J.E., Lamberti G.S.E., Lopes M.J.A., Hagen D.E., Moraes J.G.N. Generating bison-cattle hybrids to evaluate parental contributions to embryonic development using in vitro procedures. 2023 Undergraduate Research Symposium, Oklahoma State University, April 18, 2023, Stillwater, USA.</li> <li>Van Rhijn S., Lamberti G.S.E., Lopes M.J.A., Melton C., Moraes J.G.N. Unraveling the Biology of Conceptus Elongation. 2023 Undergraduate Research Symposium, Oklahoma State University, April 18, 2023, Stillwater, USA.</li> <li>Lopes M. J. A., Lamberti G.S.E., Stein D., Moraes J.G.N. First assessment of in vitro embryo production metrics using Angus semen after 29 and 39 years of cryopreservation. 2023 Undergraduate Research Symposium, Oklahoma State University, April 18, 2023, Stillwater, USA.</li> <li>G. N. Moraes, T. Gull, A.C. Ericsson, C. A. Bottoms, S. E. Poock, A. L. Patterson, W. Warren, T. E. Spencer, M. C. Lucy. Uterine disease programing of long-term infertility in dairy cattle. 56th Annual Meeting of the Society for the Study of Reproduction (SSR), July 11-14, 2023, Ottawa, Canada.</li> <li>C.C. Silva, J. G. N. Moraes, I. Sellmer Ramos, M. O. Caldeira, S. E. Poock, T. E. Spencer, and M. C. Lucy. Vaginal and uterine microbiome in metritic versus healthy dairy cows at disease diagnosis (day 7 to 14 postpartum), after clinical cure (day 28 to 35 postpartum) and at mid-lactation (day 80 to 165 postpartum). 11th International Ruminant Reproduction Symposium, May 28th to June 1st, 2023, Galway, Ireland.</li> <li>Sellmer Ramos, J. G. N. Moraes, M. O. Caldeira, S. E. Poock, T. E. Spencer, and M. C. Lucy. Postpartum uterine disease reduces Forkhead box A2, a critical regulator of uterine gland function, within the uterus of lactating dairy cows. 11th International Ruminant Reproduction Symposium, May 28th to June 1st, 2023, Galway, Ireland.</li> <li>O. Caldeira, J. G. N. Moraes, S. E. Poock, T. E. Spencer, and M. C. Lucy. The transcriptome profile of bovine uterine endometrial epithelial cells isolated from cows diagnosed with metritis early postpartum provides evidence for inflammatory memory within the uterus. 11th International Ruminant Reproduction Symposium, May 28th to June 1st, 2023, Galway, Ireland.</li> <li>Lamberti G.S.E., Sadeghi H., Fernandes M.A., Filho, S.Z., Marchizeli, J.C., Knychala, R.M., Lucy M.C., Moraes J.G.N. Effect of breed on oocyte recovery and embryo production following ovum pick up (OPU) and fertility outcomes after transferring fresh in vitro produced embryos. 49th Annual Conference of the International Embryo Technology Society (IETS), January 16-19, 2023, Lima, Peru.</li> <li>Function of MOF and H4K16ac during bovine preimplantation embryogenesis. Tri-Repro symposium, Cornell University, 2023 Apr.</li> <li>Sex-specific Early Development in Bovine Embryos. Summer Research Experience for Undergrads, Cornell University, 2023 summer.</li> <li>Transcriptome Comparison Between Cryopreservation Methods of Bovine Blastocysts, PAG conference, San Diego, 2024 Jan.</li> <li>Characterization of transposon element expression during bovine embryogenesis, TAGC, 2024 March.</li> <li>Polivanov, L.J., J. Shelton, N.E. Nordell, H. Qiu, E.G. Rogan, S.E. Bartelt-Hunt, M. Zahid, B.R. White, S.J. Sillman and A.T. Desaulniers. 2023. Maternal atrazine consumption via drinking water impairs offspring development. Proceedings of the 20th Annual Gilbert S. Greenwald Symposium on Reproductive and Developmental Sciences. November 2-3, 2023. p. 73.</li> <li>Ross, C.E., R.A. Cederberg, A.T. Desaulniers, F.H. Choat, D.H. Elsken, S.G. Kurz, G.A. Mills, C.A. Lents and B.R. White. 2023. Diminished reproductive hormone concentrations in GnRHR-II knockdown gilts during the follicular phase of the estrous cycle may be attributed to reduced GnRHR-II levels in theca cells. Mol. Reprod. Dev. 90:724.</li> <li>Shelton, J., L.J. Polivanov, N.E. Nordell, H. Qiu, E.G. Rogan, S.E. Bartelt-Hunt, M. Zahid, B.R. White, S.J. Sillman and A.T. Desaulniers. 2023. The effect of environmentally relevant atrazine exposure on maternal physiology. Proceedings of the 20th Annual Gilbert S. Greenwald Symposium on Reproductive and Developmental Sciences. November 2-3, 2023. p. 68.</li> <li>Zhao R, Liu J, Wang B, and *Tian X. 2024. Evidence Against the Role of Toll-like Receptors 7/8 in Sex Selection in the Mouse. Poster at the 50th annual meeting of the International Embryo Technology Society, January 10-13, 2023, Denver, Colorado.