Animal & Avian Sciences Theses and Dissertations

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End date: 2019Subject: search.filters.subject.Agriculture, Animal Culture and Nutrition

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    CAN CHOLINE SPARE METHIOININE FROM CATABOLISM IN LACTATING MICE AND DAIRY COWS?
    (2009) Benoit, Sarah Lee Ann; Erdman, Richard A; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Several studies have demonstrated that supplementation of rumen-protected choline (RPC) improves milk production in the lactating dairy cow; however, there are an equal number of studies failing to observe production responses. To date, there are only three studies that provide quantitative information in ruminants on the metabolic fates of methyl groups derived from choline and Methionine (Met). This has limited the ability to predict when, and under what conditions, RPC supplementation will be beneficial. The objectives of this thesis were to determine the interaction of choline and Met methyl group metabolism and the extent of methyl group transfer during lactation, and define what role, if any, is there for RPC in remethylation of homocysteine and in the sparing of Met in lactating animals. A preliminary study with lactating mice consuming a low protein basal diet (10%) was conducted. From 11 to 15 d postpartum, stable isotopes of [methyl,2H3] choline and [methyl,2H3] Met replaced the unlabeled choline and Met in the basal diet to measure the metabolic fates of choline and Met including Met remethylation and sources of Met methyl in the mammary gland. Isotopic analysis revealed that the liver is a major site of Met remethylation from dietary choline with a minimum choline methyl group contribution to Met remethylation of 35%. Mammary tissue was not a major site of Met remethylation from dietary choline (< 10% of Met methyl) as measure by Met methyl in mammary tissue and milk casein. However, there was a significant unlabeled source of methyl groups contributing at a minimum of 45% Met remethylation in the mammary tissue, presumably by de novo synthesis. This suggested that in addition to the liver, the mammary gland is an active site of methyl group transactions. In a subsequent study, lactating dairy cows were fed a total mixed ration formulated to meet the nutrient requirements with exception of metabolizable Met that was restricted to 1.49 % of metabolizable protein. Treatments included a Control (basal diet) and RPC supplemented diet where the basal diet was top dressed with 15g/d RPC, diets were fed in a single reversal design with 2 week experimental periods. Stable isotopes of Met, [1-13C] Met, [13CH3] Met, and [methyl-2CH3] choline were continuously infused on d 14 of each period to determine the metabolic fate and methyl transactions of Met methyl as measured in blood and milk casein. Treatment had no effect on milk production or composition. However, plasma free Met from choline transmethylation was shown to act as a significant contributor to casein synthesis. Fractional Met remethylation rates in the control and RPC treatments were 26 and 23%, respectively. Methionine Met methyl loss within the mammary tissue appears to be minimal. Based on casein Met enrichment, upwards of 40% of Met present in casein had undergone transmethylation with choline serving as the ultimate methyl donor. Furthermore, plasma versus casein Met methyl enrichment data suggested that a significant amount of de novo methyl group synthesis occurs in the dairy cow's mammary gland with choline serving as a major methyl donor.
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    Personality Traits in the Budgerigar, Melopsittacus undulatus
    (2008) Callicrate, Taylor; Estevez, Inmaculada; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    This study investigated bold-shy personality in the budgerigar, Melopsittacus undulatus . Adult budgerigars (14 females, 9 males) fed either a control diet, or one supplemented with docosahexaenoic acid (DHA), were subjected to seven behavioral tests and two immunocompetence assays. Behavioral responses were categorized by context: fear, feeding, or activity. Correlations were obtained within contexts and among immunocompetence variables and all behavioral variables. Kruskal-Wallis analysis was used to investigate effects of gender and DHA on all variables. Budgerigars behaved consistently within activity and feeding contexts. Males had higher feeding rates, and their feeding responses were negatively correlated with a measure of innate immunity. Cluster analysis characterized birds by activity levels; bold birds were highly active and shy birds were less active. The results of this study suggest that budgerigars exhibit consistent behaviors in two contexts, feeding and activity, which are unrelated to each other, and that activity is the predominant personality trait.
