Animal & Avian Sciences Theses and Dissertations

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Adaptation of A/Mallard/Potsdam/178-4/83 (H2N2) in Japanese quail leads to Replication and Transmission in Chickens
(2005-05-02) Sorrell, Erin Maureen; Perez, Daniel R; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Influenza is a single stranded, negative-sense RNA virus with a segmented genome that can infect avian and mammalian species. Influenza viruses from the avian reservoir do not seem to replicate efficiently in humans upon direct transmission. Therefore, an intermediate host is involved in generating mutations to create a more transmissible or an avian-human reassortant virus. Quail have been highlighted as a potential reservoir and intermediate host of avian influenza. To assess the potential of quail as an intermediate host, A/Mallard/Postdam/178-4/83, H2N2 was tested to determine if through adaptation in quail a mallard strain can replicate and transmit in quail, as well as other avian species. After six serial passages of lung homogenate a virus arose, which replicated and transmitted directly to contact quail. When chickens were infected with this quail-adapted virus replication and transmission were observed, while no replication was noted in the chickens infected with wild type H2N2 virus.
ANALYSIS OF THE ROLE OF PRDM14 IN PLURIPOTENCY AND SPECIFICATION OF THE GERM CELL NICHE IN THE DOMESTIC PIG
(2018) Sandmaier, Shelley; Telugu, Bhanu; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
The generation of functional, mature germ cells in order to pass on its genetic information to the next generation is of paramount importance to a species’ survival. As a result, primordial germ cells (PGC), the precursor cells that give rise to mature spermatozoa or oocytes, are one of the first cell types to be specified and induced in the early mammalian embryo. In mammals, this process happens during the primitive streak stage of development and PGCs continue to proliferate as they migrate from the posterior end of the embryo through the hindgut and settle in the future gonadal region. One gene in particular, PRDM14, is required for PGC specification in the mouse, but its function was unknown in the domestic pig (Sus scrofa). This dissertation project sought to determine the role of PRDM14 in the porcine germ cell program as well as in early embryo development. The first study used the mouse as a model using CRISPR/Cas9 technology to direct pluripotent stem cells toward the germ cell pathway by aggregation with a host embryo that was unable to form PGCs. Chimeras generated by this technique exhibited 100% occupation of the germline by donor control stem cells. In the second study, mRNA transcripts for a suite of germ cell-related genes were analyzed to determine their expression in various stages of the early porcine embryo: from zygote to blastocyst and embryonic day 28. Unlike in the mouse and human systems, several genes that were predicted to be expressed in the early embryo (PRDM14, TET1, and PRDM1, among others) were found to be extremely low in abundance. In the final study, the function of PRDM14 in the pig was disrupted using gene editing, and the resulting phenotype was characterized. Loss of PRDM14 does not seem to be critical for the specification of PGCs in the domestic pig, as evidenced by the existence of germ cells following knockout. Together, these studies have applications for spermatogonial stem cell transplantation technology in order to expand the genetic reach of superior animals by producing animals whose germ cells are derived from a genetically valuable donor.