</li> <li>Salman S, Gungor O, Ranjitkar S, Zhang D, Balsbaugh JL, Liddle J, Zaidi F, Ramamoorthy P, *Tian XC. Metabolomic and Proteome profiles of bovine follicular fluid during the window of in vivo oocytes maturation. Poster at the 50th annual meeting of the International Embryo Technology Society, January 10-13, 2023, Denver, Colorado.</li> </ol> Miscellaneous publications (semi-technical/lay publications) <ol> <li>Ross, C.E., and B.R. White. 2023. Ain’t no lie: Understanding the porcine estrous cycle is the key to ensuring your breeding program is N’Synch. Nebraska Pork Talk, Nebraska Pork Producers Association. (Accepted).</li> <li>US Patent: 11,774,454B2 Title: NON-INVASIVE ANALYSIS OF EMBRYO METABOLITES</li> <li>APPLICATION: Non-invasive sex selection of male and female bovine</li> <li>Jiang Z, Wu J, Wang Y, Pinzon-Arteaga C, Wei Y. Bovine blastocyst like structures (blastoids) and uses thereof. Oct 6, 2022, US Patent No: 63/370,192.</li> <li>Jiang Z, Wang Y. Bovine trophoblast stem cells and uses thereof. Oct 6, 2022. US Patent No: 63/370,068.</li> </ol>

Impact Statements

IMPACTS Objective 1 1. Elucidation of the cell populations in the ovary and the roles of FSH in early folliculogenesis is useful to devise better in vitro systems for follicle development and improves the efficiency of assisted reproduction. 2. Presence of the SLICK1 allele in the prolactin receptor gene makes cows more thermotolerant, but the underlying mechanisms are not well understood. Elucidating the differences in prolactin signaling as a result of this genetic mutation improves our understanding of the mechanisms involved in thermotolerance. 3. Investigating the impact of genetic and pharmacological approaches to modulate NRF2-mediated oxidative stress response in bovine preimplantation embryos will provide a unique opportunity to enhance survival and mitigate the long-term impact of preimplantation period oxidative damage on fetal development and offspring health. Moreover, the study will identify genes and genome regions which can be targets for future interventions to enhance viability of embryos from assisted reproductive technologies. Moreover, the research on extracellular vesicles-mediated molecular signaling in ovarian follicle and oocytes in Dr. Tesfaye lab will facilitate the development of diagnostic markers associated with maternal physiology and embryo developmental competence and future potential therapeutic application of EVs in assisted reproductive technologies. The use of bovine oviduct organoid technology will allow the generation of physiologically relevant EVs as molecular cargo in embryo oviduct communication for future application in ART to enhance in vitro embryo production yield and quality. 4. Changes in metabolites and proteins during the windows of follicle development and oocyte maturation will help guide us improve the in vitro oocyte maturation conditions. 5. While numerous methods have been tested to separate bovine X- and Y-bearing sperm, most of them are either unpractical or have failed in repeatability. We tried to confirm a reported new method and started the question on the value of this sperm separation technology. 6. Naïve pluripotency in the bovine has major application values in agriculture. Their culture condition, however, is very complex. Improving the culture medium and feeders will allow the cells to be more user-friendly for their applications. 7. Equine studies have demonstrated ideal sperm isolation procedures and holding conditions that promote capacitation while preventing precocious acrosome activation. Optimization of such conditions will help to advance the commercialization of equine in vitro fertilization. 8. We have also optimized conditions for maintaining cooled stallion sperm for up to 6 days, which will have an impact on the equine artificial insemination industry with the adoption of new devices that can maintain stallion sperm for longer than 48 hr. 9. Our sheep studies have determined that Florida Native Sheep may be mismanaged and could benefit from redirecting the breeding season to periods of increased day light where reproductive parameters suggest that reproductive efficiency could be increased. 10. Maternal periconception sheep studies indicate that short-term increased caloric dietary intake of dams prior to conception may increase pubertal testosterone rates of singleton rams which may be beneficial for advancing the onset of puberty with impacts on management strategies for increased efficiency. 11. We developed a novel system to study the impacts of environmental stressors on post-ejaculatory sperm function that can be used to advance human infertility research using a bovine model which is not restricted by gamete availability or genome editing. 