Browsing by Subject "Bovine"
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Item An updated object oriented bovine QTL viewer and genome-wide bovine meta-analysis(2009-05-15) Salih, HanniWaves of bovine genomic data have been produced as a result of the bovine genome sequencing projects. In addition to the massive amounts of genomic sequence, significant annotation including single nucleotide polymorphisms, sequence tagged sites and haplotype blocks have been produced by the Bovine HapMap Project. Furthermore, many agriculturally significant traits in cattle such as milk yield and carcass weight are measured on a quantitative scale and have been genetically mapped as quantitative trait loci (QTL). QTL data can be used to generate another form of bovine annotation linking phenotype to genotype. However, it is impossible for humans to be able to analyze genomic scale data without computer based tools. Bioinformatic tools have been shown to greatly increase productivity and improve efficiency when dealing with large data sets. My dissertation presents an integrated, extensible database that houses SNPs, STSs, haplotypes, and QTL. The database is presented to researchers through a restructured, object oriented Bovine QTL Viewer that displays multiple levels of bovine annotation synergistically. Evaluation of use of the viewer was performed using a survey based approach and measured quantitatively. In addition, the QTL data from the database was used to analyze the frequency of gene ontology (GO) annotations within QTL regions. QTL regions were divided into 8 trait based groups. GO terms were counted within each category of QTL and in non- QTL regions of the genome. Top level GO term frequencies were generated from the counts and these frequencies were compared between QTL and non-QTL portions of the genome. Furthermore, specific sets of GO terms believed to be related to QTL categories were also used to determine if QTL regions were enriched for genes annotated with such GO terms. As a result, we determined that gene density varied significantly across QTL regions and that many QTL categories showed GO term frequency differences that could be related to the trait?s biology. Furthermore, our selected GO term sets were shown to be significantly enriched in some QTL categories.Item Comparison of gene expression in pre-implantation bovine embryos either injected or transfected with a siRNA targeted against E-cadherin(2009-05-15) Hanna, Carol Bailey McCormickThe ability to create transgenic livestock is a tremendous benefit in scientific research for many disciplines including functional genomics, pharmaceutical synthesis and development of enhanced production animals. Transgenes can either be stably or transiently expressed to alter gene function and obtain a specifically engineered phenotype. To create a transgenic bovine embryo, genetically altered somatic cells must be used in somatic cell nucleus transfer, or early 1-cell embryos (zygotes) must be microinjected with plasmid DNA or small interfering RNA (siRNA). Given the cost and skill associated with both methods, a preliminary investigation exploring alternative delivery techniques of siRNA (transient expression) into bovine zygotes with a nonhomologous Cy3 labeled siRNA (Cy3-siRNA) was first performed. It was discovered that zygotes injected with more than 50 Bmol L-1 of Cy3-siRNA fail to form a blastocoel and that, although bovine zygotes are not susceptible to chemical transfection, the trophectoderm cells of the blastocyst are. Based on this information, bovine E-cadherin gene expression was compared in day 9 blastocysts derived from either injected zygotes (day 1) or transfected blastocysts (day 7) with a Cy3 labeled E-cadherin specific siRNA (Cy3-siEcad) to determine 1) if gene suppression in zygotes injected with 25 Bmol L-1 Cy3-siEcad continues during embryo development up to hatching, and 2) if blastocysts transfected at a ratio of 9:6 with GeneJammer? truly experience gene knock down after siRNA transfection capable of maintaining suppression to day 9. Quantitative PCR indicated blastocysts transfected with Cy3-siEcad had a significant 15.3% decrease (P < 0.05) in E-cadherin mRNA at day 9 compared to the injected zygotes. Protein fluorescence analysis from immunocytochemistry of whole mounted day 9 blastocysts revealed injected zygotes accumulated significantly less E-cadherin protein (67.7%) than the transfected blastocysts (P < 0.05). From these data, it can be concluded that although siRNA injection may be capable of knocking down gene expression for the first 7 days of embryonic development, it does not persist to the hatching stage; however, blastocysts transfected at day 7 do express altered gene expression in the trophectoderm which can continue through embryonic hatching events.Item Effect of semen parameters of bovine spermatozoa after using a contemporary collecting receptacle(2007-05) Langdon, Wendee C.; Prien, Samuel D.; Brady, Heidi A.; Johnson, Jay W.Bovine seminal parameters (motility, forward progression, and acrosome reaction) were tested using three different collection methods. To represent a traditional collection method, one method collected into a dry, standard 15 mL conical tube. The other two