Animal & Avian Sciences Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/2741
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Item MEDIATION OF CORTICOSTERONE-INDUCED GROWTH HORMONE GENE EXPRESSION IN CHICKEN EMBRYONIC PITUITARY CELLS: IDENTIFICATION OF TRANS-ACTING FACTORS AND A NOVEL PITUITARY CELL TYPE(2024) Liu, Kuan Ling; Porter, Tom E.; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Growth hormone (GH) is responsible for up to 30% of growth in broiler chickens. Somatotrophs, or GH secreting cells, begin to differentiate around embryonic day (e)14, in conjunction with an increase in the primary plasma glucocorticoid (GC) corticosterone (CORT). CORT treatment of e11 chicken embryonic pituitary (CEP) cells induces premature GH secretion. This GC-induced process involves trans-acting factors because the GH gene lacks a canonical GC response element (GRE). In addition to the binding of ETS1 and the GC receptor (GR) to the GC-responsive region (GCRR; 1045/ 964), we hypothesize that there are other regulatory factors necessary for CORT responsiveness. By modifying the pGL3_-1742/+25 GH-luciferase reporter, we have constructed various other GH-luciferase reporters and analyzed them for promoter activity in response to CORT treatment. We identified a putative distal (d) ETS-Like 1 (ELK1) binding site that is necessary. The proximal (p)PIT1 site and pTATA box were also identified to be critical for CORT induction of the GH gene. Interestingly, cloning multiple copies of the extended GCRR (eGCRR; -1067/-900) further increased promoter activity in an additive manner under both basal and CORT treated conditions. Through single-cell RNA sequencing (scRNAseq), 8 members of the ETS family of transcription factors were identified in > 5% of the somatotroph population. Commercial antibodies were validated, and human (h)ETV1, hELF2, hELK3, and hETV6 antibodies were confirmed to recognize their recombinant chicken ortholog and to identify their corresponding protein in e11 CEP cells. Results from chromatin immunoprecipitation quantitative PCR suggest that multiple ETS members are involved in CORT induction of the GH gene with more evidence pointing towards ELF2 and ELK3. Identifying trans-acting factors for the GH gene inducible by CORT allows for better understanding of endogenous GH regulation in chickens. Further analysis of the scRNAseq data from e11 CEP cells revealed a cluster of cells expressing genes for more than one hormone-producing cell type (“premature nebulous” cluster). Within the premature nebulous cluster, a large population (~30%) was co-expressing proopiomelanocortin (POMC) and growth hormone (GH). We named this novel cell population the cortico-somatotrophs. Through RNA fluorescent in-situ hybridization (RNA-FISH) and dual label immunofluorescence, we verified the existence of the cortico-somatotrophs at both the mRNA and protein level, respectively. Cortico-somatotrophs were also shown to share genes for receptors normally specific to both corticotrophs (CRH-R1) and somatotrophs (GHRHR). Additionally, in response to CORT treatment, the cortico somatotrophs showed an increase in GH as well as a decrease in POMC mRNA levels. The discovery of the cortico-somatotrophs suggests a modification to the current dogma on pituitary cell lineages, where corticotrophs and somatotrophs may have overlapping developmental pathways. In conclusion, our discovery of the cortico somatotrophs has furthered our understanding of CEP development and opened the door for further exploration of the cell lineages during pituitary development.Item Phenotypic and Genetic Analysis of Reasons for Disposal in Dairy Cattle(2024) Iqbal, Victoria Audrey; Ma, Li; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Reasons for disposal are defined as why a cow has left the herd during lactation and are documented as termination codes. Dairy cattle termination codes were collected by Dairy Records Processing Centers and stored in the National Cooperator Database maintained by the Council on Dairy Cattle Breeding for analysis. The list of possible termination codes is as follows: code 0 is cow lactation that ended typically without an abortion, code 1 is locomotion problems, code 2 is female transferred or sold, code 3 is low milk yield, code 4 is reproductive problems, code 5 is unspecified reasons, code 6 is death, code 7 is the presence of mastitis, code 8 is abortion, code 9 is udder problems, code A is an unfavorable phenotype, and lastly code B is undesirable temperament. Understanding termination codes is the key to understanding and improving farm management. Unfortunately, the secondary termination codes are not utilized, despite studies saying one reason is too limited. Heifer termination codes should be more utilized, and studies show that this could improve heifer management. The four processing centers' principal termination codes deviated a little from year to year, but processing center D had the most variation in principal termination codes. There were few records with termination codes 9, A, and B. There was low lameness found for Jersey cattle but more fluctuations for their termination codes 6, 7, and 8. Jersey's main reason for disposal was sold and low milk yield. As for Holstein, the main reasons for disposal were low milk production and