12. New knowledge on the roles of PPARG in early embryo development suggest that this gene could serve as a potential embryo therapeutic target and dietary intervention strategy to prevent embryo loss by the addition of pharmacological agents that directly target the PPARG receptor. 13. Our work filled a significant knowledge gap in the study of translational regulation over a period of rapid developmental change and provided an extensive database that can be mined for more detailed insights into bovine oocyte and preimplantation development. 14. Our work provides foundational information to discover essential biological pathways underpinning bovine pre- and peri-implantation development and the molecular causes of the early pregnancy failure during this critical period. 15. Dominant follicle removal (DFR) and a shorter interval of OPU following DFR in Bos taurus X Bos indicus cattle increases the number of higher quality cumulus-oocyte-complexes (COCs). This allows for the optimization of in vitro embryo production (IVP) from Bos indicus crossbred cattle. 16. Large numbers of embryos will be needed to improve the dairy cattle genetics in Africa. The efficiency of the present IVF system will enable that large-scale embryo production. 17. Overall, the adapted protocol produced blastocyst and cleavage rates for WTD similar to those achieved when using the protocol on cattle. With continued adjustments, the protocol could be optimized for IVF embryo production in WTD. 18. Time-lapse monitoring and artificial-intelligence-based automated image analysis have the potential for accurate embryo evaluation; however, further research is warranted to innovate economically feasible options for in-field applications. 19. Ramen spectroscopy has the potential of providing valuable information about metabolic functions and protein structure in oocytes and embryos on a single cell basis. This information will be useful for improvement of assisted reproductive technologies utilized for breeding of elite cattle including those resulting for genetic modification. 20. Improved outcomes from bovine intra cytoplasmic sperm injection will provide an additional method for incorporation of gene editing techniques and fertilization with spermatozoa for which there are limited numbers such as with sex sorted sperm. 21. The methods and advanced mathematical models developed in this project will help extend the application of MFA framework to complex mammalian systems. Unraveling the metabolic basis of normal early embryo development will provide significant benefits to human and animal reproductive health. Improved embryonic competency following in vitro production and cryopreservation would stimulate the industry by lowering costs, especially those related to recipient management. 22. Raman spectroscopy provided a spectral profile that effectively identified Good or Poor survival semen upon collection/extension to enable strategic storage for permanently maintained high fertility after artificial insemination. 23. Obesity and other physiological factors affect epigenetic changes in oocytes and sperms, which affect embryonic and fetal development, generating long-term impacts on offspring performance. 24. Improvement in proper meiotic spindle configuration at MII stage in bovine oocytes in FLI-supplemented IVM could have a significant impact on improving development and quality of embryo derived from in vitro production, and animal production. 25. Although low oxygen level does not improve oocyte maturation and embryo development, improved oocyte quality would be potential beneficial for embryo development in vivo. 26. Improvement in the quality of sheep oocytes and embryos in commercial serum-free medium could have a significant impact on improvement of IVP embryos and reduce LOS in the lambs. 27. Promoted cellular reprogramming and post-EGA procession in iSCNT embryo supplemented with donor cell mitochondria and/or demethylase mRNA allow for use of them in endangered species rescue. 28. Understanding the role of the paternal histone epigenome in embryogenesis is critical as we further appreciate how the health, diet, and environment of the sire can program the embryo for implantation and long-term health and development. These studies utilizing mutant mouse sperm that produce embryos with poor developmental competence are important to identify chromatin pathways that regulate the molecular mechanisms in other mammalian species. Identifying the genomic location of altered H3K27ac in sperm with abnormal paternal histones, particularly at chromatin modifying enzymes, genes involved in zygotic gene activation, and cell differentiation provide key insight into how paternal histones regulate early embryogenesis. 