Animal & Avian Sciences Theses and Dissertations

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THE STUDY OF ENERGY METABOLISM IN THE PERIPARTURIENT DAIRY COW: INVESTIGATING THE ROLE OF KETONES AND NAD+
(2024) da Silva, Mariana; Rico, Jorge Eduardo; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Dairy cows typically experience increased energy demands during the transition from gestation to lactation, and a myriad of metabolic adaptations are set in place to facilitate this transition. This thesis integrates findings from two experiments exploring energy metabolism during this critical phase. In the first experiment, the effects of hyperketonemia on dairy cow health and productivity were investigated. Eight Holstein cows (19.62 ± 5.44 days postpartum) were intravenously infused with either Na-BHB (KET) or NaCl (CON) for 48 hours. The study aimed to assess the impact of elevated beta-hydroxybutyrate (BHB) levels on blood biomarkers and immune response. Despite no significant differences in dry matter intake or overall milk yield, KET cows exhibited lower milk yield post-immune challenge (LPS bolus) and higher plasma NEFA levels. While plasma glucose concentrations remained unchanged, but BHB concentrations were significantly higher in the KET group (P < 0.001). The second experiment focused on the role of Nicotinamide Adenine Dinucleotide (NAD) coenzymes in energy metabolism during the transition from gestation to lactation. Twenty-six peripartal dairy cows were enrolled in an observational study to examine the NAD metabolome in liver, blood, and milk. Liver biopsies were performed 21 days before and 7 days after parturition, and blood samples were collected weekly. The study hypothesized that hepatic NAD levels deplete while blood and milk NAD pools increase postpartum, with a possible influence of residual feed intake (RFI). Measurements of plasma glucose, free fatty acids, BHB, and insulin, along with comprehensive NAD metabolome profiling using HPLC coupled to mass spectrometry, aimed to elucidate the relationship between NAD metabolism, metabolic biomarkers, and production performance. After calving, animals showed decreased glucose and insulin levels, and increased NEFA and BHB levels, with no significant group differences (P = 0.53). Oxidative stress markers (protein carbonyl, 8-OHdG) and total antioxidant capacity were measured on plasma. The DNA and protein oxidative stress markers remained unchanged relative to parturition, but the Low-RFI group tended to display higher antioxidant capacity (P = 0.08). NAD metabolites increased, and NAD precursor concentrations decreased in the liver. Nicotinamide Mononucleotide was higher in the High-RFI group (P = 0.04) and tended to decrease post-calving (P = 0.06). The liver NAD metabolome remained stable (P = 0.83). Both studies underscore the complexity of energy metabolism during the peripartum period in dairy cows. Collectively, our findings expand our understanding of novel aspects of energy metabolism, with potential implications for health, productivity, and disease resilience in dairy cows. Further research is essential to fully understand these mechanisms and improve management strategies for dairy cows during this critical period.
COORDINATED TRAFFICKING OF HEME TRANSPORTERS BY CARGO SORTING COMPLEXES IS ESSENTIAL FOR ORGANISMAL HEME HOMEOSTASIS
(2025) Dutt, Sohini; Hamza, Iqbal IH; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Heme, an iron-containing organic ring, is a vital cofactor responsible for diverse biologicalfunctions and is the major source of bioavailable iron in the human diet. As a hydrophobic and cytotoxic cofactor, heme must be transported in a highly controlled manner through membranes via specific intra- and inter-cellular pathways. However, the genes and pathways responsible for heme trafficking remain poorly understood. Unlike other metazoans, Caenorhabditis elegans cannot synthesize heme but requires heme for sustenance. Thus, C. elegans is an ideal animal model to identify heme trafficking pathways as it permits organismal heme homeostasis to be directly manipulated by controlling environmental heme. Heme is imported apically into the intestine by HRG-1-related permeases and exported basolaterally by MRP-5/ABCC5 to extra- intestinal tissues. Loss of mrp-5 causes embryonic lethality that can be suppressed by dietary heme supplementation raising the possibility that MRP-5-independent heme export pathways must exist. Here we show, by performing a forward genetic screen in mrp-5 null mutants, that loss of the vesicular cargo sorting Adaptor Protein complexes (AP-3) fully rescues mrp-5 lethality and restores heme homeostasis. Remarkably, intestinal heme accumulation due to mrp-5-deficiency causes a concomitant deficit in the lysosomal heme importer HRG-1 abundance and localization. Loss of both MRP-5 and AP-3 subunits resurrects HRG-1 levels and localization, thus underscoring the crucial role of HRG-1 in dictating mrp-5 mutant phenotypes. In the absence of MRP-5, heme is exported by SLC49A3 homolog, a previously uncharacterized transporter. Live- cell imaging reveals vesicular coalescence that facilitates heme transfer between the importers and exporters at the interface of lysosomal-related organelle. These results define a mechanistic model for metazoan heme trafficking and identifies SLC49A3 as a promising candidate for heme export in mammals.