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    Regulation of milk fat synthesis by dietary fatty acids
    (2008-08-04) Kadegowda, Anil Kumar Gopalapura; Erdman, Richard A.; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    The objectives of this thesis research were to determine the molecular mechanisms by which dietary fatty acids (FA) regulate lipogenic gene expression and milk fat synthesis. Principal component and multivariate analyses were conducted to establish the relationship between milk fat and FA concentrations in lactating cows fed milk fat depressing (MFD) diets. This analysis showed that in addition to the established inhibitory effect of t10c12 CLA, t7-18:1 and t7c9-CLA isomers might be involved in MFD. Lactating mice were used to test the effects of several individual trans-18:1 isomers and t10c12-CLA on milk fat synthesis, lipogenic genes in liver and mammary tissues. Both MFD and extensive conversion of t7-18:1 to t7c9-CLA in mammary and liver tissues were shown in mice fed the t7-18:1. As expected, t10c12-CLA feeding caused MFD and reduced the expression of lipogenic transcription factor (TF) SREBP-1C. Potential roles of the TF ChREBP, PPARG, and INSIG1 were also established. A subsequent study aimed to establish whether these mechanisms operated in lactating dairy cows. Compared with un-infused controls and a t10c12 CLA negative control, post-ruminal infusion of butterfat which contains all fatty acids in the same proportion to those found in milk fat to a mixture of fats containing only the long chain fatty acids (LCFA) were examined. Milk fat content, milk yield and mammary lipogenic gene expression were increased by butterfat but not by the LCFA mixture. This suggested that rates of short and medium chain fatty acid synthesis might be limiting for milk fat production. The effects of individual FA and a PPARγ-specific agonist (Rosiglitazone) on mRNA expression via qPCR of 19 genes with roles in de novo synthesis, FA uptake and transport, desaturation, triacylglycerol synthesis, transcriptional regulation, and nuclear receptor signaling in a MACT cell culture system were examined. The FA regulated mammary lipogenic gene expression to different extents. PPAR-γ activation of de novo lipogenesis coupled with exogenous FA availability might play a role in regulating milk fat synthesis. These experiments demonstrate the role of FA in regulating mammary lipogenic pathways, highlighting the complexity and multiple transcriptional factor involvement in milk fat synthesis.
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    The potential role of butyrophilin and xanthine oxidoreductase in controlling the amount and size of milk-fat droplets
    (2008-08-05) Jacob, Jaison; Mather, Ian H; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    The secretion of fat droplets from mammary epithelial cells requires the expression of two major proteins, butyrophilin1A1 (BTN) and xanthine oxidoreductase (XOR). Ablation of the BTN or XOR gene in mice results in the accumulation of large fat droplets suggesting a reciprocal relationship between BTN/XOR concentration and milk-fat droplet size. We tested this hypothesis by correlating BTN/XOR concentrations in cow and mouse with their fat droplet size. The amount of BTN in mouse was 75 times less than in bovine samples. The size of fat droplets in mice was larger than in cow, but no correlation was found between fat-droplet size and the amount of BTN/XOR. Experimental reduction in fat-droplet size in mice did not change the concentration of BTN. We propose that a low amount of BTN is sufficient to mediate its role in milk-fat secretion and that it may have additional functions to its potential role as a structural protein.
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    Towards the derivation of bovine embryonic stem cells
    (2008-01-28) Pant, Disha; Keefer, Carol; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    The ability of embryonic stem cells (ESCs) to self-renew and differentiate into a wide range of cell types has encouraged researchers to attempt to isolate ESCs from embryos of domestic species for the past two decades. Success has been limited. The aim of the current study was to investigate whether colonies derived from inner cell masses (ICMs) of bovine blastocysts expressed the same markers of pluripotency and candidate genes representing the various signaling pathways as those found in human or mouse ESCs. The ability of selected cytokines to maintain the major transcription factors associated with pluripotency (NANOG, POU5F1 and SOX2) in the ICM explants was also tested. The results of the study showed that the three major transcription factors (NANOG, POU5F1 and SOX2) were expressed initially in culture but were lost with continued culture and passaging. Markers of differentiation (BMP4, HNF4, NCAM, and CDX2) were also expressed in the initial days of culture. The candidate genes representing the various signaling pathways were expressed in the initial days of culture as well as in subsequent passages. Noggin, a cytokine inhibiting the BMP4 pathway successfully up-regulated the relative expression of NANOG in the ICM explants with respect to controls. The results indicate that signaling pathways associated with regulating pluripotency are expressed in ICM explants and that with cytokine supplementation pluripotency may be maintained. An alternate approach in which differentiating cells in the primary colonies were selectively ablated to eradicate cells secreting pro-differentiation signals was tested. Bovine embryos that carried the hygromycin resistance gene driven by the NANOG promoter were generated by SCNT. Any pluripotent colonies generated from these embryos should survive in the presence of hygromycin. When cultured in the presence of Noggin and hygromycin, colonies were generated; however they failed to proliferate on passaging. This suggests that the culture conditions were not optimal for the NANOG promoter to remain active over extended culture.