ANTAGONISTIC MECHANISM OF METABOLITES FROM LACTOBACILLUS CASEI AGAINST FOODBORNE ENTEROHEMORRHAGIC ESCHERICHIA COLI
(2022) Aditya, Arpita; Biswas, Debabrata; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Enterohemorrhagic Escherichia coli O157: H7 (EHEC), a foodborne enteropathogen, remains a significant public health concern since its discovery in 1982. With an incredibly low infectious dose (10-100 bacteria), this pathogen can cause self-limiting diarrhea, vomiting, and abdominal cramps. However, more complicated disease conditions such as bloody diarrhea or hemolytic colitis have been known to develop depending on the serotype involved in the infection, and on immune status and/or age of the patients. Due to its Shiga toxin (Stx) production ability, EHEC infection may lead to a kidney-related problem known as hemolytic uremic syndrome (HUS), which requires advanced medical care. Unlike other bacterial illnesses, therapeutic administration of antibiotics to treat EHEC infections is not recommended due to their controversial association with Stx production. As a result, only preventative/prophylactic and immune-supportive strategies are followed for EHEC infections. Using the antibacterial properties of probiotic bacteria and the metabolites they produce are promising alternative strategies for preventing EHEC infections. We have targeted the probiotic bacteria Lactobacillus casei to determine the mechanism of this alternative strategy. In our study, we have executed microbiological, molecular, chromatographic, and metagenomic approaches to determine the antagonistic mechanisms of action of their metabolites, specifically conjugated linoleic acid (CLA) produced by Lactobacillus casei, against the growth and metabolism of EHEC. The metabolites of wild-type L. casei (LCwt) were augmented by supplementing it with a prebiotic-like dietary component, namely peanut flour (PF) (LCwt+PF), while another LCwt was also genetically engineered (LCCLA) to over convert CLA from linoleic acid (LA). These modifications showed effective results in controlling EHEC both in vitro and in ex vivo conditions. Total metabolites present in cell-free culture supernatant (CFCS) of LCwt, LCwt+PF, and LCCLA were able to control the growth of EHEC without negatively hampering the relative abundance of Firmicutes and Bacteroidetes present in rumen fluid (RF). Among these CFCSs, CFCSCLA exerted the most desirable outcome by eliminating EHEC. In vitro studies demonstrated that, a lower concentration of purified CLA worked synergistically with other metabolites of LCwt and augmented their inhibitory activity against EHEC. The orchestrated effect of metabolites has been observed to downregulate the virulence genes, disrupt the cell membrane, interfere with cell division, and damage their genomic DNA. The probable effect of these metabolites, specifically CLA, on Stx production and neutralization was also investigated by assessing host cell cytotoxicity. Total metabolites of Lactobacillus spp. as well as CLA itself, showed improvement in cell survivability when exposed to Stx. Our findings established a ground to explore the effect of specific metabolites obtained from probiotic bacteria in control and prevention of EHEC. The findings also showed a promising association of purified CLA in neutralizing Stx which can be further explored to use it in therapeutic purposes.
ANTIBIOTIC ALTERNATIVES FOR TREATMENT OF MASTITIS IN DAIRY CATTLE
(2019) Scholte, Cynthia Madeleine; Moyes, Kasey M; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Mastitis is one of the most costly diseases to the dairy cow industry. Implementation of management protocols have decreased the incidence rate of contagious mastitis pathogens, while the incidence of cases caused by environmental pathogens, such as Escherichia coli (E. coli) and Streptococcus uberis (Strep. uberis), remains unchanged. Currently, antibiotics are the primary therapy utilized to control mastitis. However, growing concern for antibiotic overuse, improper use and bacterial resistance have led to the examination of alternative strategies. Two promising alternative mitigation strategies were explored in the research leading to this dissertation. The first is a potent endolysin, PlyC, which has demonstrated bactericidal activity against several streptococcal species in a variety of applications. However, it is unknown how PlyC interacts with the bovine immune system. Varying doses of PlyC were non-toxic and did not alter the oxidative burst response of bovine neutrophils, the first immune cells recruited to the intramammary infection. The second alternative to an antimicrobial drug explored was citrus oil dissolved in ethanol. This was tested against E. coli strain P4. Prior antimicrobial investigations with citrus oil utilized dimethylsulfoxide as a carrier solvent, which is currently prohibited in the dairy industry. Citrus oil and its primary components were tested to determine minimum inhibitory, bactericidal and biofilm eradication concentrations. Citrus oil components, citral and linalool, demonstrated better antimicrobial activity than the native oil in vitro. In addition, citral and linalool were minimally toxic to bovine neutrophils and did not hinder their oxidative burst response in vitro. Because citral demonstrated greater antimicrobial activity than linalool, it was the logical candidate to test for the efficacy as an intramammary therapy against experimentally-induced E. coli mastitis. Unfortunately, citral treatment did not differ from a common intramammary antibiotic therapy or sterile buffered solution. In conclusion, the use of PlyC as an alternative therapy for Strep. uberis mastitis is promising as PlyC may not interfere with immune response during mastitis. The antimicrobial effects of citral may better serve as a disinfectant than a therapy for E. coli mastitis.