29. This body of work shows that photobiomodulation treatment is a promising candidate for improving the bovine in vitro maturation system. 30. Conceptus elongation is a critical period for maternal recognition of pregnancy in cattle. 31. Understanding the normal mechanisms regulating embryonic and placental development is necessary to acquire basic knowledge that can serve as a foundation to diagnose abnormal embryogenesis. The long-term goal of this research is to improve cattle production systems and reduce the impact of reproductive problems in cattle operations. 32. Results from our studies in the bovine uterus help elucidate the long-term impact of postpartum metritis on reproductive performance. Further understanding of the biological processes regulating uterine inflammation, remodeling and repair are necessary to develop alternative strategies for the treatment of uterine diseases to reduce its impact on fertility. 33. Fills a fundamental knowledge gap by establishing protocols to profile an epigenetic regulator MOF and H4K16ac during bovine embryonic development. 34. Identify the underlying mechanisms behind the developmental rate difference between male vs. female embryos. 35. Characterize the functional role of transposon elements during bovine early development. Overall, our results will also provide a unique opportunity to develop a targeted embryo biomarker assessment system for improving animal fertility and reproduction efficiency. 36. As a result of these studies, we acquired a change in fundamental knowledge regarding how the GnRH-II/GnRHR-II system regulates 17ß-estradiol levels and follicular dynamics in porcine females, representing a potential avenue for future reproductive therapies. 37. Together, these data strongly suggest that GnRH-II and its receptor play a crucial role in regulating follicular recruitment and development during the estrous cycle, impacting overall fertility outcomes of gilts. 38. As a result of these experiments, we expect to better understand how the GnRH-II receptor is regulated within extra-pituitary tissues related to reproduction. 39. New pharmacological agents may be developed to manipulate the reproductive axis, leading to enhanced fertility rates and reduced incidences of endocrine disorders impacting reproduction. 40. Data from these experiments could lead to novel swine-specific contraceptive methods to manage feral pig populations. 41. As a result of this project, we expect to see a change in knowledge regarding the mechanisms underlying regulation of ovarian steroidogenesis by GnRH-II and its receptor and how this hormone-receptor complex is involved in determining ovulation rate and subsequently, litter size, in pigs. 42. A genetic test to identify fertile and/or subfertile female gametes could enhance traditional selection procedures and novel pharmacological agents could improve swine production practices. Ultimately, this will lead to a change in condition, increasing profitability and therefore, sustainability for pork producers. 43. Identification and flux quantification of the novel sulfur amino acid-based redox homeostasis mechanisms revealed previously unknown mechanisms cells can use to support redox homeostasis. These are likely to be important in embryonic development, which we plan to investigate in future studies. 44. Manipulating cathepsin L level in oocytes and early embryos is a promising approach to improve the efficiency of IVP technology in cattle. 45. Potentially improve the success of SCNT. Objective 2 1. CRISPR editing of porcine embryos 2. Electroporation of oocytes provides a path to create monoallelic edits as a biomedical porcine model. 