collection methods utilized the revolutionary technology patented under the trade name BreedMaXtm; one dry BreedMaXtm (designated as BreedMaXtm-) tube was used, and one BreedMaXtm tube as designed was used (designated as BreedMaXtm+). BreedMaXtm was developed with the intention of allowing the breeder to breed with less semen to obtain more breedings from a single ejaculate. The collected data suggest that presence or absence of media in the BreedMaXtm appeared to have little effect on motility and forward progression; yet there was a trend toward delayed acrosome reaction in the BreedMaXtm+. The results of this study suggest that the BreedMaXtm+ maintains semen parameters for longer periods of time as compared to the conventional collection techniques.Item Expression of Candidate Genes for Horn Growth in Early Bovine Development(2011-02-22) Vitanza, Sarah M.Bovine horns develop primarily after birth and the presence or absence of horns is due to a single gene. It has been reported that the horn bud appears in the bovine embryo at d 60 of gestation. Our hypothesis is that the gene that determines the presence of horns is expressed in osteoprogenitor cells of the early fetus and will affect the expression of RUNX2, MSX1, MSX2, and/or TWIST1. To test this hypothesis, bovine fetal samples were collected from commercial females at the Caviness Packing Company in Hereford, Texas. Fetuses ranged from d 28 to d 80 of gestation. A survey of the expression of genes from the region on bovine chromosome 1 known to contain the locus that causes horns (IFNAR1 to SOD1), was conducted using qualitative and quantitative RT-PCR, and in situ hybridization. Genes with known roles in osteogenesis and chrondrogenesis (MSX1, TWIST1, RUNX2 and SOX9) were included as positive controls. With the exception of OLIG1, which was only expressed in the brain, all of the genes investigated were expressed in fetal frontal and parietal bones by qualitative RT-PCR. The level of expression of C21orf59, C21orf66, IL10RB, and SFRS15 increased in the frontal bone of horned samples from d 55 to d 70 of gestation. At d 60 of gestation, a change in the shape of the frontal bone was observed, which has been reported to be the developmental stage when the horn bud appears. At this time point, MSX1, TWIST1, RUNX2 and SOX9 were detected in frontal bone, in cells from the osteoblast lineage, as expected. Furthermore, C21orf59, C21orf62, C21or66 and SFRS15 from the polled interval were localized to developing mesenchyme, osteoblasts and/or osteoclasts of the frontal bone, suggesting that each of these genes has a role in intramembranous bone formation. In addition, gradients of expressed C21orf66 and SFRS15 were detected in developing endochondral bone. There was evidence of an antisense transcript of C21orf66 expressed in the same cell types as the sense transcript. Further characterization of this antisense transcript demonstrated that it covered the entire sense transcript. Based on observed expression in the mesenchyme, rather than just in mature osteoblasts or osteoclasts, C21orf66 and/or its antisense transcript become the most likely candidates for the polled locus.Item Expression of the bovine DNA (cytosine 5) methyltransferase family during preimplantation development and aberrations induced by somatic cell nuclear transfer(Texas A&M University, 2005-02-17) Golding, Michael CameronBovine preimplantation embryos derived from nuclear transfer experiments exhibit a global state of genomic hypermethylation that likely account for the large number of developmental abnormalities observed to date. The central hypotheses of this work is that the genomic hypermethylation and improper epigenetic reprogramming reported in studies of bovine nuclear transfer, are in large part due to abnormal expression and regulation of the DNA methyltransferase proteins. Bovine Dnmt mRNAs display strong sequence homology to those of human and mouse and similar to other species, exist as multiple isoforms. Two of these splice variants, which have been termed Dnmt2γ and Dnmt3a4 represent previously unreported sequence combinations. Investigation of bovine DNA methyltransferase expression in the bovine oocyte and early preimplantation development has revealed an intricate system divergent from observations previously reported in the mouse. Specifically, the somatic version of Dnmt1 along with Dnmt2, 3a and 3b are all expressed during these initial stages of bovine development. Further, real time analyses of the Dnmt transcripts in cloned and in vitro produced embryos reveal significant differences in the mRNA expression levels of Dnmt1 and 2 but not Dnmt3a and 3b suggesting that the de novo methyltransferases may be functioning normally while Dnmt1 and Dnmt2 are aberrantly methylating the genome during a critical time when methylation levels should be receding. Real time PCR analysis of the Dnmt transcripts in fetal and adult tissues has revealed a developmental and tissue specific expression pattern suggesting that proper expression and function of these enzymes is a key element in the process of differentiation. These results are further supported by studies of Dnmt expression in aging bovine fibroblast cultures, which suggest that the Dnmts may play