College of Agriculture & Natural Resources
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The collections in this community comprise faculty research works, as well as graduate theses and dissertations.
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Item COMPARATIVE STUDY OF LIPOPROTEIN METABOLISM IN MAREK'S DISEASE SUSCEPTIBLE AND RESISTANT LINES(2010) Yuan, Ping; Song, Jiuzhou; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Marek's disease virus (MDV) infection causes atherosclerosis, and prior vaccination prevented the development of this disease. Two main strategies to resist Marek's disease (MD) have been demonstrated: vaccination and genetic resistance. However, little is known about the role of genetic resistance in the progression of MDV induced atherosclerosis. Atherosclerosis is primarily associated with lipoprotein metabolism. The purpose of this study was to investigate whether lipoprotein metabolisms are different in distinct MD susceptible and resistant chicken lines. Here, we studied different backgrounds of lipoprotein metabolism in the two lines and the changes of lipoprotein levels in response to MDV infection. The results showed that during chicken growth, the increase in total cholesterol was mostly due to the increasing (LDL+VLDL) in MD susceptible line, whereas it was mainly due to the elevating HDL in MD resistant line. These results suggested that different lipoprotein metabolisms exist in MD susceptible and resistant lines.Item IDENTIFICATION OF A NON-CLASSICAL GLUCOCORTICOID-RESPONSIVE ELEMENT IN THE 5'-FLANKING REGION OF THE CHICKEN GROWTH HORMONE GENE(2010) Knubel, Kristina Heuck; Porter, Tom E; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Growth hormone (GH) effects growth and contributes to a lean phenotype in broiler chickens. GH secretion by the anterior pituitary begins on embryonic day (e) 14, concomitantly with a rise in adrenal glucocorticoids (GC) or corticosterone (CORT) secretion. CORT treatment of chicken embryonic pituitary (CEP) cells induces GH secretion prematurely. GC induction of the GH gene requires on-going protein synthesis or an intermediary protein, but the gene lacks a classical GC-response element. We hypothesized that a GC-responsive intermediary protein is necessary for the CORT induced increase in GH. Characterization of the upstream region of the gene may help identify such a protein. To this end, a fragment of the GH gene (-1727/+48) was cloned into a luciferase reporter and characterized in e11 CEP cells. CORT treatment increased luciferase activity and mRNA. Inclusion of CHX blocked CORT induction of luciferase mRNA. Through deletion analysis, we found that a GC-responsive region (GCRR) is located at -1045 to -954. By defining the GC-responsive region and cis-acting elements located within, trans-acting proteins involved in GC induction of the GH gene may be identified. The GCRR is CORT-responsive in either orientation, but it is context-dependent. Potential transcription factor motifs in the GCRR include ETS-1 and a degenerate GRE (GREF). Nuclear proteins bound to a GCRR probe in a CORT-regulated manner and unlabeled competitor DNA competed off binding. Mutation of the central portion of the DNA probe resulted in a significant decrease in protein binding. Mutation of the ETS-1 site or GREF site in the -1045/+48 GH construct resulted in ablation of luciferase activity. ETS-1 and GR are associated with the endogenous gene under basal and 1.5 h CORT-treated conditions, while GR recruitment increased after CORT treatment. GC regulation of the GH gene during chicken embryonic development requires cis-acting elements located 1 kb upstream from the transcription start site and includes recruitment of ETS-1 and GR. This is the first study to demonstrate involvement of ETS-1 in GC regulation of the GH gene during embryonic development. Characterization of GC regulation of the GH gene during embryonic development enhances our understanding of growth regulation in vertebrates.Item 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.Item The effect of sperm mobility phenotype on fertility persistence in layer and broiler hens(2008) Baczynski, Kathleen; Estevez, Inmaculada; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Domestic fowl (Gallus gallus domesticus) were studied to identify accurate predictors of potential fertility in two lines of broiler breeder males along with fertility persistency in layer and broiler hens. Sixty-four Hy-Line layer and thirty-seven broiler breeder hens were AI with identical amounts of high or low mobility sperm from FG males. Morphological measurements were taken to determine relationships of these with semen volume, concentration, and mobility. We hypothesized that 1) semen quality would