ASSESSMENT OF MALE GIANT PANDA SEASONAL REPRODUCTION, SEXUAL MATURITY AND COMPARATIVE SPERM CRYOTOLERANCE
(2010) Aitken-Palmer, Copper; Ottinger, Mary Ann; Howard, JoGayle; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
The giant panda is unique as one of the most studied endangered species. Interestingly, despite this, little is known about basic male reproductive physiology. Behavioral problems have posed challenges for reproductive success in the captive population, while geographical isolation and habitat fragmentation threaten the wild population. Techniques such as sperm cryopreservation can improve genetic management and reproductive success within captive populations. But without comparative freeze-thawing studies, diverse methods used for cryopreservation result in inconsistent post-thaw quality, potentially compromising sperm quality and fertility for artificial insemination. This, compounded by limited knowledge of male reproductive physiology, has challenged conservation goals for the giant panda. Specific objectives of this study include: 1) assess the influence of season on male giant panda reproduction by determining annual changes in testis size, body size, testosterone production, sperm quality and production, and reproductive behaviors throughout the year; 2) characterize the onset of sexual maturity in the male giant panda by documenting the timing of initial sperm production, increasing testicular size and body weight, rising androgen production and initiation of reproductive behaviors; and 3) identify factors influencing giant panda sperm cryopreservation by comparing the effect of freeze-thawing on sperm motility, acrosomal integrity, forward progressive movement and longevity. Results provide evidence that 1) reproductive seasonality in the male giant panda is more protracted than the female, with coordinated increases in testes size, androgen production, sperm density, and sexual behaviors; thereby maintaining reproductive competence to prepare for, and accommodate, a brief unpredictable female estrus; 2) sexual maturity in the male giant panda is first observed with consistent sperm production at 5.5 yr, then accompanied by increasing body weight, fecal androgen concentrations, and culminating with the onset of reproductive behaviors, such as scent marking between 7.5 - 8.5 yr; and 3) the giant panda spermatozoon has the capacity to withstand cold stress applied by multiple means, specifically after freezing and thawing using a unique field friendly dry shipper approach. These data greatly improve our understanding of male giant panda reproductive physiology, specifically seasonal rhythms, the cascade of changes associated with sexual maturation, and insights into improved comparative approaches for sperm cryopreservation.
ASSOCIATION OF SINGLE NUCLEOTIDE POLYMORPHISMS WITH PHENOTYPIC PRODUCTION TRAITS IN BROILER CHICKENS
(2009) Liu, Xuan; Porter, Tom E; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
This research investigated the association between SNPs and phenotypic production traits in fat and lean chicken broiler lines. In previous research, eleven SNPs in the promoter regions of four candidate genes were selected. In this study, significant associations were detected between AKR1B10 SNP1 and SDC1 SNP1 and fat yield. SDC1 SNP1 was significantly associated with fat weight. SOD3 SNP2 was associated with breast yield. Five sire-SNP interactions and one sex-SNP interaction were significant. There was a significant interaction between sex and SDC1 SNP3 on muscle-related factor. GPC3 SNP1 interacted with time period on body weight from week 1 to week 9. QTLs on chromosomes 1, 3 and 4 for body fat were refined by incorporating these SNPs into QTL analysis. These genetic markers may be of great value for marker-assisted selection (MAS) for chickens with less abdominal fat as well as genetic markers for body fat accumulation in humans.
