Brief Summary of Minutes of Annual Meeting

OBJECTIVE 1: Understand the biology and underlying mechanisms of gamete development, fertilization, and embryogenesis.

1. Relationships between anti-mullerian hormone (AMH) and first exposure pregnancy rate (PR) to spring or fall breeding, number of lambs born to first lambing, age at first lambing (using lifetime records), and estimated breeding values (EBVs) for number of lambs born (NLB), number of lambs weaned (NLW), maternal weaning weight (MWT), weaning weight (WWT), and maternal index were determined. While AMH did not appear to consistently predict fertility in ewes, higher EBVs (NLB, NLW, Index) were associated with younger ewes and greater prolificacy at first lambing.
2. Pregnancy rate was greater in fall than spring bred Katahdin ewes. Ewes that became pregnant compared with non-pregnant had a greater concentration of AMH regardless of season bred but tended to have a lower concentration of prolactin. The overall lower prolactin in spring suggests a reduction associated with fescue toxins.  Selecting ewes more tolerant to fescue toxins may improve success of out-of-season breeding.
3. The vaginal and fecal microbiota of beef heifers was characterized to explore a relationship between bacterial signatures and fertility. Our study shows that bovine vaginal and fecal microbiome could be used as biomarkers of bovine reproduction.
4. ATAC-seq was used to identify changes in chromatin structure genomewide at the time of bovine genome activation.
5. Increased testicular cortisol decreased Sertoli cell numbers in juvenile boar testis.
6. Addition of specific concentrations of the fatty acid transport inhibitor FABP3-I to oocyte maturation medium reduces lipid accumulation in bovine oocytes and embryos without impairing embryonic development.
7. The DNA methylome of bovine in vivo developed pre-implantation embryos was systematically characterized.
8. Previous confusions of different types of stem cells in the porcine testis were clarified.
9. In vitro follicle development can be paired with in vitro oocyte maturation with specific individual follicles. Using this approach, it was possible to stablish a non-invasive marker for the efficiency of IVFC based on follicle daily growth rate and diameter. In addition, 18 days seems to be the more suitable culture time for caprine early antral follicles (EAFs).
10. Pretreatment with low concentrations (3%) of D2O improved the cleavage rate of bovine vitrified-warmed oocytes, suggesting a potential beneficial effect, whereas deleterious effects were observed using the higher concentrations. Therefore, further studies are required to assess a potential use of D2O to improve oocyte cryotolerance, likely testing different incubation times.
11. Higher numbers of total and viable COCs were aspirated from Holstein lactating cows than heifers. Furthermore, cleavage and blastocyst rates were higher in lactating cows than in the heifers. Conventional semen gave more embryos than did sexed semen in heifers but not in cows. Overall, oocytes from Holstein lactating donors are more suitable for in vitro embryo production than are those from heifer donors.
12. Using NMR, it was confirmed that sugar metabolism is different between sexes. The new information obtained identifies markers that can be used to predict the embryo sex. These results open a new, non-invasive method to effectively evaluate sex of the embryos before transfer.
13. A method for isolation of spermatozoa from the highly viscous alpaca ejaculate was developed.
14. It was confirmed that stimulation with FSH prior to ovum pick up (OPU) results in greater developmental competence compared to non-stimulated OPU or slaughterhouse derived oocytes. RNA seq data identified differentially expressed genes in both the germinal vesicle and metaphase II stages of oocytes retrieved by these methods and could serve as molecular markers of developmental competence.
15. RNA seq analysis identified genes differentially expressed as a result of heat stress at the germinal vesicle (GV) stage and metaphase II (MII) stages of oocyte development. This information provides new insight into the molecular mechanisms of heat stress on bovine oocytes which may help to reveal master regulators controlling oocyte competence.
16. Effects of heat stress on DNA methylation and DNA hydroxymethylation of bovine oocytes at the GV and MII stage. In this study there was no difference between cold and hot months in DNA methylation and DNA hydroxymethylation of GV stage and MII stage oocytes.