decline as males aged, 2) morphology would be positively correlated with semen quality, and 3) females AI with high mobility sperm would have a longer duration of fertility. Results revealed a significant age*line interaction for semen volume (p=0.0307), sperm concentration (p=0.0003), and sperm mobility (p=0.0405). Morphological measurements were correlated with different semen parameters in both lines. Fertility was positively correlated with semen quality. Sperm mobility influenced fertility in layer hens but not in broiler breeders.Item Priming with Oral Progestin Before Ovulation Induction Facilitates Ovarian Function in the Cat (Felis catus)(2007-11-21) Bauer, Rosemary Aileen; Ottinger, Mary Ann; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Artificial insemination (AI) has been developed in multiple felid species as a tool for retaining gene diversity in threatened or endangered populations. Yet, pregnancy success remains low (< 5%) following AI in most felids, particularly in species that spontaneously ovulate. This failure has been attributed to variable ovarian status at the time of insemination and adverse residual effects caused by exogenous gonadotropins used to induce ovulation. Using the domestic cat as a research model, a new AI regimen that incorporated short-term ovarian suppression with oral progestin (altrenogest; ALT) before ovulation induction was investigated. The hypothesis was that oral progestin priming would prevent spontaneous ovulation, improve ovarian responsiveness to exogenous gonadotropins and mitigate adverse effects caused by persistent gonadotropin actions. Specific objectives were to: (1) increase fundamental understanding of the mechanisms controlling ovarian function; and (2) characterize how oral progestin priming prior to exogenous gonadotropin treatment influences ovarian responsiveness, fertilization, early embryonic development and luteal function in the cat. Fecal hormone monitoring was used to establish an ALT dosage that provides rapid, reversible ovarian suppression with no residual effects on estrous cyclicity. With this information, the influence of progestin priming on ovarian responsiveness to exogenous gonadotropin dosage was investigated. Priming increased ovarian sensitivity to gonadotropins, supporting the use of lower dosages for ovulation induction. Next, in vivo fertilization success and in vitro early embryonic development was characterized following laparoscopic, intrauterine AI in cats treated with ALT. Progestin-primed females demonstrated a good ovarian response to ovulation induction and more consistent embryonic development, compared to cats treated with gonadotropins alone. Furthermore, endocrine data revealed that normal luteal progesterone levels were maintained only in queens primed with the oral progestin. Finally, histology and quantitative RT-PCR were used to characterize the differential effects on luteal function observed. Aberrant CL progesterone production was not associated with changes in ovarian morphology, or the expression of six specific genes associated with luteal function and progesterone biosynthesis. Overall, these studies increased knowledge of domestic cat reproductive physiology and improved understanding of ovarian suppression for enhanced AI efficiency in felids.Item Effects of Aging and Moderate Calorie Restriction on the Reproductive Axis of the Male Rhesus Macaque (Macaca mulatta)(2007-04-25) Sitzmann, Brandon Dale; Ottinger, Mary Ann; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Calorie restriction (CR) has been established as the only non-genetic method of altering longevity and attenuating biological changes associated with aging. This nutritional paradigm has been effective in nematodes, flies, rodents, dogs and possibly non-human primates. Its long history notwithstanding, little is known regarding the exact mechanism(s) of CR action or its potential impact on the hypothalamic-pituitary-gonadal (HPG) axis. The objectives of this project were to: 1) analyze neuroendocrine changes to the HPG axis that occur with aging and 2) evaluate the effects of moderate CR on reproductive function in male rhesus macaques. Pituitary gene expression profiling, semi-quantitative RT-PCR (sqRT-PCR) and immunohistochemistry showed circadian clock mechanism components present in three age categories of macaques, demonstrated age differences in expression for Per2, indicated differential expression of Per2 and Bmal1 at opposing time points and revealed daily rhythmic expression of REV-ERBα protein. These data indicate the ability of the macaque pituitary to express core-clock genes, their protein products, and to do so in a 24-hour rhythm. Young Adult CON and CR pituitary gene expression profiles detected potential