OVARIAN STROMAL CELLS IMPROVE SURVIVAL, BUT NOT GROWTH, IN PRE- AND EARLY ANTRAL FELINE FOLLICLES
(2024) Marks, Batsheva Naomi; Keefer, Carol; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Ovarian stromal cells act as crucial support and regulators for in vivo folliculogenesis; however, less is known about their effect on in vitro grown follicles. The objective of this study was to investigate the impact of ovarian stromal cell co-culture or conditioned medium (CM) on survival and development of cat pre-, early, and antral follicles in vitro. Ovaries were obtained from cats older than six months (n = 3), then enzymatically digested to release stromal cells. The ovarian stromal cells were allowed to grow to confluency in a T75 flask, before being cryopreserved for long term storage in liquid nitrogen. Cells were thawed one week prior to follicular culture onset, and passaged once before CM collection. CM was subsequently removed 24 - 48 hours after feeding, and stored at -80C until used. Ovarian follicles were mechanically isolated from cats older than six months (n = 23 cats, 155 follicles), encapsulated in 0.5% alginate hydrogel. The isolated follicles were then divided into five treatment groups (control, ovarian stromal cell co-culture, 20% CM, 50% CM, and 100% CM in Endothelial Cell Growth Medium), and classified based on initial diameter as preantral (224.4 + 4.7 m), early antral (394.8 + 7.4 m), or antral (592.2 + 18.8 m). Culture subsequently lasted for 13 days, and survival and growth of the follicles were evaluated on Days 0, 4, 6, 8, 11 and 13. At the end of culture, follicles were assessed via qRT-PCR for expression of CYP19A, FSHR, and GDP9 to further quantify development. Statistical analysis was done in R software. Follicles in 100% CM had higher survival up to Day 11 of culture as compared to other treatment groups (Cox proportional hazards model, p < 0.01). Initial stage also influenced survival, with antral follicle survival significantly lower than that of pre- and early antral follicles (p < 0.0001). However, no differences in growth were detected across the treatment groups, nor across initial size classifications (Kruskal-Wallis test, p > 0.05). Post culture qRT-PCR analysis of the three selected genes showed upregulation of CYP19A in 50% CM follicles compared to the control (ANOVA, p < 0.05). However, there were no differences in CYP19A expression between the control and other treatment groups, or in GDF9 and FSHR expression among culture groups (p > 0.05). In summary, the findings demonstrated that conditioned medium collected from primary culture of ovarian stromal cells improves in vitro survival and modulates CYP19A expression of isolated cat follicles. Further research to identify paracrine factors present in conditioned medium will elucidate the roles of ovarian stromal cells pertaining to follicle survival during in vitro folliculogenesis.
IDENTIFICATION OF KEY MOLECULES IN PLACODE-DERIVED NEURONS THAT COORDINATE CHICK TRIGEMINAL GANGLIOGENESIS
(2024) Hines, Margaret; Taneyhill, Lisa; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
The trigeminal nerve is the largest of the cranial nerves, possessing three main branches (ophthalmic, maxillary, and mandibular) and relaying sensations of pain, touch, and temperature from the face and head to the brain. Cell bodies of this nerve are positioned in the trigeminal ganglion, which arises from the coalescence of neural crest cells and placode cells. These progenitor cells give rise to trigeminal sensory neurons, with placode cell differentiation occurring first. While the dual cellular origin of the trigeminal ganglion has been known for decades, the molecular mechanisms controlling trigeminal ganglion development remain obscure. To elucidate molecules involved in this process, we performed RNAsequencing on the forming chick trigeminal ganglion when only placode cells contribute neurons and identified Neurogenin2 (Neurog2), Neuronal Differentiation 1 (NeuroD1), and Elongator acetyltransferase complex subunit 1 (Elp1) for further study. While Neurog2, NeuroD1, and Elp1 have established roles in neurogenesis in other systems, their functions in placode cells during trigeminal gangliogenesis had yet to be investigated. To address this, we used the chick embryo due to experimental advantages afforded by this model for the study of trigeminal placode cells and trigeminal ganglion development. Using morpholino antisense oligonucleotides, we depleted Neurog2, NeuroD1, or Elp1 from trigeminal placode cells and demonstrated each are essential for proper trigeminal ganglion development. Knockdown of Neurog2, NeuroD1, or Elp1 reduced trigeminal ganglion size and led to aberrant innervation of the eye by the ophthalmic branch. While depletion of Neurog2 and NeuroD1 had opposite effects on the width of the ophthalmic branch, Elp1 reduction appeared to have no effect. However, Elp1 knockdown led to less compact trigeminal ganglion nerve branches, decreased axon projections, and general disorganization of neurons and neural crest cells. Taken together with prior findings, our results suggest a novel interrelationship among Neurog2, NeuroD1, and Elp1 during trigeminal gangliogenesis. Our results have potential high significance for providing new insights into the function of Neurog2, NeuroD1, and Elp1 in trigeminal ganglion development and the etiology of human and animal diseases arising from defects in neural crest cells and/or placode cells.