BIOACTIVE LACTOBACILLUS CASEI IN REDUCING GROWTH AND COLONIZATION OF CAMPYLOBACTER JEJUNI
(2018) Tabashsum, Zajeba; Biswas, Debabrata; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Campylobacter jejuni (CJ) is one of the pre-dominant causative agents of acute gastroenteritis in the US and occurs commonly through handling/consumption of contaminated poultry products. Probiotics with enhanced bioactive metabolites such as conjugated linoleic acids (CLAs) play crucial role in improving host health and act as antimicrobials. Further, prebiotic like components such as bioactive phenolics from berry pomace extract (BPE) can stimulate growth of beneficial microbes including Lactobacillus casei (LC) and inhibit bacterial pathogens in vitro. In this study, we aimed to assess efficiency of CLA overproducing LC (LC+mcra) alone or in presence of BPE against CJ. LC+mcra alone or LC+mcra with BPE reduced CJ growth, adhesion and invasion efficiency to cultured cells and also altered physicochemical properties, gene expressions related to virulence. These findings suggest, BPE and LC+mcra in combination may able to prevent CJ colonization in poultry and reduce cross-contamination, hence control foodborne infections with CJ in human.
BIOLOGY AND MANAGEMENT OF REPRODUCTION IN CAPTIVE CRANES
(2017) Brown, Megan Elizabeth; Keefer, Carol L; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
There are 15 species of cranes found throughout the world, 11 of which are listed as vulnerable or endangered. All 15 species are currently managed in captivity; however, with increased threats to wild crane habitats and populations, ex situ management becomes increasingly critical as a hedge against extinction. Reproduction and the production of offspring is required to ensure self-sustaining populations managed in ex situ conservation breeding programs. However, current reproductive success of the endangered whooping crane (Grus americana), as well as other species, maintained ex situ is suboptimal and hinders population sustainability and reintroduction goals. The objectives of this dissertation were to 1) develop a cryopreservation protocol for crane semen to improve genetic management in endangered cranes, 2) investigated seasonal hormone patterns and measured the impact of captive environment on hormone production and reproductive behaviors, and 3) retrospectively examine the effect of bird as well as management variables on egg fertility in whooping cranes. The findings demonstrated that 1) sperm of both whooping and white-naped crane performed better following cryopreservation when dimethyl-sulfoxide is utilized as a cryoprotectant, 2) seasonal fluctuations occur in hormone production in both sexes, while addition of a water feature to captive enclosures stimulated reproduction in females, and 3) female specific variables had the greatest influence on probability of egg fertility. Overall findings will help whooping crane management Continued research into the mechanisms controlling sperm sensitivity to cryo-damage, egg production, and fertilization are necessary to mitigate reproductive problems in captive crane species.
BOVINE NEUTROPHILS RELEASE EXTRACELLULAR TRAPS UPON STIMULATION WITH OSTERTAGIA OSTERTAGI
(2018) Mendez, Jonatan; Xiao, Zhengguo; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Ostertagia ostertagi is a widespread parasite that causes significant production losses in the cattle industry. Recently discovered neutrophil extracellular traps (NETs) have been demonstrated as important effector mechanisms of neutrophils against various pathogens including parasitic worms. Exposure of neutrophils to O. ostertagi extract resulted in a significant release of extracellular DNA and co-localization of NET associated proteins histone and neutrophil elastase confirmed these structures of DNA as NETs. In response to both live and heat-killed O. ostertagi larvae, there was a similarly strong release of NETs. O. ostertagi induced NETs were significantly diminished by inhibition of enzymes NADPH oxidase, neutrophil elastase, and myeloperoxidase. Interestingly, NETs were also released in response to non-pathogenic nematode C. elegans indicating a potential conserved response to nematodes. Mouse neutrophils demonstrated a similar NET response to O. ostertagi however there was no response to C. elegans. Surprisingly, these NET responses did not appear to be dependent on production of reactive oxygen species (ROS) as has been previously reported. This is the first report indicating O. ostertagi-induced NET formation and indicates a potential role for NETs in the response against O. ostertagi infection.