death. Recommendations include removing termination code 5 (other reasons) and enforcing a secondary termination code for code 2 (sold). Also, including the percentage of animal records used in traits developed at the CDCB was recommended to encourage farmers to add more records to improve breeding selections. Overall, the top main reasons for disposal were low milk yield, death, and reproduction across breeds from 2011 to 2022. To determine whether health traits correlate to termination codes and how health traits change the probability of survival, a multinomial logistic regression was developed, where twelve health traits, breeds, and other factors were used as an independent variable for the termination code, the dependent variable. The output is a regression coefficient list that conveys the effect of each health trait for each termination code. The results show the apparent impacts of animal breeds on different termination codes, such as dairy crossbreeds negatively affecting termination due to reproductive advantages that follow the literature. Lastly, using termination codes as phenotype, this study focuses on developing a genome-wide association study (GWAS) using the Weighted single-step Genomic Best Linear unbiased prediction (WssGBLUP) model to find significant SNPs related to survival in Holstein cows. In summary, this study provided an understanding of reasons for disposal trends, modeled the reasons for disposal, determined the likelihood of termination post-incidence, and found the heritability and important SNPs of each termination code.Item A WIDE SCALE INVESTIGATION INTO LNCRNA IN BOS TAURUS(2023) Marceau, Alexis; Ma, Li; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Although the history of genetic research has focused on genes and gene products, there is an interesting emerging subclass of genetic elements: long noncoding RNAs (lncRNAs). These are portions of the genome that are longer than 200 base pairs in length and are transcribed from DNA to RNA but do not yield a protein. The function of lncRNA is wide reaching and difficult to define; however, they are predominantly linked to the regulation of gene expression. This is done via transcriptional control, translation control, pre- and post- transcriptional and translational control, epigenetic modifications, RNA processing,as well as other methods. In this dissertation, multiple Bos taurus tissues across various life conditions were investigated in order to identify lncRNA and to begin making predictions about the role and function of identified transcripts. First, lncRNA were identified and analyzed in Bos taurus rumen tissue in pre-weaning and post-weaning cattle. lncRNA were implicated in the weaning process and demonstrated enrichment in complex traits, indicating the continued impact rumen-associated lncRNA have on dairy cattle. Following this study, mammary tissues from dry and lactating cattle were used for lncRNA analysis, in relation to the lacta-tion processes. This study revealed both the presence and impact of mammary lncRNA, and identified lncRNA associated with genes and biological processes that are strongly linked to lactation and mammary tissue function. Subsequently, immune system related tissues were analyzed for lncRNA and their roles. This investigation demonstrated lncRNA to be present in all investigated tissues, including transcripts being repeatedly present. Further analysis into identified lncRNA associated transcripts with genes and functions that are crucial to immune response. Finally, a tutorial was created to make lncRNA identification research more easily accessible to future researchers. The findings and creations of this dissertation increase the knowledge base of lncRNA and their role, allowing for further research endeavors and improvements in Bos taurus husbandry.Item The Genetic Architecture of Complex Traits and Diseases in Dairy Cattle(2022) Freebern, Ellen; Ma, Li; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Genetic architecture refers to the number and locations of genes that affect a trait, as well as the magnitude and the relative contributions of their effects. A better understanding of the genetic architecture of complex traits and diseases will be beneficial for analyzing genetic contributions to disease risk and for estimating genetic values of agricultural importance. In particular, genetic and genomic selection in dairy cattle populations has been well established and exploited through genome-wide association studies, sequencing studies, and functional studies. The objective of this dissertation is to understand the genetic architecture of complex traits and apply the understanding to investigate the biological relationship between genetics and diseases in dairy cattle. First, we performed GWAS and fine-mapping analyses on livability and six health traits in Holstein-Friesian cattle. From our analyses, we reported significant associations and candidate genes relevant to cattle health. Second, we evaluated genome-wide diversity in cattle over a period of time by running GWAS and proposed a gene dropping simulation program. From this study, we identified candidate variants under selection that are associated with biological and economically important traits in cattle. Also, we demonstrated that gene dropping