USING TRIBUTYRIN, A BUTYRATE PRODRUG, DURING GESTATION TO ALTER PERFORMANCE IN PIGS
(2024) Cooper, Elizabeth Jean; Salem, Mohamed; Taneyhill, Lisa A; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Prenatal and postnatal muscle growth and development occurs in a series of three waves. Every stage of the process requires the coordinated actions of myogenic precursor cells (MPC) or satellite cells (SC). Fetal programming of the muscle resident stem cells through maternal dietary intake is a potential method by which to influence muscle fiber growth throughout life. Butyric acid is a potent histone deacetylase (HDAC) inhibitor and has demonstrated strong effects on improving activation and differentiation of skeletal muscle satellite cells. It has also been implicated in serving as an important regulatory mechanism in intestinal cells. In animal models, this activity has resulted in direct promotion of muscle growth and improved resistance to pathogen induced illness. Whether butyrate supplementation during gestation can influence fetal and then lifelong muscle development has not yet been examined. Tributyrin, a pro-drug form of butyrate was utilized in all studies to address technical difficulties associated with using butyric acid as a feed supplement. In the first experiment, we aimed to investigate whether tributyrin at various concentrations in the sow diet could enhance fetal development and the degree to which inclusion levels might be most ideal (1% or 2% butyric acid). We found that tributyrin had a dose-dependent effect on fetal myogenic precursor cell activity and muscle structural organization. An increased proportion of primary fibers identified in the muscle of 2% treated fetal pigs indicated a greater capacity for an increased number of myofibers. Given these results, we repeated the gestational feeding study with more sows which were then allowed to farrow naturally with just one dietary level of tributyrin used (2%). Piglet tissues were collected between postnatal days 3 and 5, and sow milk samples were collected once on day 5 post farrowing to test the fatty acid composition. We found significant changes in the sow’s transition- milk in response to tributyrin. There was an indication of differences in expression of genes associated with SC differentiation in whole Longissimus dorsi (LD) muscle. However, there was no notable change in the muscle fiber size or numbers by day 3 or 5 post farrowing. In our final study, we performed a collaborative study with a contract research organization (CRO) based in the Midwest. This study followed the same design as our first two studies, but post-farrowing offspring growth was monitored. We saw significant improvements to pre-weaning average daily gains in tributyrin treated piglets. A subsample of gilts were selected once they reached 6 months of age for evaluation of carcass traits. Of those selected gilts, there was a significant reduction in overall fat accumulation. Future research is needed to confirm whether this is a universal response for pigs that had prenatal exposure to tributyrin. These findings help support the idea that prenatal tributyrin can enhance muscle growth and lifelong performance in pigs.
INVESTIGATION OF DISRUPTED INSULIN SIGNALING IN A SWINE MODEL
(2024) Markley, Grace Irene; Stahl, Chad H; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Insulin is an anabolic hormone involved in glucose uptake and synthesis of fats, proteins, and glycogen. Domesticated livestock species such as swine require efficient insulin signaling to meet production demands across the world. Insulin signaling is tightly regulated and acts on metabolic tissues such as the liver, skeletal muscle, and adipose tissue. The most well characterized disruption of insulin signaling is insulin resistance that is often caused by obesity induced inflammation. However, insulin signaling can be disrupted via atypical mechanisms such as immune response to a pathogen and adaptor proteins. We aimed to evaluate the impact of pathogen exposure and intrinsic adaptor proteins on insulin signaling in pigs. The first study focuses on the impact of growth factor receptor bound protein 10 (GRB10) as an inhibitor of insulin signaling. In commercial swine, the presence of GRB10 has been linked to growth, reproduction, feed efficiency and lean muscle growth. While insulin induces glucose uptake in typical tissues, such as the liver and skeletal muscle, insulin also acts on other cell types including mesenchymal stem cells (MSC). MSC are adult multipotent stem cells that can self-renew and differentiate into multiple cell types including adipocytes. The process of adipogenesis requires insulin signaling to synthesize new triglycerides and store them in lipid droplets. While GRB10 has been established as a regulator of insulin signaling, the role of GRB10 in swine MSC has yet to be firmly established. We generated GRB10 knockdown (GRB10-KD) MSC to evaluate the impact of GRB10 on insulin signaling and glucose uptake. We observed reduced glucose utilization under basal conditions and reduced insulin signaling when incubated with insulin over 48 hours. We also noticed a two-fold reduction in proliferation rate among GRB10-KD MSC. When differentiated into adipocytes, we observed an increase in transcript abundance with genes associated with insulin signaling and adipogenesis. GRB10 has the potential to regulate insulin signaling in swine MSC and contribute to overall growth and development. The second chapter focuses on the impact of lipopolysaccharide (LPS), an endotoxin produced by gram-negative bacteria, which can induce a severe, systemic immune response. In pigs, chronic LPS exposure has induced insulin resistance. However, the effects of acute exposure to LPS on insulin signaling and resistance have not been elucidated. We found that acute exposure to LPS in crossbred post-weaning pigs induced changes in insulin signaling and glucose metabolism. There was an LPS induced decrease in insulin two hours after injection which was paired with hyperglycemia. At 24 hours post LPS there was a marked insulin resistance indicated by hyperinsulinemia and hyperglycemia. We also noted that there were liver specific decreases in genes associated with glucose metabolism, insulin signaling and fatty acid metabolism. As well as reduction in protein abundance such as protein kinase B (AKT) and phosphoinositide-3 kinase (PI3K) in the liver after LPS administration. During an acute exposure to endotoxin, insulin signaling, and glucose metabolism is reduced in the liver. These results highlight that insulin signaling is a complex and dynamic process that can be controlled through a variety of mechanisms and swine can serve to model these disruptions.