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    Integrating Macronutrient Metabolism In Developing Chicken Embryos
    (2007-12-19) Sunny, Nishanth Edakulathur; Bequette, Brian J; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    The objective of this thesis research was to determine the pathways of glucose metabolism and utilization in small and large egg embryos during the latter half of development, and whether in ovo nutrient supplementation alters glucose use. A further objective was to determine the contribution of glutamate, glutamine and glycerol to glucose, glycogen and non essential amino acid (NEAA) synthesis during embryo development. In ovo stable isotope ([U-13C]glucose, [U-13C] glutamate, [U-13C]glutamine and [U-13C]glycerol) injection approaches were developed along with mass isotopomer distribution analysis of metabolic intermediates and end-products to acquire a metabolic phenotype of the fluxes and partition of these substrates through central pathways. Embryos developing in small and large eggs maintained similar rates of glucose metabolism. Thus, glucose entry and utilization gradually increased from day 12 to 18 embryonic. By embryonic day 20, gluconeogenesis accounted for >80% of glucose entry, a part (65%) of which was represented by glucose carbon recycling. Glutamate and glutamine were not found to be significant gluconeogenic precursors in day 19 embryos. However, catabolism of these amino acids contributed to ~25% of proline flux in the liver. By contrast, there was significant [M+3] 13Cisotopomer abundance in blood glucose and in liver and muscle glycogen when [U- 13C]glycerol was injected in ovo. These observations clearly confirmed that glycerol derived from triacylglycerides is a significant precursor for glucose and glycogen synthesis. In ovo supplementation on day 9 embryonic of glucose and/or amino acids (5 non-essential amino acids) did not alter gluconoegenesis. However, these supplemental treatments significantly reduced catabolism of glucose via glycolysis. 13C-Mass isotopomer abundances of most substrates differed when each was individually compared in blood and in the various tissues, indicating differences in substrate utilization between tissues. In summary, this thesis research has provided new information on the degree and pathways of nutrient (glucose, glycerol, amino acids) use by the developing embryo and the rapid adjustments in the activity of networks of enzymes involved in non-essential amino acid, glucose and glycogen metabolism to support embryo survival. Most importantly, this work has systematically evaluated the potential substrates that the embryo utilizes for glucose synthesis, in particular, the significant role of glycerol.
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    Urea-N Recycling in Lactating Dairy Cows
    (2007-12-11) Dinh, Sarah; Kohn, Richard A; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    This study was designed to determine the effect of rumen degradable protein (RDP) and rumen undegradable protein (RUP) on urea-N recycling and microbial N flow. Eight mid-lactation Holstein cows were assigned to a repeated 4 x 4 Latin square. The diets were isoenergetic with RDP and RUP concentrations arranged in a factorial design (10.0 and 12.5% RDP and 5.6 and 8.1% RUP as a percentage of DM). The 10.0% RDP diets resulted in greater milk yield and lower milk protein concentration than the 12.5% RDP diets. High RUP diets tended (P = 0.1) to increase bacterial N flow in the liquid fraction. The NRC 1989 model predicted flow of microbial N and total N from the rumen more accurately than the NRC 2001. The NRC 2001 model predicted a higher RDP requirement and a lower RUP requirement for all four diets compared with the NRC 1989 model. Both models reflect the dietary changes that were intended by increasing the RDP and RUP in a factorial manner. There was no effect of RUP or RDP on the g/d of urea-N transferred from the blood to the gut or returning to the ornithine-urea cycle. However, plasma urea-N (PUN) incorporated into rumen microbial protein tended (P = 0.14) to increase with the low RDP diets. The rate of transfer of PUN to the gut appeared to be independent of PUN concentrations. The gene expression for urea transporters (bUT-B2) in the rumen did not change due to diet. As dietary protein intake increased, a constant amount (g/d), or decreasing fraction (g/g), of PUN was transferred to the gut. The apparent saturation of urea transporters in the gut prevented excess PUN from recycling to the gut on high-protein diets sparing the energy cost for N excretion that would have resulted from a constant percentage of PUN being recycled.