some as yet unidentified role in cellular senescence. Recently, it has been postulated that the cause of abnormal methylation observed in cloned embryos may be due in part to misexpression of the Dnmt1o isoform during preimplantation development. Work presented here raises new and significant hypotheses that must be considered both regarding the cadre of DNA methyltranferases that direct epigenetic programming during normal development and regarding the implication of abnormal DNMT expression in cloned embryos. Bovine preimplantation embryos derived from nuclear transfer experiments exhibit a global state of genomic hypermethylation that likely account for the large number of developmental abnormalities observed to date. The central hypotheses of this work is that the genomic hypermethylation and improper epigenetic reprogramming reported in studies of bovine nuclear transfer, are in large part due to abnormal expression and regulation of the DNA methyltransferase proteins. Bovine Dnmt mRNAs display strong sequence homology to those of human and mouse and similar to other species, exist as multiple isoforms. Two of these splice variants, which have been termed Dnmt2γ and Dnmt3a4 represent previously unreported sequence combinations. Investigation of bovine DNA methyltransferase expression in the bovine oocyte and early preimplantation development has revealed an intricate system divergent from observations previously reported in the mouse. Specifically, the somatic version of Dnmt1 along with Dnmt2, 3a and 3b are all expressed during these initial stages of bovine development. Further, real time analyses of the Dnmt transcripts in cloned and in vitro produced embryos reveal significant differences in the mRNA expression levels of Dnmt1 and 2 but not Dnmt3a and 3b suggesting that the de novo methyltransferases may be functioning normally while Dnmt1 and Dnmt2 are aberrantly methylating the genome during a critical time when methylation levels should be receding. Real time PCR analysis of the Dnmt transcripts in fetal and adult tissues has revealed a developmental and tissue specific expression pattern suggesting that proper expression and function of these enzymes is a key element in the process of differentiation. These results are further supported by studies of Dnmt expression in aging bovine fibroblast cultures, which suggest that the Dnmts may play some as yet unidentified role in cellular senescence. Recently, it has been postulated that the cause of abnormal methylation observed in cloned embryos may be due in part to misexpression of the Dnmt1o isoform during preimplantation development. Work presented here raises new and significant hypotheses that must be considered both regarding the cadre of DNA methyltranferases that direct epigenetic programming during normal development and regarding the implication of abnormal DNMT expression in cloned embryos.Item Fatty Acid Carcass Mapping(2010-01-14) Turk, Stacey N.We hypothesized that subcutaneous (s.c.) adipose tissue would differ in monounsaturated (MUFA) and saturated fatty acid (SFA) composition among different depots throughout a beef carcass. To test this, 50 carcasses from a variety of breed types and backgrounds were sampled. External fat samples were collected from eight different carcass locations: round, sirloin, loin, rib, chuck, brisket, plate and flank. Samples were used to provide information on slip points, fatty acid composition and MUFA:SFA ratios. Lipids were extracted from s.c. adipose tissue by a modified chloroform:methanol procedure, and fatty acid composition and slip points were measured. The brisket was significantly lower in palmitic (16:0) and stearic (18:0) acid than the other seven sampling sites (P = 0.001). The brisket demonstrated the highest values of MUFA (P = 0.001) with the exception of possessing the lowest value of transvaccenic (18:1t11) acid (P = 0.002). There were also significant differences in the amounts of PUFA among the eight sampling sites. The lowest values were from the brisket with a mean of 25.1. The flank had the highest slip point with a mean of 39.0 (P < or = 0.001). There was a high negative correlation shown between palmitoleic and stearic acid (R2 = 0.827). The brisket displayed the highest values for MUFA:SFA ratios (P = 0.001), whereas the flank was the lowest. Due to the significant differences amongst fat depots within bovine carcasses in their fatty acid composition we conclude that substantial differences exist across fat depots.Item Host and pathogen transcriptional profiles of acute Brucella melitensis infection(2009-05-15) Rossetti, Carlos AlbertoThe parallel gene expression profiles of Brucella melitensis and the host have not been elaborated. In this study, I analyze and discuss the transcriptional profiles of B. melitensis invasive-associated genes, the expression profile of intracellular B. melitensis and B. melitensis-infected non-phagocytic cells in the first 12 h post-infection (PI), and the in vivo temporal global transcriptome of both B. melitensis and the infected bovine host in the first 4 h PI. The initial study found that B. melitensis at late-log phase of growth were more invasive in non-phagocytic cells than at early-log or stationary growth