DIFFERENTIATION AND REGULATION OF BOVINE TH2 CELLS
(2024) Kandel, Anmol; Xiao, Zhengguo Zhengguo; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Memory CD4+ T cells, specifically type-2 (Th2) cells, are pivotal in defending against infections caused by extracellular pathogens, including several economically important parasites. However, whether interleukin-4 (IL4) expression is a signature feature of bovine Th2 cells likewise in mice and humans is unclear. Pasture-raised cattle, routinely exposed to extracellular parasites such as Ostertagia ostertagi (OO), are likely to develop a typical Th2 memory response. Therefore, using cytokine induction assay, we evaluated the circulatory memory bovine T cell profile of these cattle and also analyzed if the expression of presumptuous memory marker, CD45RO, is reliable in identifying memory bovine T cells. Surprisingly, the majority of the memory CD4+ T cells dominantly produced interferon-gamma (IFNγ), with only a small fraction co-expressing IL4, and memory bovine T cell identification did not correlate with CD45RO expression. Results suggested that cattle naturally exposed to extracellular parasites do not develop typically IL4 dominant Th2 response. To further investigate these results, resting CD4+ T cells isolated from healthy cattle blood were cultured under simple in vitro Th2 culture. Analysis of differentiated cells through flow cytometry revealed limited IL4 protein detection, which was in line with the lack of upregulation of IL4 and its master regulator GATA3 transcripts shown by the quantitative polymerase chain reaction (qPCR) assay. To validate whether differentiated cells were actually Th2, unbiased proteomic analysis was conducted. Based on differentially expressed 397 proteins between differentiated cells and naïve phenotype, bovine Th2 differentiation was validated; nonetheless, the process was not found to be associated with IL4 induction. Moreover, despite using published strategies from mice and humans, such as reducing T cell receptor (TCR) stimulation strength and adding exogenous recombinant bovine IL4, the expression of IL4 could not be significantly enhanced. Interestingly, differentiated bovine Th2 cells proliferated in the presence of OO antigens, suggesting that extracellular parasites could influence bovine Th2 differentiation, at least in vitro. To validate the results from pathogen-infected tissues and in vitro culture, a panel of anti-parasitic CD4+ single T cell clones was established from five pasture-raised cattle that were infected with OO. Evaluation of memory responses exhibited by the anti-parasitic CD4+ single T cell clones strongly supported IFNγ dominant memory response, and only 20% of them co-expressed IL4 through a small subset of IFN γ + cells. All the data pointed out that bovine CD4+ T cell differentiation is partially distinct from those in mice and humans, and IL4 expression is not a hallmark feature of the bovine Th2 cells.
Variations in Vocalizations of Fin Whales, Balaenoptera physalus, in the St. Lawrence River
(1980) Edds, Peggy Louise; Buchler, Edward; Animal & Avian Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, MD)
Recordings were made of vocalizations from fin whales, Balaenoptera physalus from a fixed hydrophone in the St. Lawrence River between 28 June and 27 September 1979. Land-based observers monitored activity from a hillside hut while recordings were being made from a shoreline site. Photographs of dorsal fin variations were used to identify distinctive individuals. The 1522 sounds recorded were classified into 11 categories based on frequency and temporal characteristics. The predominant call was a descending sweep of frequencies. Parameters measured for this downsweep exhibited a dichotomy of characteristics which indicates calls with initial frequencies below 40 Hz have less variability than calls with initial frequencies above 40 Hz. In general, solitary animals produced primarily the lower frequency downsweeps. Higher frequency downsweeps were recorded from pairs or trios of fin whales. Solitary individuals did not exhibit unique variations in downsweep parameters. No clusters of values which might indicate uniquely individual ranges were consistently present in multiple animal recordings. The data suggest that the variability of fin whale vocalizations is primarily contextual rather than individual.
Infant and Juvenile-Directed Care Behaviors in Adult Toque Macaques, Macaca Sinica
(1985) Baker-Dittus, Anne; Animal & Avian Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, MD)
The identity of care-givers, and the distribution of care to young are examined in a free-ranging polygynous primate, Macaca sinica. Care behavior is used as one measure of investment in young. There is no evidence that dominance rank influences offspring sex, but high-ranking mothers provide more care to newborn infants than do low-ranking mothers. Mothers provide more care to infant and first year sons than to daughters. This supports Dittus's suggestion (1979, 1980) that mothers should schedule care to sons and daughters differently, providing high levels of care to sons before they emigrate from the natal group. Care to daughters is low per unit time, but continues over a long period because daughters remain in the natal group. Adult females, other than the mother, tolerate, groom and support young. These behaviors are low cost, relative to the high cost behaviors of nursing and carrying, which only mothers provide. Young tend to associate with adult female relatives. Unlike male-biased maternal care, adult female care is biased towards female young; and female young return care more than do male young . Adult female rank is positively correlated with the amount of support adult females provide to female young, and the amount of grooming they receive from female young. Adult female rank has no effect on any measure of association between adult females and male young. Taken together these findings suggest that reciprocal altruism has been important in shaping adult female interactions with young. Adult and subadult M. sinica males direct the low cost, affiliative behaviors of hugging, carrying and grooming towards male infants; and direct aggressive behaviors towards female infants. I suggest that the distribution of behaviors reflects their value to male and female young. Affiliative behaviors to male infants, and aggressive behaviors to female infants serve to channel infants into association with adults from whom they can learn appropriate sex-typical behaviors, and as such benefit both male and female infants. High cost care behaviors of support in agonistic interactions, and protection are provided only by the adult male, and are provided to male and female young equally.