3. Thyroid hormone is necessary to support both fetal and placental development during pregnancy. Maternal supply of TH to the fetus early in pregnancy is necessary before the fetus can produce TH itself. The placenta uses thyroid hormone to regulate metabolic processes, which includes using 60-80% of the oxygen and glucose it takes up from the maternal circulation. Regulation of placental function by thyroid hormone during pregnancy is complex with the potential to regulate proliferation, differentiation, hormone production, invasion and angiogenesis. Major gaps exist in our understanding of how T3 and T4 are transported through the placenta to the fetal circulation, and how the placenta uses TH to regulate placenta metabolism and function. The placenta has relatively high DIO3 activity, which inactivates T4. One theory suggests that TTR secreted on the maternal side of the placenta, complexes with T4 and is believed to shuttle protected T4 to the fetus. Therefore, production of TTR by the placenta may be necessary for the delivery of TH to the fetus however, this theory has not been tested in vivo. 4. Impaired placental development and function is an underlying cause of fetal growth restriction. The causation and progression of placental insufficiency is not well understood and requires additional studies that investigate the mechanisms of placental function. Our second objective is to reduce DIO2 protein in trophoblast cells to determine the impact on placenta function. It is our long-term goal to determine the causes behind impaired placental function, and how placental-insufficiency manifests itself in FGR. We will test if impairment of trophoblast expression of either TTR or DIO2 will result in significant placental and fetal growth restriction by mid-gestation, setting the stage for functional placental insufficiency and FGR. Understanding how thyroid hormones are regulated by the placenta and specifically how TTR transports of thyroxin (T4) is critical to understanding placental function during FGR. 5. The modification of a system to generate multiple embryos by passage embryo constructs will help to advance the production of genetically superior animas by avoiding costly cloning procedures. Results obtained from these studies will also impact the human biomedical field of placental research by developing embryo models conducive to trophoblast stem cell lines. 6. The bovine TSCs not only serves a model to study the unique placentation process in the ruminants and early pregnancy failure, but also enables the first generation of blastocyst-like structures (blastoids) from a large livestock species. 7. The bovine blastoids represent a valuable model to study early embryo development and understand the causes of early embryonic loss. Upon further optimization, bovine blastoid technology could lead to the development of new artificial reproductive technologies for cattle breeding, which may enable a paradigm shift in livestock reproduction. 8. For the first time, it was demonstrated that mature bioactive human insulin can be produced in cows milk, which could potentially revolutionize how insulin is administered to diabetic patients. 9. Addition of Sr on tissue engineering scaffolds may increase in vivo osteogenic differentiation of pASCs. 10. Platlet rich-plasma (PRP) has similar effects as FBS, potentially making it a suitable substitute for in vitro expansion of ASC populations. 11. Using calcium phosphate resulted in a fibrin scaffold that coagulated faster, had a rougher surface, higher stiffness, and desirable properties for practical use during surgical operations and scaffolds used in bone tissue engineering. 12. Preliminary research indicates that patch repair of intestinal perforations is likely inferior to sutures alone. 13. A topology-optimized PCL cage with BMP-2 is capable of resulting in an intervertebral fusion, similar to a conventional ring-based design of the same bioresorbable material. This should allow for more rapid spinal fusion with out using ectopic bone or bone substitute. 14. The RapidVent Emergency Ventilator withstands continuous use over an extended period and allows for the control of physiological parameters of the pig. These data have allowed for an EUA from FDA for the RapidVent emerency use for COVID-19 patients. 15. The Gal KO genetically engineered sheep, like humans, produce anti-Gal antibody and mirror current clinical immune discordance, thus, improving translational value of this new model. 16. Refine methods to produce animals by genetic engineering or genome editing for the improvement of livestock production efficiency and development of human biomedical models. 17. Develop a new reproductive biotechnology center with gene editing facilities for ruminant (TAMU Station). 18. The AAV8-CRISPR-Cas9 liver-targeting genome editing systems developed here provide novel tools, assays, and approaches that not only validate and advance the redox systems at hand now, but could also help improve these type of tools for use in livestock. 19. Rapid detection of off-targeting events can assess accuracy of designed genome editing systems.