17. Using both single cell/embryo reduced representation bisulfite sequencing (RRBS) and single-cell/embryo whole genome bisulfite sequencing (WGBS), stage-specific genome-wide dynamics of DNA methylation was characterized in bovine sperm, immature, in vivo and in vitro matured oocyte as well as in vivo developed early embryo stages. This study provides the first comprehensive analysis of the global dynamics of DNA methylation in bovine gametes and single early embryos. These data will serve as an important reference base for embryos produced by assisted reproduction such as in vitro fertilization and cloning as well as models for human early embryo development. 
18. Metabolites expelled into the medium were accurately monitored and identified from a single blastocyst thru metabolic flux analysis of heavy isotope labeling. This is tremendously lower number of embryos than the hundreds of embryos used in more traditional metabolic approaches. Measurement of mass isotopomer distributions of metabolites, chiefly pyruvate, lactate, citrate and amino acids, followed by correction for natural abundances and metabolic modeling revealed several metabolic insights.
19. CRISPR/Cas 9 gene editing was utilized to knockout pig conceptus IL1B2 expression and the secretion of IL1B2 during the time of conceptus elongation. This study demonstrates that IL1B2 is involved with the rapid transformation of pig conceptuses from spherical to long filamentous form.
20. CRISPR/Cas 9 gene editing was utilized to knockout pig conceptus CYP19A1 gene expression. Estrogen production by CYP19A1-/- conceptuses was inhibited on Day 14 and 17 of pregnancy. Trophoblast elongation and attachment occurred normally in both Day 14 CYP19A1+/+ and CYP19A1-/- conceptuses indicating that conceptus estrogen production was not essential to early development and attachment in the pig. CYP19A1-/- conceptuses maintained functional CL and progesterone production beyond Day 15 and embryo development continued to Day 25 of pregnancy. However, gilts carrying CYP19A1-/- embryos abort before or after Day 30 of pregnancy. Endometrial RNA-Seq analysis indicated that gene expression was altered in recipient gilts containing CYP19A1-/- conceptuses on Day 14 and 17. Co-transfer of IVF wild type conceptuses rescued the pregnancy of CYP19A1-/- conceptuses.
21. CRISPR/Cas 9 gene editing was utilized to knockout pig conceptus PTGS2 gene expression. Results indicate that conceptus PTGS2 expression is not essential for blastocyst development, uterine migration, conceptus elongation, and establishment and maintenance of pregnancy.
22. Serum testosterone concentrations responded differently to hCG treatment in GnRHR-II KD compared to littermate control boars; hCG-stimulated testosterone levels were significantly lower in GnRHR-II KD vs. control males.
23. Treatment with a GnRH analogue that simultaneously agonizes GnRHR-II and antagonizes GnRHR-I revealed that circulating testosterone levels in control males tended to increase above baseline concentrations, whereas testosterone secretion was not stimulated in GnRHR-II KD animals.
24. Concentrations of 10 different gonadal steroid hormones were either significantly lower or tended to be lower in serum from GnRHR-II KD compared to littermate control males.
25. Mass spectrometry analysis of serum from mature GnRHR-II KD and littermate control boars indicated that concentrations of 10 different gonadal steroids did not differ between lines following castration.
26. After castration, concentrations of all 10 gonadal steroids plummeted in control barrows, whereas only DHEA-S, testosterone and androstenedione production were decreased in GnRHR-II KD castrates.
27. Semen analysis of mature males from each line suggested that number of artificial insemination doses and total motility were reduced in GnRHR-II KD boars, whereas total sperm per ejaculate, progressive motility and percentage of rapid cells tended to be reduced.
28. On Day 10 of the estrous cycle, circulating progesterone concentrations were lower in GnRHR-II KD vs. littermate control gilts.
29. Ovulation rate was reduced in GnRHR-II KD compared to control gilts, despite no difference in weight of the ovaries between lines.
30. The active transposition into active chromatin (ATAC-seq) protocol was optimized to allow for mapping of open chromatin regions genome-wide in small numbers of cells. To reveal differences in chromatin status between in vitro fertilized (IVF) and SCNT embryos genome-wide, we have generated high-quality open chromatin maps from stage-specific preimplantation embryos generated from IVF or from SCNT embryos.