The interaction between a parasitic barnacle, Loxothylacus panopaei (Cirripedia, Rhizocephala), and three of its crab host species (Brachyura, Xanthidae) along the east coast of North America.
(1993) Alvarez, Fernando; Reaka-Kudla, Marjorie; Animal & Avian Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, MD)
Rhizocephalan barnacles parasitize primarily decapod crustaceans (Bocquet-Vedrine 1968, Overstreet 1983). The principal effect of the parasites is often the permanent sterilization of the hosts (Bocquet-Vedrine 1972, O'Brien & Van Wyk 1984). Regardless of the processes involved, the end result of the infection is the creation of non-reproductive individuals that will use resources that otherwise would be available to the healthy traction of the host population. The potential impact on the host population then resides in the accumulation of sterile resource-using individuals in each generation. Prevalences of barnacles on anomuran and brachyuran crabs have been reported in a number of studies (e.g., Walker 1985, Hawkes et al. 1986, Johnson et al. 1986, Wardle & Tirpak 1991 ). Rhizocephalan infections can reach very high levels locally, suggesting that entire host populations may have a greatly reduced reproductive capacity. However, in most host species, the pattern of distribution of parasites is not uniform throughout the host's range. I examined the crab-rhizocephalan interaction using the system composed by the barnacle Loxothylacus panopaei and three of its crab host species: Panopeus lacustris, Eurypanopeus depressus, and Rhithropanopeus harrisii, occurring along the east coast of the North America. Through experimental infections conducted in the laboratory, it was determined that B. harrisii of a wide range of sizes could be infected by L. panopaei. The complete life-cycle of the parasite was manipulated in the laboratory. Neither host molting frequency nor host molt increments differed significantly between parasitized and control crabs. Host survival was significantly reduced during the parasite's developmental period; the heaviest mortality of the host occurred in the megalopal stage.
MOLECULAR ANALYSIS OF CYCLOPHILIN FUNCTION IN THE YEAST SACCHAROMYCES CEREVISIAE
(1994) Davis, Edward S.; Brennan, Miles S.; Digital Repository at the University of Maryland; University of Maryland (College Park, MD)
The cyclophilins are a family of proteins first identified as receptors for cyclosporin A (CsA), a cyclic peptide of fungal origin. CsA inhibits T-lymphocyte activation, and is thus a potent immunosuppressant. Although cyclophilins are ubiquitous, and highly conserved, among eukaryotes, their normal physiological functions are unknown. As the receptors for CsA, cyclophilins might be involved in regulating signal transduction pathways. Cyclophilns also have peptidyl-prolyl, cis-trans isomerase (PPIase) activity in vitro, suggesting a role in protein folding in vivo. While CsA inhibits cyclophilin's PPIase activity, this inhibition is insufficient to account for the pharmacological activity of CsA. Therefore, previous results cannot be readily synthesized into a model for cyclophilin function. The goal of this project was to define and characterize physiological roles of cyclophilins using the yeast S. cerevisiae. Three S. cerevisiae cyclophilin genes were cloned and inactivated by insertional mutagenesis. I demonstrated that one, CPR3, is necessary for the efficient metabolism of non-fermentable carbon sources. The CPR] gene product, Cpr3, is localized to the mitochondrial matrix, and a truncated version of Cpr3 expressed in bacteria binds CsA. CPR3 inactivation does not significantly compromise the induction of transcription of two nuclear cytochrome genes. Thus, Cpr3 is not necessary for the signal transduction pathway governing cytochrome gene expression. To identify biochemical targets of Cpr3, I demonstrated that inactivation of a mitochondrial lactate dehydrogenase is insufficient to account for the growth defect of cpr3 mutants. An exhaustive search for high-copy suppressors of the growth defect of cpr3 mutants led to the identification of a novel gene, JEN1, that suppresses the growth defect at elevated temperature. JEN1 encodes a protein that is probably a lactate transporter, and thus not a direct biochemical target of Cpr3. A dominant mutation in a nuclear gene, JEN2, suppresses the growth defect of cpr3 mutants on lactate at 30°C and 37°C. JEN2 might encode a direct biochemical target of Cpr3. In summary, the cyclophilin, Cpr3, plays a general role in the efficient function of yeast mitochondria, and presents an excellent model system for studying cyclophilin function.