phase. Microarray-based studies identified 454 Brucella genes differentially expressed between the most and the least invasive growth phases. Additionally, B. melitensis strains with transposon interrupted in loci BMEII0380 (acrA) and BMEI1538 (hypothetical protein) were found to be deficient in internalization compare with the wild-type strain. A second experiment was designed with the goal of characterizing host and pathogen transcriptome in parallel. For detecting intracellular Brucella gene expression, a combined protocol consisting of a linear amplification of sense-stranded RNA biased to pathogen transcripts to the previously enriched host:pathogen RNA mixed sample, was developed. RNA samples were hybridized on human and Brucella cDNA microarrays, which analysis revealed a common down-regulation transcriptional profile at 4 h PI that was reverse at 12 h PI. The integrity of B. melitensis virB operon and the expression of host MAPK1 were confirmed as critical for early B. melitensis intracellular survival and replication in non-phagocytic cells. Finally, a temporal morphological and molecular characterization of the initial B. melitensis:bovine host interaction using a calf ileal loop model was performed. B. melitensis was isolated from intestinal Peyer?s patches as soon as 15 min and from systemic blood after 30 min postintra luminal inoculation. Microarray results revealed a common transcriptional profile in Brucella, but two different transcriptional profiles were identified in the host in the first 4 h PI. The importance of differentially expressed biological processes, pathways and individual genes in the initial Brucella pathogenesis is discussed.Item Role of leptin in regulating the bovine hypothalamic-gonadotropic axis(Texas A&M University, 2004-09-30) Amstalden, MarcelThe physiological mechanisms through which nutrition mediates its effects in controlling reproduction are not well characterized. Both neural and endocrine components have been implicated in the communication of nutritional status to the central nervous system. Leptin, a hormone synthesized and secreted mainly by adipocytes, is heavily involved in this communication network. The objectives of studies reported herein were 1) to determine the effects of short-term restriction of nutrients on circulating leptin, leptin gene expression in adipose tissue, and leptin receptor (LR) gene expression in the adenohypophysis of ovariectomized cows; and 2) to investigate the responsiveness of the hypothalamic-adenohypophyseal (AP) axis of fasted and non-fasted cattle to leptin. Studies demonstrated that circulating concentrations of leptin and leptin gene expression in subcutaneous adipose tissue are decreased by fasting. Although 2 to 3 days of fasting did not affect patterns of release of luteinizing hormone (LH), cerebroventricular infusions of leptin increased mean circulating concentrations of LH in fasted, but not normal-fed cows, without affecting frequency or amplitude of pulses of LH. In vitro studies were conducted to determine whether the in vivo effects of leptin could be accounted for at the hypothalamic and/or AP levels. Leptin did not affect the release of gonadotropin-releasing hormone (GnRH) from hypothalamic-infundibular explants from either normal-fed or fasted cattle. Moreover, leptin did not affect the basal release of LH from bovine AP cells or AP explants from normal-fed cows. However, leptin induced a higher basal release of LH from AP explants of fasted cows and increased GnRH-stimulated release of LH from AP explants of normal-fed cows. Results demonstrate that leptin acts directly at the AP level to modulate the secretion of LH, and its effects are dependent upon nutritional status. Cellular mechanisms associated with the increased responsiveness of gonadotropes to leptin in fasted cows were investigated. Expression of LR and suppressor of cytokine signaling-3 (SOCS-3) in the adenohypophysis did not account for the increased responsiveness of fasted cows to leptin. Therefore, although leptin clearly stimulates the hypothalamic-gonadotropic axis in nutrient-restricted cattle, it is unclear why cattle maintained under neutral or positive energy balance are resistant to leptin.Item Structural and functional characterization of the polled interval on bovine chromosome 1(Texas A&M University, 2008-10-10) Wunderlich, Kris RakowitzThe horned condition in cattle is believed to be the wild type with morphogenesis primarily occurring after birth. The polled condition has existed since domestication and has been selected for its economic importance. The polled locus has previously been mapped by genetic linkage analysis to the proximal region of bovine chromosome 1. In order to help us eventually identify the causative mutation, the objective of the study was to structurally and functionally characterize the polled interval from IFNAR1 to SOD1 on BTA1. Our hypothesis was that the polled locus is a tissue specific transcription factor that is expressed in the developing horn buds and acts directly or indirectly upon SOX9. A 2.5 Mb BAC contig and STS content map of the polled interval was constructed. Three candidate genes encoding transcription factors were identified within this region but only C21orf66 was