is an applicable method to investigate changes in the cattle genome over time. Third, we investigated the effect of maternal age and temperature on recombination rate in cattle. We provided novel results regarding the plasticity of meiotic recombination in cattle. Additionally, we found a positive correlation between environmental temperature at conception and recombination rate in Holstein-Friesian cows. Collectively, these studies advance our understanding of the genetic architecture and the biological relationship between complex traits and diseases in dairy cattle.Item MRP5 AND MRP9 PLAY A CONCERTED ROLE IN MALE REPRODUCTION AND MITOCHONDRIAL FUNCTION(2021) Chambers, Ian George; Hamza, Iqbal; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Heme is an essential iron-containing cofactor in proteins that perform diverse functions in biology. Free heme is not only hydrophobic but also generates cytotoxic peroxide radicals. In eukaryotes, heme synthesis occurs in the mitochondria but must be transported to different intracellular organelles to be utilized by hemoproteins, a process that remains poorly understood. In Caenorhabditis elegans, MRP5/ABCC5 is an essential heme exporter as mrp-5 knockout worms are unviable due to their inability to export nutritional heme from the intestine to extra-intestinal tissues. Heme supplementation restores viability of these mutants but fails to restore male reproductive deficits. By contrast, MRP5 in mammals regulates heme levels in the secretory pathway but shows no reproductive phenotypes. Phylogenetically, the closest homolog of MRP5 in vertebrates is MRP9/ABCC12, which is absent in C. elegans raising the possibility that MRP9 may genetically compensate for MRP5 lossin vertebrates. Here, we show that MRP5 and MRP9 double knockout (DKO) mice are viable but reveal significant male reproductive deficits, reminiscent of mrp-5 worms. Although MRP9 is highly expressed in sperm, MRP9 knockout mice show reproductive phenotypes only when MRP5 is absent. Unlike other ABCC transporters, these proteins localize to mitochondrial-associated membranes (MAMs), dynamic scaffolds that associate the mitochondria and endoplasmic reticulum. Consequently, combined loss of both transporters results in abnormal sperm mitochondria and reduced fertilization rates in DKO mice. Untargeted metabolomics show striking differences in metabolite profiles in the DKO testes, consistent with the localization of these transporters to MAMs where inter-organellar metabolite exchange occurs. RNA-seq results show significant alterations in genes related to mitochondria function and energy production, EIF2 signaling, and retinoic acid metabolism. Targeted functional metabolomics reveal retinoic acid levels are significantly lower in the DKO testes. These findings establish a model in which MRP5 and MRP9 play a concerted role in regulating normal male reproductive functions and mitochondrial sufficiency.Item MODULATING KEY GENES INVOLVED IN PANCREAS FORMATION AND INSULIN SIGNALING USING CRISPR/CAS9 IN THE PIG(2019) Sheets, Timothy P; Telugu, Bhanu P; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Among the metabolic diseases, diabetes remains a “pressing problem” as recognized by World Health Organization, not only due to the impact on individuals’ lives, but also because of the rapid increase in newly diagnosed patients. To better understand the mechanisms of diabetes, this dissertation investigates the role of NGN3 in pancreas development using CRISPR/Cas9 gene targeting in the pig model. NGN3 was selected for study because of its critical role in endocrine pancreas formation. Our research demonstrates that the targeted ablation of NGN3 blocks development of the endocrine pancreas, a finding supported through gene expression analysis. Furthermore, follow-up studies show that clonal piglets derived from NGN3-ablated animals lack the major endocrine islet cell types and subsequent expression of key endocrine hormones. This porcine model provides valuable insights into the study of type 1 diabetes in early post-natal life and future applications of human-to-pig chimeric organ development for transplant surgery. Expanding upon this porcine model for diabetes, we sought to apply this approach to the study of type 2 diabetes using a novel pig model, thus bridging the gap between mouse and human. For this endeavor, we identified GRB10 as a potential critical mediator in insulin signaling, development, and growth potential following an extensive literature review. The potential for dual applications in both agriculture and medicine was also identified as an objective. Analysis of qPCR data from in vitro overexpression studies supports that GRB10 modulates insulin signaling through the canonical insulin pathway. Additional data from two in vivo gene editing trials targeting the GRB10 locus in both Ossabaw and domestic pig breeds show a supportive qualitative trend towards growth regulation in the Ossabaw pig breed. Further evidence is required to determine whether GRB10 plays the same role in the domestic pig, as a limited cohort size of mutants precluded an extensive analysis of phenotypes. Together, our assessment of NGN3 and GRB10 offer significant potential for modeling of both type 1 and type 2 diabetes as well as modeling of growth traits