CAUSATIVE AGENTS FOR FOWL TYPHOID AND PULLORUM DISEASE IN POULTRY AND APPROACH TO CONTROL
(2023) Julianingsih, Dita; Biswas, Debabrata; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
In response to escalating consumer demand, a significant number of conventional US poultry farms have transitioned to antibiotic-free and chemical-free practices, particularly those adopting pasture/organic methods. However, recent reports highlight a resurgence of bacterial diseases in both conventional and pasture poultry farms, resulting in elevated bird mortality rates and reduced profitability. This comprehensive study investigates the prevalence of S. Gallinarum and S. Pullorum, causative agents of fowl typhoid and pullorum disease, in integrated crop-livestock/pasture farm environments and meat products. A total of 1,286 samples from 7 farms and 13 retail markets were examined, revealing that S. Pullorum and S. Gallinarum are common in both farm poultry environments and market products. Antibiotic resistance patterns, determined through an antibiogram assay, indicated high resistance to multiple antibiotics. S. Pullorum and S. Gallinarum were discovered in 2.7% and 1.5% of samples, respectively, at the pre-harvest stage. Only 1.6% of the meat samples recovered from retail markets had S. Gallinarum detected in them at the post-harvest level. Concurrently, a different study investigates the possibility of Orange Cold-press Valencia Terpeneless, a citrus oil variety, acting as a natural antimicrobial in poultry farming. This study tackles the problems caused by a decline in the usage of antibiotics, which has resulted in an increase in bacterial infections. Citrus oil exhibits potential as an antimicrobial agent, inhibiting the growth of S. Pullorum and S. Gallinarum, with consistent MIC and MBC values. Time-dependent experiments with 0.4% citrus oil show total suppression of bacterial growth, which is confirmed by environmental simulations. Furthermore, the study reveals that both Salmonella strains have downregulated their virulence genes, which may indicate a change in the pathogenicity of the bacteria. Overall, the findings highlight the crucial importance of surveillance programs and preventive measures. Citrus oil is presented as a promising natural alternative for antibiotics in the treatment of Salmonella-related infections in the poultry farming industry.
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.
Metagenomics and molecular tools for monitoring stress conditions and disease susceptibility in rainbow trout
(2023) Januario, Fabiane C; Salem, Mohamed; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Examining gene transcription alternative splicing can distinguish gene-splicing events associated with stress and identify biomarkers that can be used to monitor stress conditions and decrease sampling invasiveness. Previously, we in silico predicted that a gene called prolyl 4-hydroxylase subunit alpha-2 (P4HA2) with retained intron is expressed under stress conditions. In this study, we aimed to validate the intron's retention and hypothesized that it would continue to show differential expression in fecal and mucus samples. The in vitro expression of the intron verifies the in silico data, where the intron of interest was upregulated during stress conditions. Separately, a microbiome study using the same samples identified bacterial taxa predictive of stress. Higher levels of 3 bacterial biomarker taxa species belonging to the genera Romboutsia and Cetobacterium were more abundant in the stressed fish. However, a pathogenic class of bacteria (Gammaproteobacteria) was significantly higher in the control group.
The Life Cycles, Ecology, and Evolution of the Witch-hazel Leaf Gall Aphid, Hormaphis hamamelidis (Fitch) (Homoptera: Aphidida)
(1987) von Dohlen, Carol Dean; Gill, Doulgas E.; Zoology; Digital Repository at the University of Maryland; University of Maryland (College Park, MD)
Two divergent life cycles based on geographic location have been documented for the witch-hazel leaf gall aphid, Hormaphis hamamelidis (Fitch, 1851). At low elevations in northern Virginia, the aphid was found to have seven distinct generations alternating between the primary host, Witch-hazel (Hamamelis virginiana L.), and a secondary host, river birch (Betula nigra L.). These findings confirm the original published life cycle description from the same locality. A second, abbreviated life cycle consisting of only three generations restricted to witch-hazel was discovered at high (1000 m) elevations in north central and northwestern Virginia. Aphids with each life cycle were sympatric at an intermediate elevation site. Based on available life cycle and geographic data, a preliminary Phylogeny of the tribe Hormaphidini is proposed that suggests an unusual polarity in the evolution of aphid life cycles. Several features of intraspecific interactions and host-plant relations were examined in both lowland and highland populations of Hormaphis. In contrast to previous publications documenting severe competition, density effects, and habitat heterogeneity for another galling, host-alternating aphid, Pemphigus betae on Populus angustifolia, the effects of density and host-plant qualities on Hormaphis hamamelidis were fewer and more benign. Aphids did not compete for gall sites, and gall Position and final leaf area did not influence reproduction. High gall densities negatively affected gall growth and aphid fecundity. Factors accounting for the differences in Population dynamics between Hormaphis and Pemphigus are hypothesized and discussed.
The study of hyperketonemia in the dairy cow.