OBJECTIVE 2: Refine methods for production of genetically modified animals to improve livestock production efficiency.

1. A method to corroborate genotype/phenotype changes in bovine CRISRP mutated embryos was developed.
2. CRISPR induced SOCS2-KO was optimized for sheep embryos.
3. Embryo biopsy followed by embryo transfer identified issues of mutation chimerism after injection of CRISPR/Cas9 in sheep embryos.
4. Targeting and genome editing constructs for KDM1A and FATP4 were designed, tested in vitro, and used to study their function in placenta in vivo.
5. LIN28A and LIN28B have been knocked down in vivo and tissues collected. RNA and protein have been collected from the trophectoderm samples and knockdown confirmed. Let-7 miRNA levels have been measured and are confirmed to be altered in the knockdown samples and several let-7 target genes have been investigated.
6. Preliminary data targeting FATP4 in the trophectoderm suggest significantly increased fetal muscle and fatty acid oxidation capacity. Several transfers have been completed during the past breeding season to establish pregnancies.
7. Based on preliminary data generated involving KDM1A, a previously submitted NIH/USDA Dual Purpose award was funded (provided by USDA), and is starting January 2019. This 5-year grant proposal focuses on the role of KDM1A in regulating AR and ESR1 signaling in placental development and function, and involves both in vitro and in vivo experimental approaches.
8. We generated lentiviral GFP fluorescent reporters controlled by bovine OCT4 and NANOG full-length enhancers, OCT4 distal enhancer, and OCT4 proximal enhancer regions, respectively. These reporters will help us to identify and enrich the reprogrammed cells during bovine iPSC generation. We also developed a reprogramming system that increases the TRA-1-60 positive colony formation ~50-100x at day 10 of reprogramming for human iPSC induction. The successful development of bovine fluorescent reporters and the highly efficient reprogramming system we developed provided the much needed tools to help identify the bovine-specific events that are under-regulated in reprogramming process compared with human iPSC induction.
9. Naked DNA has been shown to bind naturally to the sperm, a method called sperm-mediated gene transfer (SMGT). In order to determine if the liposome-DNA complex had bound to sperm, real time PCR was used to detect GFP DNA and images of the sperm were analyzed using the Spatial Light Interference Microscopy (SLIM). SLIM confirmed the presence of liposomes on the sperm head and tail.
10. Transgenic cows, one for the production of human proinsulin and another for factor IX of human blood coagulation were produced and are being submitted to induction of hormonal lactation for the evaluation of the expression of recombinant proteins in milk.
11. Tissue-engineered cartilage was successfully produced on 3D-printed bioresorbable scaffolds using an adipose-derived stem cell and chondrocyte co-culture technique. This potentiates co-culture as a solution for several key barriers to a clinically translatable cartilage tissue engineering process.
12. Co-culturing GFP-positive adipose-derived mesenchymal stem cells (ASC) with porcine satellite cells demonstrated enhanced myogenic capability of ASC, as the percentage of labeled myotubes increased compared to mouse co-cultures. Enhancing myogenic potential of ASC through soluble factor treatment or expansion of ASC with innate myogenic capacity should allow for their therapeutic use to regenerate muscle tissue lost to disease or injury.
13. Novel collagen-glycosaminoglycan hydrogel (CG) scaffolds have shown promise for supporting bone and cartilage growth from mesenchymal stem cells. We have shown that there is a differential ability of ASC and BMSC to break down and metabolize the scaffold matrix and may indicate that one cell type may be preferable to the other for repairing osteogenic defects using these scaffolds. Current experiments underway will analyze expression of matrix-degrading enzymes to determine the source of the difference between cell types in scaffold shrinkage during differentiation.
14. Higher concentrations of magnesium can improve nodule formation into osteogenic differentiation in vitro; the 2 mM concentration showed the highest nodule formation compared with the other groups. Low concentrations of copper (0.1 and 1 mM) and selenium (0.1, 0.5, and 1.0 mM) have positive effects on bone formation in vitro. These results showed the value of magnesium, copper and selenium in bone physiology.
15. Monoallelic and biallelic targeted cells were used for cloning by somatic cell nuclear transfer (SCNT), resulting in 55% pregnancies per embryo transfer (30/55 recipients). Our findings demonstrate that site directed insertion of a large transgene construct can be efficiently attained by using CRISPR/Cas9 along with the suppression of the NHEJ-dedicated Ku70 protein.
16. A tissue engineering approach to address craniofacial defects requires a biomaterial that balances macro-scale mechanical strength with the micron-scale features that promote cell expansion and tissue biosynthesis. We report an analytical approach to quantify radial, angular, and depth bone infill from micro-computed tomography data. The collagen-PCL composite showed improved overall infill, and significantly increased radial and angular bone infill versus the PCL cage alone. Bone infill was further enhanced in the composite for defects that penetrated the medullary cavity, suggesting recruitment of marrow-derived cells. These results indicate a multi-scale mineralized collagen-PCL composite offers strategic advantages for regenerative repair of craniofacial bone defects.
17. Pregnancies were established from frozen-thawed and from vitrified-warmed alpaca preimplantation embryos.
18. It was shown that oocytes collected by ovum pick-up (OPU) had decreased mitochondrial density, and increased incidence of irregularly shaped mitochondria as well as decreased vesicle density. FSH oocytes had a decreased density of cortical granules, especially noticeable following maturation. Oocytes recovered by conventional OPU had the poorest developmental rates, beginning with poor cleavage rates. Follicles collected from OPU are a heterogenous population of follicular sizes potentially resulting in a high variability in oocyte competence.
19. Successfully knocked-out 5 Ig genes in goat fetal fibroblasts using CRISPR/Cas9 and SCNT
20. Confirmed that human artificial chromosome (HAC) is functional in goats. HAC was introduced into goat fetal fibroblasts that were consecutively used for cloning. Human IgG were produced in the transchromosomal goats.
21. A CFTR-/- sheep model was produced using CRISPR/Cas9 manipulation of sheep fetal fibroblasts that were subsequently reprogrammed and used to clone the CFTR null animals.
22. A novel genetically modified line of sheep with an inactivated type I interferon receptor (IFNAR) was produced. These sheep: (1) should be more susceptible to viral infections than wildtype sheep and will make an excellent model for studying viral pathogenesis, and the effectiveness of viral vaccines and antivirals; and (2) female, IFNAR deficient sheep should be sterile due to an inability to respond to interferon-tau, the pregnancy recognition factor in ruminants.
23. Immunodeficient pigs were used for human stem cell transplantation. The pigs could support proliferation and differentiation of lineage-specific organoids derived from human. These immunodeficient pigs were produced without SCNT or breeding step.

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