Project Number: 8042-31440-001-013-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Jun 1, 2020
End Date: Jul 22, 2022
With an economic impact of nearly $700 billion annually, the cattle industry in the U.S. has the greatest economic value among all livestock production systems. Improving production efficiency in the bovine while maintaining good animal health has always been the goal of our laboratory. The ability to selectively remove undesirable production traits or introduce beneficial traits is of even greater interest as unprecedented challenges associated with climate change, increased pollution and water consumption from agriculture, increased resistance to antibiotics, movement of humans and animals that increases exposure to novel disease and projected increased meat consumption due to population increases and diet preference changes in developing countries. A specific long-term and primary approach of our laboratory is to use bioengineering to improve reproductive efficiency. In both dairy and various stages of beef production, only a single sex is desired. Beltsville technology patented in 1980 sorts sperm based on the minor DNA content difference of the X- and Y-bearing sperm. It is only able, however, to distinguish less than 10% of all sperm. Genetic engineering can improve this inefficient technology developed 40 years ago. Selectively editing of a gene or genes within a cell, whose nucleus will then be used towards nuclear transfer (cloning), is currently the most effective approach to specifically modify the bovine genome. Nuclear transfer with cells of “pluripotency” (i.e., can give rise to all three germ layers, as present in the early embryos) would be advantageous because the chromatin is more “zygotic-like” thus more easily reprogrammed. Additionally, the oocyte, which is used to reprogram the donor cell, needs to be properly matured so it contains all necessary reprogramming factors. Only about one third of in vitro matured oocytes, therefore, the establishment of the “gold standard” metabolomic and proteomic profiles of the follicular fluid and granulosa cells of the preovulatory follicles, as well as those of the immature and matured oocytes will provide us reference bases to improve nuclear transfer and the success of genome modification. With prior support from the UCONN-USDA collaborative agreement, we have made tremendous progress in nuclear transfer, reprogramming and bovine pluripotency. Another primary aim of the lab is to improve cattle health while reducing antibiotic use. We propose to conduct animal trials in this new 3-year agreement with the USDA. Major Goals: a) Objective 1: Generate and characterize bovine naïve induced pluripotent stem cells (iPSCs) for use as donor cells to improve nuclear transfer. b) Objective 2: Establish metabolomics of bovine gamete and embryo development. c) Objective 3: Complete characterization of the hormonal profiles, protein/metabolite dynamics of the bovine oocyte in vivo maturation process. d) Objective 4: Determine differential sperm protein expression. e) Objective 5: Genetically engineer the bovine genome for efficient sperm sorting. f) Objective 6: Plant-derived antimicrobials as alternative to antibiotics for prevention and treatment of mycoplasma-induced bovine diseases.
Objective 1: Generate and characterize bovine naïve induced pluripotent stem cells (iPSCs) for use as donor cells to improve nuclear transfer. a. iPSC will be generated through the reprogramming of bovine somatic cells. b. Verify expression of known stem-cell markers and culture conditions for long-term propagation. c. Test candidate iPSCs for differentiation ability and propensity to form teratomas. d. Compare nuclear transfer efficiency and normality of cloned embryos/offspring using iPSCs and differentiated somatic cells for nuclear transfer. Objective 2: Establish metabolomics of bovine gamete and embryo development. a. Investigate developmental pathways, functional genes and novel gene expression. Compare to other species/embryos by assisted biotechnology. b. Generate inner cell mass and trophectoderm of in vivo and in vitro produced blastocysts and compare transcriptomes. c. Investigate metabolome of media from oocytes/embryos and analyze (ABBL). Objective 3: Characterize hormonal profiles, protein/metabolite dynamics of the bovine oocyte in vivo maturation process. a. Proteomics and metabolomics will be conducted in pre-ovulatory bovine follicles. b. Develop new in vitro maturation media based on follicular fluid components investigated (ABBL). Oocytes matured in new medium will be compared to those matured in vivo by staining, fertilization, embryo development, and protein expression/metabolite profiling. Bioinformatics will be conducted (ABBL). c. Compare oocyte maturation and embryo development under low and ambient O2 conditions in epigenetics and metabolomics. Data analysis of metabolomics will be conducted (ABBL). Objective 4: Determine differential sperm protein expression. a. ABBL scientists will train a University of Connecticut graduate student in sperm protein preparation followed by tandem mass spectrometry analysis performed either at ARS or the University. Conduct analyses in collaboration. b. Investigate X- or Y-linked proteins and their expression in bovine sperm by Western blot. c. Identify differential proteins expressed by the X- and Y-bearing sperm and edit these genes in the bovine genome. Genome-edited cells will be used for Objective 5 by nuclear transfer. Objective 5: Genetic engineering of the bovine genome for efficient sperm sorting. a. Additional candidate genes in mice will be identified and tested. b. Test sperm sorting process using a regular cell sorter in the mouse model. c. Hire post-doctoral fellow at the University of Connecticut to conduct bovine nuclear transfer using gene-edited somatic cells/iPSCs from Objective 1. Objective 6: Plant-derived antimicrobials (PDAs) as alternative to antibiotics in cell culture for contamination eradiation and in prevention/treatment of mycoplasma-induced bovine diseases. a. Test gene expression, morphology, viability and functionality of mammalian cells after co-culturing with PDAs. b. Investigate PDA bactericidal vs. bacteriostatic nature against M. bovis. c. In vivo testing of the effectiveness of PDA treatment in cattle. M. bovis will be inoculated in newborn calves fed +/- PDAs. Calf microbiomes will be analyzed by ABBL scientists.