in the pig through application of advanced genome engineering technology.Item Genetic Architecture of Complex Traits and Accuracy of Genomic Selection in Dairy Cattle(2018) Jiang, Jicai; Ma, Li; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Genomic selection has emerged as an effective approach in dairy cattle breeding, in which the key is prediction of genetic merit using dense SNP genotypes, i.e., genomic prediction. To improve the accuracy of genomic prediction, we need better understanding of the genetic architecture of complex traits and more sophisticated statistical modeling. In this dissertation, I developed several computing tools and performed a series of studies to investigate the genetic architecture of complex traits in dairy cattle and to improve genomic prediction models. First, we dissected additive, dominance, and imprinting effects for production, reproduction and health traits in dairy cattle. We found that non-additive effects contributed a non-negligible amount (more for reproduction traits) to the total genetic variance of complex traits in cattle. We also identified a dominant quantitative trait locus (QTL) for milk yield, revealing that detection of QTLs with non-additive effect is possible in genome-wide association studies (GWAS) using a large dataset. Second, we developed a powerful Bayesian method and a fast software tool (BFMAP) for SNP-set association and fine-mapping. We demonstrated that BFMAP achieves a power similar to or higher than existing software tools but is at least a few times faster for association tests. We also showed that BFMAP performs well for fine-mapping and can efficiently integrate fine-mapping with functional enrichment analysis. Third, we performed large-scale GWAS and fine-mapped 35 production, reproduction, and body conformation traits to single-gene resolution. We identified many novel association signals and many promising candidate genes. We also characterized causal effect enrichment patterns for a few functional annotations in dairy cattle genome and showed that our fine-mapping result can be readily used for future functional studies. Fourth, we developed an efficient Bayesian method and a fast computing tool (SSGP) for using functional annotations in genomic prediction. We demonstrated that the method and software have great potential to increase accuracy in genomic prediction and the capability to handle very large data. Collectively, these studies advance our understanding of the genetic architecture of complex traits in dairy cattle and provide fast computing tools for analyzing complex traits and improving genomic prediction.Item DYNAMIC ANALYSIS OF CD4+ T CELL EPIGENETIC STATUS IN CHICKENS FOLLOWING MDV INFECTION AND DURING DIFFERENTIATION IN MICE(2018) Ding, Yi; Song, Jiuzhou; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Epigenetic modifications constitute a complicated regulatory network controlling various biological processes from cell development to immune responses. The mechanisms through which CD4+ T cells react to environmental stimuli, including virus intrusion and differentiation signals, represent the fundamental cell biological question of how the external microenvironment influences intrinsic transcriptional networks. This dissertation investigates the epigenetic status changes in CD4+ T cells induced by Marek’s disease virus (MDV) infection in chickens and during differentiation in mice. First, a genome-wide gene expression analysis in the immune organs from resistant line 63 and susceptible line 72 chickens was performed to explore Marek’s disease (MD) resistance mechanisms. MDV infection influences both cytokine-cytokine receptor interaction and cellular development in resistant and susceptible chickens. Second, we examined the epigenetic status of CD4+ T cells induced by MDV infection, including chromatin accessibility and chromosome organization. Our results revealed extensive epigenetic modification changes caused by MDV infection. Only resistant line 63 chickens could initiate robust adaptive immune responses at the transcription level, and the increase in chromatin accessibility and chromosome reorganization represented by A/B compartment flipping were related to up-regulated genes induced by MDV infection at 10 days post-infection in line 63 chickens. Finally, we investigated CD4+ T cells plasticity during Th1 helper cell differentiation. We showed “early” (48 hours) CD4+ T cells were plastic for cellular reprogramming while “late” (72 hours) cells lost reprogram plasticity and became committed to Th1 cell fate. T-bet, the Th1 cell master regulator, was not the direct determinant of Th1 cell plasticity. Our integrative analysis of multiple “omics” datasets revealed dynamic and genome-wide changes of chromatin accessibility associated with the process of cellular differentiation and commitment. We predicted that several candidate regulators could contribute to cellular plasticity, including Mxi1, JunB, BATF, IRF4, and Hif-1α. We observed that substantial alterations of chromatin interactions occurred at the IRF4 locus across differentiation time. Conditional deletion of IRF4 in CD4+ T cells impacted the expression of T cell activation and differentiation genes, including T-bet, and extended Th1 cell plasticity during the differentiation