expressed in the horn buds from 1 d old Bos indicus influenced calves. The C21orf66 gene has 18 exons, spans 30,976 bp of genomic DNA, and 144 SNP were identified. No single SNP discovered in C21orf66 can be attributed as the causative mutation. None of the genes from the polled interval were differentially expressed in skin and horn from 1 d old Bos indicus influenced calves. However, there were significant differences in the levels of expression of RUNX2, SOX9, BMP4, PRKCA, and FOXL2 in these samples. Expression of RUNX2 was localized to the osteoblasts, and both RUNX2 and SOX9 were expressed in sebaceous glands of the horn at 1 d of age. Histological examination of horns and scurs from newborn, 5 to 6 mo, and ~1.5 yr old Bos indicus influenced cattle suggest that horns form through intramembranous ossification. Based on the data presented herein, we propose that the polled locus is upstream of RUNX2 and SOX9 in the osteogenic pathway, and could have its primary effect on the differentiation of mesenchymal condensations. The genes IL10RB, SFRS15, C21orf66, OLIG1, OLIG2 and HUNK remain candidates for the polled locus and warrant further investigation.Item The use of laser-assisted hatching in bovine in vitro produced embryos to improve pregnancy rate(2009-05-15) Menges, Suzanne LynnIn vitro produced (IVP) embryos not hatching from the zona pellucida (ZP) after transfer is one possible contributing factor of a lower pregnancy rate when compared to in vivo embryos. This study evaluates using a microscope objective mounted laser to cut the ZP prior to transfer into the recipient to assist hatching. The preliminary data evaluated the effect of laser treatment on IVP embryos and subsequent blastomere survival. In six replicates, bovine oocytes were in vitro produced according to the standard laboratory procedures of TransOva Genetics, Sioux Center, IA. On days 5, 6, and 7 of in vitro culture, embryos were randomly divided into 3 groups: no treatment (Control; n=63), sham ZP cut (Sham; n=68), or ZP cut (Cut; n=70). Control embryos were immediately returned to the incubator. Sham embryos were exposed to all conditions as Cut except laser assisted hatching. The XyClone? system was used to treat the Cut group using pulse strength of 90% and pulse length of 600 ?sec. Embryos were returned to culture until day 8 when embryonic development and the percentage of live cells were determined and analyzed with Chi square. The number of developing embryos and the percentage of live cells per embryo showed no significant difference. Mean live cells ranged from 89-96% regardless of day of treatment. The laser assisted hatching effect on IVP embryo viability was evaluated by randomly dividing commercially produced embryos obtained from TransOva Genetics into two groups on day of transfer, Control or Cut. The ZP of treated embryos were cut with the laser using 80% pulse strength and pulse length of 500 ?sec on day 7, immediately prior to transfer into estrous synchronized recipients. Ultrasonagraphy determined pregnancy rates. Thirty day pregnancy rates were 49.2% and 54.1% for Control (n= 189) and Cut (n=148) embryos, respectively, and were not statistically different (p > 0.05). However, 60 day Control pregnancy rate was 45.7% (n= 166) and the Cut group rate was 57.7% (n= 123) revealing a statistical difference (p < 0.05). These results demonstrate that the XyClone? system assisted hatching can improve 60 day pregnancy rates for IVP embryos by approximately 11 %.Item Use of unique collection device improves conception rates of bovine and equine(Texas Tech University, 2008-12) Graves-Evenson, Kory K.; Prien, Samuel D.; Albin, Robert C.; Jackson, Samuel P.; Thompson, Leslie D.While the practice of artificial insemination may date back eight centuries, there is still a need for improved techniques for semen handling. Previous research from this laboratory using a canine or equine model, demonstrated that semen collected in a modified collection device, the Device for Improved Semen Collection (DISC), remained fertile for longer periods as compared to samples collected using standard techniques. The object of the present study was to perform controlled breeding trials involving cattle and horses comparing semen collected in the DISC to a traditional control (TC). All sires were collected in both the DISC and the TC. Following collection, all semen samples were processed using standard techniques designed to produce breeding doses consistent with industry standards. Cells were then held a minimum of 24 hrs prior to breeding. In two separate trials, cattle were synchronized with a standard 2-shot prostaglandin protocol. Horses were bred using cells that had been held for periods of 24, 48 or 72hrs post extension. Data collected from the present study supports earlier work, demonstrating extended motility (and in theory fertility) from semen collected in the DISC. Pregnancy data from all three fertility trials demonstrate higher conception rates in animals bred with sperm collected in the DISC unit. Further, to date, no birth defects have been recorded. These data indicate that the DISC to be a superior system for semen collection, resulting in higher conception rates without increased risk of birth defects.