(2023) Barrientos-Blanco, Mario Alberto; Rico, Eduardo; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
The metabolic phenomenon of ketosis in dairy cows has remained ambiguous, casting uncertainty over our understanding and its real implications. Ketosis, commonly defined as blood β-hydroxy-butyrate (BHB) ≥ 1.2 mM (i.e., hyperketonemia), has been observationally connected to the onset of peripartal metabolic disorders (e.g., infectious diseases, fatty liver), and reduced milk yield in dairy cows. Although BHB is currently used as standard biomarker for the prediction of negative health and performance outcomes during the peripartum, the nature of this relationship is ambiguous. In contraposition, recent discoveries in mammalian biology indicate BHB as therapeutic metabolite (e.g., alleviation of inflammation and oxidative stress). Our overreaching goal was to study the effects of BHB on dairy cow metabolism and health. In our first study, 6 multiparous (parity = 2.8 ± 0.9) Holstein mid-lactation dairy cows (128 ± 52 days in milk; DIM), were enrolled in a study to evaluate a ketogenic diet using calcium butyrate (—CaBu—; a ruminal ketone precursor) against an un-supplemented control (Control) in a crossover arrangement of treatments. The CaBu resulted in nutritional ketosis (P < 0.05) with blood BHB levels of 0.2 mM higher relative to Control. Although CaBu resulted in reduced dry matter intake (DMI; P < 0.05), milk production was not affected (P > 0.40), and feed efficiencies were improved (P < 0.05) relative to Control. No differences in glucose, NEFA, respiration rates, pain scores, or rectal temperatures were observed between treatments. In the second experiment, 8 multiparous Holstein (2.75 ± 0.89) mid-lactation dairy cows (140 ± 48 DIM), feed ad libitum, were enrolled in a in a crossover arrangement of treatments. The aim of the study was to evaluate the effect of ketones by intravenous infusion of either Na-BHB solution (2.5mM; EK) to sustain hyperketonemia —BHB > 1.2 mM and < 3.0 mM—, or NaCl as a control (2.5mM; Control) over a 72h period. A systemic lipopolysaccharide (LPS) challenge (E. coli 055:B5; 0,085 g/kg BW,) was intravenously administered at h 60 from infusion start. Cows sustained hyperketonemia throughout the 72h experimental period (1.4 BHB mM vs. 0.7 BHB mM in EK vs. Control, respectively). While DMI and milk production were not affected by the BHB infusion, the combination with the LPS challenge resulted in reductions of 20.8% (P < 0.05) and 40.1%, (P = 0.14) for both measurements in EK vs. Control, respectively. No differences were detected in the glucose and NEFA concentrations, but insulin was higher 46.6% (P < 0.05) in EK group. Among the immune markers, IL-1 was 30.8% higher (P < 0.05) in the EK group, and not differences were detected in TNF, IL-10, CRP, and caspase-1. As expected, the LPS challenge induced increased respiration rates, temperature, and pain scores over the time course of the evaluation (P < 0.001); however, respiration rates tended to be reduced in 8.4% (P < 0.1) and rectal temperature increased in 0.3% (P < 0.05) by the BHB treatment (P < 0.05). Our results are indicative that, in the absence of an immune challenge, hyperketonemia results in no negative impact on cow productivity and health. These data add support to our hypothesis that cofactors other than ketones may be necessary for the development of negative trajectories of health and performance of lactating dairy cows. Future studies will be required to confirm that BHB hyperketonemia metabolic effects could differ from ketosis disorder in dairy cows.
PITUITARY-TARGETED KNOCKOUT OF GLUCOCORTICOID RECEPTORS SUPPRESSES GROWTH HORMONE EXPRESSION DURING MOUSE EMBRYONIC DEVELOPMENT
(2023) Klug, Scott; Porter, Tom E; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
The pituitary, an endocrine gland primarily regulated by the hypothalamus, secretes multiplehormones and regulates the release of several other hormones from multiple glands within the body. Pituitary development is conserved across vertebrate species and involves a complex temporal and spatial balance of multiple transcription and signaling factor gradients, which control cell commitment, differentiation, and proliferation. Proper pituitary development is critical to survival, since several essential physiological processes are regulated by the pituitary during embryogenesis and throughout life, including metabolism, milk production, stress, reproduction, and growth. Glucocorticoids (GCs) produced by the adrenal glands during embryogenesis play an important role in the differentiation of somatotrophs, the endocrine cell type within the pituitary that produces growth hormone (GH), as well as GH mRNA expression in both avian and mammalian species. In chickens, somatotrophs differentiate between e12 and e16, while mouse somatotrophs differentiate between e15.5 and e17.5. The establishment of the hypothalamic-pituitary-adrenal (HPA) axis and a rise in circulating levels of corticosterone(CORT), the primary GC in rodents and chickens, coincides with somatotroph differentiation and GH mRNA expression in both rodents and chickens. Furthermore, exogenous synthetic GCs such as dexamethasone have been shown to prematurely differentiate somatotrophs in rats and chickens in vitro and in vivo. GCs generate physiological reactions by binding to target cells that express the glucocorticoid receptor (GR). GR, in turn, acts as a transcription factor and regulates the expression of several genes. Despite numerous studies on GH mRNA regulation and somatotroph differentiation by GCs, the mechanism is not completely understood. To better understand the role GR plays in GH regulation by GCs in a mammalian model, mouse embryos with pituitary-targeted GR knockout were generated utilizing the Cre-LoxP Recombinase system under control of the pituitary-specific αGSU promoter. GH mRNA was significantly decreased in GR(-/-) embryos, while GR(+/-) embryos expressed intermediate levels of GH mRNA in comparison to wild-type GR(+/+) embryos. Significant differences in expression of other pituitaryhormones in GR(-/-) embryos were not observed, indicating that GR must not play an essentialrole in regulating the expression of any other pituitary hormone gene. Furthermore, all GR(-/-) embryos died at birth, or soon after. To our knowledge, this is the first study to report homozygous GR knockout in the pituitary suppresses embryonic GH expression and results in a neonatal lethal phenotype.