process. Our findings provided deeper understanding of CD4+ T cell commitment and responses toward viral infection.Item FUNCTIONAL CHARACTERIZATION OF HEME TRANSPORTERS IN ZEBRAFISH(2017) Zhang, Jianbing; Hamza, Iqbal; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Hrg1 and Mrp5 are identified as eukaryotic heme importer and exporter, respectively. Two Hrg1 paralogs have been annotated in zebrafish genome, Hrg1a (Slc48a1b) and Hrg1b (Slc48a1a) with 84% homology in protein sequences. Hrg1a and hrg1b are widely expressed in embryonic and adult zebrafish. Yeast growth assays reveal that zebrafish Hrg1a and Hrg1b are both capable of heme import. However, hrg1a and hrg1b double knockout (hrg1 DKO) zebrafish generated by CRISPR/Cas9 has no overt defects in differentiation and maturation of erythroid cells. Knockdown of hrg1a in hrg1b mutants or vice versa does not impair erythroid lineage in zebrafish embryos. These genetic results suggest that Hrg1 is not required for maturation and hemoglobinization of primitive erythroid cells. Hrg1a and hrg1b mRNA are upregulated in adult kidneys and spleens upon PHZ-induced hemolysis, together with hmox1, a downstream heme degrading enzyme, suggesting that Hrg1 is involved in adult heme-iron recycling during erythrophagcytosis in kidney and spleen of adult zebrafish. DAB-enhanced Perl’s iron staining reveals that iron is accumulated in macrophages in the kidney and spleen in adult wild-type zebrafish. However, macrophages with positive Perl’s staining are rarely found in the kidney of hrg1 DKO and instead large amount of iron is deposited in renal tubules, suggesting defects in heme-iron recycling by kidney macrophages in hrg1 DKO under PHZ-induced hemolysis. Whole transcriptome sequencing of mRNA extracted from spleens and kidneys reveals massive differentially expressed genes in hrg1 DKO involved in immune response, lipid transport, oxidation-reduction process and proteolysis. These indicate that hrg1 DKO are deficient in recycling heme-iron derived from damaged RBCs in the absence of functional Hrg1. Phylogenetic analysis reveals that Mrp5 and Mrp9 are closed homologs in the zebrafish genome. Yeast growth assays reveal that both zebrafish Mrp5 and Mrp9 are capable of heme export. Morpholino knockdown of mrp5 and mrp9 in zebrafish showed severe anemia in developing embryos indicating their involvements in erythropoietic development. Subsequent generation and characterization of mrp5 and mrp9 mutants by CRISPR/Cas9 will further define the function of Mrp5 and Mrp9 during zebrafish development.Item Integrative genomic, epigenetic and metabolomic characterization of beef from grass-fed Angus steers(2016) Carrillo Tabakman, Jose Adrian; Song, Jiuzhou; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Beef constitutes a main component of the American diet and still represent the principal source of protein in many parts of the world. Currently, the meat market is experiencing an important transformation; consumers are increasingly switching from consuming traditional beef to grass-fed beef. People recognized products obtained from grass-fed animals as more natural and healthy. However, the true variations between these two production systems regarding various aspects remain unclear. This dissertation provides information from closely genetically related animals, in order to decrease confounding factors, to explain several confused divergences between grain-fed and grass-fed beef. First, we examined the growth curve, important economic traits and quality carcass characteristics over four consecutive years in grain-fed and grass-fed animals, generating valuable information for management decisions and economic evaluation for grass-fed cattle operations. Second, we performed the first integrated transcriptomic and metabolomic analysis in grass-fed beef, detecting alterations in glucose metabolism, divergences in free fatty acids and carnitine conjugated lipid levels, and altered β-oxidation. Results suggest that grass finished beef could possibly benefit consumer health from having lower total fat content and better lipid profile than grain-fed beef. Regarding animal welfare, grass-fed animals may experience less stress than grain-fed individuals as well. Finally, we contrasted the genome-wide DNA methylation of grass-fed beef against grain-fed beef using the methyl-CpG binding domain sequencing (MBD-Seq) method, identifying 60 differentially methylated regions (DMRs). Most of DMRs were located inside or upstream of genes and displayed increased levels of methylation in grass-fed individuals, implying a global DNA methylation increment in this group. Interestingly, chromosome 14, which has been associated with large effects on ADG, marbling, back fat, ribeye area and hot carcass weight in beef cattle, allocated the largest number of DMRs (12/60). The pathway analysis identified skeletal and muscular system as the preeminent physiological system and function, and recognized carbohydrates metabolism, lipid metabolism and tissue morphology among the highest ranked networks. Therefore, although we recognize some limitations and assume that additional examination is still required, this project provides the first integrative genomic, epigenetic and metabolomics characterization of beef produced under grass-fed regimen.