differential expression in <150 probesets. A decline in>TSHR and CGA was detected in CR macaques as measured by sqRT-PCR. Other genes investigated showed no diet-induced changes. Young Adult CON and CR testicular gene expression profiles detected potential differential expression in <300 probesets although mRNA expression was not altered based on sqRT-PCR and real-time RT-PCR. Age-related>and/or diet-induced changes in HSD17β3, INSL3, CSNK1E and CGA were observed in a separate experiment with CGA in Old Adult CR subjects returning to youthful levels. Semen samples were collected from Young Adult CON and CR macaques. Normal spermiogram measures, ZP-binding, AR assay and SCSA® were conducted and indicated no differences between CON and CR-treated animals. Both groups exhibited similar daily testosterone profiles with no differences in mean or maximum levels; however, daily minimum testosterone levels were lower in CON animals. It appears that moderate CR had limited impact on neuroendocrine or reproductive function in male rhesus macaques based on our selected endpoints. Thus, advantageous CR health benefits can be achieved without obvious negative consequences to the HPG axis.Item Characterization of Glucocorticoid-Induced Changes in Gene Expression in the Embryonic Pituitary Gland(2006-04-27) Jenkins, Sultan Ali; Porter, Tom E; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)We have previously shown that corticosterone (CORT) can induce premature differentiation of somatotrophs in the chicken embryo. CORT induction of GH mRNA is indirect, in that protein synthesis inhibition blocks its inducing effect. In this study, we used a custom chicken microarray to analyze global gene expression in the embryonic pituitary gland and to identify potential direct targets of CORT that may regulate its induction of somatotroph differentiation. Dispersed embryonic (e) day 11 pituitary cells were pretreated with cycloheximide then with CORT or treated with CORT alone for 24 hrs. Amplified RNA from these samples was then used on our microarray to analyze gene expression and in quantitative real-time PCR (qRT-PCR) analysis to determine relative gene expression levels. qRT-PCR from these samples showed that GH was induced within 1.5 hrs and continued to significantly increase until 3 hrs. Our microarray analysis revealed 25 direct early targets of CORT. From these 25 we chose 3 genes, Dexras1, Ras-dva, and Prostaglandin-D Synthase to transfect into e11 pituitary cells and measure their effect on GH mRNA. Neither of these genes had a direct effect on GH mRNA; however Dexras1 acted synergistically with CORT to induce GH mRNA. Dexras1 was discovered in murine AtT-20 corticotroph cells because its expression was rapidly induced in response to glucocorticoids. We report here a chicken Dexras1 cDNA that is 1631 bp in length and encodes a protein of 278 amino acid residues. Comparison of the consensus chicken Dexras1 amino and nucleic acid sequence with those of human, mouse, and rat Dexras1 showed high homology among the species. Expression of Dexras1 mRNA was detected in various tissues by Northern analysis, but was highest in the pituitary. RT-PCR analysis showed expression of Dexras1 only in the pituitary. The precise role of DexRas1 is unidentified at the present time; however, its distribution in a range of tissues suggests a possible role in glucocorticoid action within a variety of systems.Item Effects of Moderate Calorie Restriction on Ovarian Function and Decline in Rhesus Monkeys(2006-04-27) Wu, Julie Mei-Fen; Ottinger, Mary Ann; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Calorie restriction (CR) has long been heralded as the only proven nutritional intervention for life extension. Recent data demonstrated that moderate CR also extended reproductive lifespan in female rats. The objectives of this project were to: 1) analyze general hormonal changes that occur with aging and menopause and 2) evaluate the effects (whether beneficial or detrimental) of moderate (30%) CR on ovarian function and decline in rhesus monkeys (Macaca mulatta). Hormone analyses demonstrated elevated FSH and reduced INHB in Old monkeys, prior to menstrual cycle irregularity and alterations in E2 or P4. Our data are the first demonstration of this hormonal event occurring in monkeys. Furthermore, moderate CR did not impair normal ovarian function or aging. Evaluation of three clinically available tests: day 3 FSH, the Clomiphene Citrate Challenge Test (CCCT) and the Exogenous FSH Ovarian Reserve Test (EFORT), demonstrated that CCCT is efficacious in monkeys, especially with the use of E2 and INHB. As such, CCCT is the most cost effective and best predictor of ovarian response. Responses were similar between CON and CR. Oocytes