MECHANISMS CONTROLLING VOLATILE FATTY ACID AND FERMENTATION GAS PRODUCTION IN THE RUMEN
(2022) Scott, Jarvis G; Kohn, Richard A; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Atmospheric methane accounts for less than approximately 16% of global anthropogenic greenhouse gas emission. However, it is significantly greater and trapping heat when compared to atmospheric CO2 on a molar lever and any reduction in atmospheric abundance in warranted. Enteric methane from ruminant species accounts for a fraction (< 30%) of the total atmospheric methane however its production also accounts for major dietary energy loss in ruminant species and affects feed efficiency and overall production. Major studies have investigated numerous feed additives and supplements with highly variable finding on the antimethanogenic property of these compounds or feeding strategies, however the findings have raised other questions regarding shifts in VFA profiles accompanying methane inhibition. Higher inclusion levels of concentrate and other nonstructural starch in the diet of ruminants have been shown to decrease methane production and shift volatile fatty acid (VFA) profiles in the rumen. Additionally, many studies have suggested that inhibiting methane production avails as a reducing equivalent to fuel the propionate producing pathway and therefore results in shift in VFA profiles in the rumen. However, very little is understood regarding how these VFA shifts come about. Microbial Kinetics and thermodynamics are physiochemical principles that can be used to study how concentrate inclusion in ruminant diets can change the substrate concentrations and ultimately lead to shifts in fermentation profile in the rumen. Substrate availability supports and/or limits the growth of microbial population in the rumen, while the accumulation of the products or reactants for major fermentation reactions dictate the profile of the VFA. Understanding the role of these physiochemical principles and ultimately the mechanisms involved with changes the profile of VFA and fermentations gas in the rumen would help researchers understand how VFA profiles are shifting during methane inhibition as well as possibly identifying a more targeted approach for inhibiting enteric methane production. Therefore, the objectives of this projects are: to develop an in vitro method to understand the basal kinetic parameters of metabolism in the rumen, to evaluate the effects of increasing forage-to-concentrate ratio on performance and change in in VFA and fermentation gas in vivo, and to test the effect of various perturbations (fermentation metabolites e.g. sodium acetate, sodium lactate etc.) on the fermentation profile of rumen fluid adjusted to different forage-to-concentrate ration. The results indicate that rumen fluid from cows on a high-concentrate diet have a greater capacity to make propionate compared to the high forages diet. The higher propionate production limits the availability of which is necessary for the synthesis of CH4. The finding also suggests that methanogenesis is process limited by substrate concentration. Finally, our studies indicate that feeding strategies targeting enzymatic activity favoring propionate production may be more beneficial than targeting methanogens in a high forage diet.
Vertical Resource Partitioning and Sexuality of Three Sympatric Species of Red Sea Sandfishes (Xyrichtys melanopus, Labridae; Trichonotus nikii, Trichonotidae; Gorgasia sp., Congridae)
(1988) Krall, Marianne Martha; Clark, Eugenie; Zoology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md)
Three species of marine sandfishes were studied from 1984 to 1986. Their inter- and intraspecific behavior was monitored during the daylight hours to examine interactions that could result in the vertical stratification of the species over the sandy bottom within the fringing and patch reefs in the north Red Sea. Horizontal plankton tows were taken at three heights and three times a day. These samples were compared to stomach contents of the fishes to determine the trophic relationships in the community and their affects on spatial relations between the species . Prey specificities of the fishes were determined by using an electivity measure. Using paraffin histology, Xyrichtys melanopus was determined to be a monandric protogynous hermaphrodite and Trichonotus nikii, a gonochorist. Previous work on the mating systems and territoriality of all three sandfish species helped in part to explain the vertical spatial arrangement of the sandfish species within the community. Effects of pollution on the b iota of the Northern Gulf of Aqaba are noted.
EFFECTS OF ENVIRONMENTAL ENRICHMENTS ON WELL-BEING MEASURES IN COLONY-CAGED JAPANESE QUAIL (COTURNIX JAPONICA)
(2022) Mathkari, Chirantana Vikas; Dennis, Rachel L; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Breeding purpose Japanese quail (Coturnix japonica) are often maintained in barren cages with little environmental complexity. These conditions can lead to unwanted social interactions and stress-related behaviors that can cause serious injury, mortality, and reduce productivity. Use of environmental enrichments has shown to improve poultry well-being; however, the optimal enrichments for quail have not been widely studied. The aim of this project was to evaluate the effectiveness of various enrichments on the well-being and productivity of breeding quail colonies (1 male, 2 females/cage). The enrichments studied were designed to reduce unwanted interactions and stress-related behaviors by either providing a shelter (protective enrichments) or by providing mental stimulation (stimulatory enrichments). In Experiment 1, using an incomplete Latin square design, each cage received one protective enrichment (Hut, Plastic leaves, or Grass), or one stimulatory enrichment (Mirror, Feeder toy, or Mat), or no enrichment (control) (average n=17/treatment). In Experiment 2, using an incomplete Latin square design, each cage received one of the following enrichment combinations: Hut + Mirror, Hut + Feeder toy, Hut + Mat, or only Hut (average n=14/treatment). Parameters measured included behaviors, body weight, Hen Day Egg Production (HDEP), egg weight, physical scores, and fecal corticosterone metabolites (FCM) levels. Measures were analyzed using a one-way ANOVA or Chi-square test on SAS 9.4. The results indicate that exposure to a single enrichment reduced stress-related damaging behaviors and increased resting; while exposure to a combination of enrichments reduced a larger variety of stress-related damaging behaviors more efficiently. Two of the three protective enrichments (Hut, Grass) exerted a notable impact on production, while combinations of protective and stimulatory enrichments were more efficient in decreasing physical scores as compared to a single enrichment. The Hut + Mat combination notably reduced the FCM levels as compared to only Hut. Our study identified enrichments which are optimal for improving breeding quail well-being, and exhibit the potential to improve the economics of the quail industry.

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