Campylobacter spp. in bulk tank milk and milk filters from US dairy farms
(2015) Del Collo, Laura P; Biswas, Debabrata; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Campylobacter spp. are a common cause of foodborne outbreaks associated with raw or unpasteurized milk, and Campylobacter spp. have also been detected on most dairies in the US. An estimate of the prevalence of thermophilic Campylobacter spp. in bulk tank milk (BTM) on US dairy operations was determined as part of the National Animal Health Monitoring System’s Dairy 2014 study. Campylobacter spp. were detected in the BTM and milk filters from 34.2% of the 234 dairies. Isolates were obtained from 18.4% of the dairies. C. jejuni was the most frequently isolated species, and this species is also the most common cause of human infection. When resistance to a panel of nine antimicrobials was tested, 68.4% of C. jejuni isolates were resistant to tetracycline. This survey suggests that BTM from US dairies can be contaminated with pathogenic Campylobacter spp., and the consumption of unpasteurized, raw milk represents a human health risk.
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.
The Captive Maned Wolf (Chrysocyon brachyurus): Nutritional Considerations with Emphasis on Management of Cystinuria
(2005-05-12) Childs-Sanford, Sara; Angel, Roselina; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
The intent of this project was to investigate options for improvement of a commercially manufactured prescription diet designed to reduce manifestation of clinical disease secondary to cystinuria in captive maned wolves in the United States. Diets high in plant-based protein, independent of sulfur amino acid content, resulted in an increase in average urine pH as well as a decrease in urine cystine crystal formation and cystine excretion in maned wolves. The rate of passage of digesta in maned wolves was very similar to that of domestic dogs on either the commercially available formula and an experimental formula. Differences in nutrient digestibility and mineral retention were seen between the wolves and the dogs, with dogs exhibiting higher digesitibility or retention in all cases. Six maned wolves maintained on these same two diets exhibited plasma taurine concentrations markedly lower than canine and feline normal reference ranges, implying that maned wolves may have a dietary requirement for taurine.
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.
CELLULAR PATHWAYS INVOLVED IN EPITHELIAL-TO-MESENCHYMAL TRANSITIONS IN NEURAL CREST CELLS
(2013) Li, Shen; Taneyhill, Lisa A; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Neural crest cells are a population of multi-potent progenitor cells in the developing vertebrate embryo that undergo an epithelial-to-mesenchymal transition (EMT) and migrate extensively to generate diverse derivatives. As such, abnormal development of neural crest cells can lead to human congenital and hereditary malformations, diseases and cancers. Both internal molecular signals and external mechanical factors play essential roles in facilitating neural crest cell EMT. How cells modulate their adhesion machinery and dynamically reorganize their actin cytoskeleton to respond to the mechanical features of their external environment during EMT is not well understood. To evaluate the role of the actomyosin cytoskeleton during neural crest cell EMT and migration, midbrain neural folds that contain premigratory neural crest cells were dissected out from chick embryos, explanted into chamber slides, and incubated to allow for the formation of migratory neural crest cells. Time-lapse imaging technique was used to record cell behaviors. To elucidate cellular pathways controlling EMT and migration, chemical inhibitors (blebbistatin, Y-27632, latrunculin-A, and nocodazole) that perturb molecular cascades regulating cellular structures were employed. Effects of these perturbations on neural crest cell EMT and migration were quantified in terms of the spreading rate of the explants, and vorticity of collectively moving cell groups. We observed that blebbistatin treatment reduced the overall velocity of migratory neural crest cells to negligible levels. Moreover, migratory neural cells developed rounder cell bodies, and lamellipodia were transformed into filopodia at the periphery of the extract. Y-27632 treatment led to more neural crest cells coming out from these explants within a shorter time period compared to control. Nocodazole treatment blocked neural crest cell EMT and the resumption was dose-dependent. Latrunculin-A caused cell death at a very low concentration. These results implicate roles for non-muscle myosin II, the target of blebbistatin, and ROCK, the target of Y-27632, as well as microtubules and actin filaments, in chick midbrain neural crest cell EMT and migration. Actin crosslinkers such as α-actinin and actin-associated proteins like palladin also participate in pathways affected by these cytoskeletal inhibitors through their regulation of focal adhesion formation and cytoskeletal organization, thereby modulating cell stiffness and migration. We are also documented the distribution of α-actinin and palladin in migratory neural crest cells in vivo. Collectively, our studies have provided insight into specific cellular pathways regulating neural crest cell EMT and migration and the impact on various biophysical parameters upon perturbation of these pathways.