from old short-term CON and CR monkeys were collected and fertilized with spermatozoa collected from normal males. Interestingly, CR appears to prolong ovarian responsiveness to exogenous gonadotropins and improve embryonic development in vitro in old female rhesus monkeys. Microarray analysis of gene expression was conducted in luteinizing granulosa cells. A subset of responsive genes were identified that will require validation by via real time polymerase chain reaction (RT-PCR). These data will provide insight into potential mechanisms of direct action of CR on the ovary. Therefore, the results of this study have provided evidence for the utility of the rhesus monkey as a model for human menopause. Additionally, moderate CR did not impair normal reproductive function or decline. We also confirmed the efficacy of the CCCT in rhesus monkeys and recommend its use as a diagnostic tool. Finally, CR improved ovarian response to exogenous gonadotropins and has beneficial effects on oocyte quality and subsequent embryo development.Item Effects of embryonic exposure to androgen-active endocrine disrupting chemicals in Japanese quail(2005-04-18) Quinn, Jr., Michael James; Ottinger, Mary Ann; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Endocrine disrupting chemicals (EDCs) are compounds that alter the production, secretion, action, and elimination of endogenous hormones. EDCs have been shown to be responsible for disrupting development, reproduction, immune function, behavior, and all other life functions mediated by hormones. In the environment, organisms are exposed to many different types of EDCs at any one time, each with different mechanisms of action, many of which are not fully understood at present. Most research done with EDCs has focused on the effects of these chemicals on the estrogen and thyroid systems, however, many of these same chemicals also exert strong effects on the androgen system. Also, many studies assessing the effects of EDCs on wildlife have focused on reproductive measures of exposure, often overlooking potential effects on the immune system. We have demonstrated that embryonic exposure to androgen-active EDCs, anti-androgenic DDE and androgenic trenbolone acetate, impairs development of the bursa of Fabricius in Japanese quail, providing a possible mechanism for EDC-induced immunosuppression. The bursa is a primary immune organ responsible for development of the humoral part of the immune system. We have also demonstrated that the bursa can be resilient to embryonic exposure to EDCs, if post-hatch exposure to these chemicals is prevented. Measures of reproduction, behavior, growth, and developmental stability were also taken in this study. Male and female rates to sexual maturity were altered by the one-time in ovo exposure to DDE and trenbolone. Male reproductive behavior, as measured by attempts to mount and successful cloacal contacts achieved, was suppressed by both chemicals. Vocalization was abolished in one and two week old chicks from the highest trenbolone acetate treatment levels. Although environmentally relevant, the levels of DDE used in this study were below those reported to affects avian reproduction. Environmental levels of trenbolone acetate are unknown, however, previous studies have concluded trenbolone acetate to be safe to wildlife and non-teratogenic. The myriad of endpoints used in this study has been compiled to provide toxicologists with a list of sensitive and persistent measures that can be used as reliable biomarkers of exposure to androgen-active EDCs in birds.Item SOMATOTROPIN RESPONSE TO CORTICOSTERONE AND THE THYROID HORMONE T3 DURING CHICK EMBRYONIC DEVELOPMENT: INVOLVEMENT OF TYPE I AND TYPE II GLUCOCORTICOID RECEPTORS(2004-05-17) Heuck, Kristina; Porter, Tom E; Animal SciencesCorticosterone (CORT) can stimulate growth hormone (GH) cell (somatotroph) differentiation and GH secretion on embryonic day (e) 12 but not e20 in the chicken. GH induction involves both glucocorticoid receptors (GR) and mineralocorticoid receptors (MR); however, this finding has been characterized only on e12. To further define changes in somatotroph responsiveness to CORT, pituitary cells obtained on e12-e20 were cultured with CORT alone and in combination with T3. GH secretion increased over basal with CORT treatment on e12, e14, e16, and e18, but not e20. Contributions of GR and MR in CORT responses were evaluated using GR and MR antagonists. Blocking both receptors was required to abolish the CORT response by e12 cells. The same treatment on e20 decreased GH secretion relative to basal. We conclude that positive somatotroph responses to CORT are lost during embryonic development and that both GR and MR mediate CORT-induced GH secretion by cultured somatotrophs.