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    Evaluation of Lef1 Transcription Factor in Mammalian Preimplantation Embryos
    (2007-09-14) Meece, Ashley Elaine; Keefer, Carol L; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    The first lineage differentiation of cells during pre-implantation embryo development is critical for continued embryonic growth. Though several transcription factors (TFs) have been identified that are involved in this transition, a specific mechanism of regulation has yet to be determined. Previous studies in our laboratory have shown the TF Lef1 is involved in differentiation of mouse embryonic stem cells into trophoblastic stem cells through the Wnt signaling pathway. Lef1 is known to be involved in lineage determination of adult skin stem cells. As we observed that two isoforms of Lef1 were expressed at different stages of pre-implantation development, we hypothesized that Lef1 was interacting with the other well-established lineage differentiation TFs, Nanog, Oct4, and Cdx2, in these early embryos. At the blastocyst stage, no significant changes in mRNA expression were seen when siRNAs, specifically designed to knockdown Lef1 expression, were injected in early embryos; however, knockdown of Lef1 did not interfere with blastocyst formation.
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    Characterization of Chicken CAT-2 Isoforms
    (2007-08-20) Kirsch, Sandra B; Hamza, Iqbal; Humphrey, Brooke D; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Lysine and arginine transport is primarily mediated by cationic amino acid transporters (CATs) in cells. The chicken CAT-2 (cCAT-2) transcript is alternatively spliced to three isoforms. Transcriptional and cellular localization experiments were utilized to study their regulation. The mRNA abundance of cCAT-2 isoforms was estimated in body tissues, and although differentially expressed, all tissues expressed each cCAT-2 isoform gene, indicating that alternative splicing was not tissue-specific. Both cCAT-2A and cCAT-2B proteins localized to the plasma membrane and cCAT-2C protein was retained in the cytosol. Chicken CAT-2A functions as a low affinity transporter with specificity for lysine and arginine. Chicken CAT-2B and cCAT-2C transporter functions were not detectable. Our data indicates that CAT-2 transporters are conserved in non-mammalian vertebrates, but cCAT-2 isoforms differ in their tissue distribution and transporter function from previously characterized CAT-2 transporters. These results also indicate a mechanism by which additional dietary lysine and arginine contribute to increased protein accretion in muscle tissue.
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    ENERGY METABOLISM IN DEVELOPING CHICKEN LYMPHOCYTES DURING THE EMBRYONIC TO POSTHATCH TRANSITION
    (2007-07-26) Rudrappa, Shashidhara Govindareddy; Porter, Tom E; Humphrey, Brooke D; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    In chickens, the primary energy substrate is lipid during embryogenesis and carbohydrate after hatch. Accordingly, chicks adapt their metabolism to utilize glucose after hatch; however, little is known about metabolic adaptation in developing lymphocytes. Therefore, the objective of this dissertation was to examine metabolic adaptation in developing lymphocytes and the associated impact on their development. The first objective examined energy substrate utilization in bursacytes and thymocytes during the embryonic to posthatch transition. Glucose metabolism increased in both lymphocyte populations during the first two weeks posthatch due to increased glucose transporter-3 mRNA abundance, glucose uptake and hexokinase activity. Additionally, some of these metabolic markers were positively correlated with the serum glucose concentration. Glutamine metabolism increased in bursacytes only, and lipid metabolism was unaltered in both populations. Collectively, glucose is a preferred energy substrate for lymphocytes posthatch, and glucose utilization by developing lymphocytes may be related to the serum glucose concentration. The second objective determined the effect of glucose availability on thymocyte metabolism, energy status and survival. Embryonic thymic lobes were grown in culture in media containing varying glucose concentrations. Thymocyte glucose metabolism and mitochondria membrane potential were highest in 15 mM glucose and apoptosis was highest in 5mM glucose. Collectively, glucose availability regulates glucose metabolism in thymocytes, and these changes in glucose metabolism were related to thymocyte energy status and survival. The third objective determined the effect of glucose availability on T cell development. Thymocyte Interleukin-7Rα (IL-7Rα) mRNA abundance and CD4+ T cell numbers over the culture period were dependent upon glucose availability. Between 12 and 24 h, thymocyte IL-7Rα mRNA abundance increased in 5 mM increased 1.74-fold, while it decreased in 15 mM by 58.6%. CD4+ numbers decreased with time in 5 mM, whereas they increased with time in 15 mM. T cell receptor (TCR) β excision circles were higher in 15 mM compared to 5 mM at 12 h. Glucose availability alters TCR β rearrangement, IL-7Rα gene expression and CD4+ T cell development, which may influence naïve T cell generation. As thymocytes develop in a low glucose environment in ovo, this may be one factor that limits T cell development until hatch.