Characterization and Control of Aggression and Reproduction in the Male Clouded Leopard (Neofelis nebulosa)
(2012) DeCaluwe, Heather Burton; Ottinger, Mary Ann; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Clouded leopards are a striking and elusive cat species whose secretive nature has made it difficult to gather information on population statistics and behavior in the wild, where the population is in decline. While captive populations are intended as a hedge against extinction, breeding clouded leopards ex situ has been a challenge, primarily due to extreme male aggression toward females. Despite the importance of aggression in this species, there has as yet been no systematic study characterizing the basis of aggressive episodes. Two mechanisms seem to underlie the aggressive behavior in clouded leopards: degree of anxiety and circulating testosterone levels. Three studies were conducted to characterize mechanisms modulating aggression in male clouded leopards. In Study 1, sixteen adult male clouded leopards were categorized as `anxious' or `calm' using a keeper questionnaire and fecal endocrine (androgen and glucocorticoid) profiles; these measures were correlated with behavior rates and frequencies before, during, and after a series of behavioral reaction tests aimed at assessing an individual's response to stress-inducing situations. In Study 2, the behavioral and endocrine responses to the same tests were compared in the same clouded leopards following three treatments: 1) an anxiety-reducing psychotropic drug (clomipramine, n = 4); 2) a gonadotropin releasing hormone agonist (deslorelin, n = 5), or 3) no treatment (n = 4). In Study 3, the long-term effects of the drug treatments on spermatogenesis and hormone concentrations were compared in clouded leopards (n = 2/treatment) and domestic cats (n = 5/treatment), a model for non-domestic felid reproduction. Studies revealed important findings about the basis of aggressive behavior in male clouded leopards. First, two of the behavioral reaction tests - `mirror image stimulation' and `unfamiliar people' - were effective tools for evaluating temperament and eliciting a behavioral response. Second, treatment with both clomipramine and deslorelin reduced anxious and aggressive behaviors (e.g. `tail flicking' and `growling') indicating multiple physiological mechanisms likely modulate aggression in this species. Finally, deslorelin temporarily suppressed hormone concentrations and reproductive function, while clomipramine had no clear effect on either. Ultimately, this information provides important tools for improving male-female pairing success and the overall management of captive clouded leopards.
The Characterization and Cryopreservation of Striped Bass (Morone saxatilis) Semen
(1999) Jenkins, Karen; Curry Woods III, L.; Department of Avian and Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md)
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.
CHARACTERIZATION OF GENETIC RECOMBINATION AND ITS INFLUENCING FACTORS IN CATTLE
(2018) Shen, Botong; Ma, Li; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Meiotic recombination is a fundamental biological process in which parental genetic materials are exchanged during egg or sperm development. Recombination is necessary for proper chromosomal disjunction during meiosis. Aberrations in this process have been confirmed as the cause of aneuploidy, leading to a potentially deleterious outcome. Along with mutation, recombination is a major force to promote genetic diversity and drive the evolution of genomes. Despite the importance of recombination, the frequency and location of recombination vary wildly within and between individuals, populations, and species. In this thesis, I characterized patterns of recombination in the cattle genome and conducted a comprehensive study of the effect of genetics, sex and age on recombination and its evolution using a uniquely large cattle database hosted at the USDA, where over a million animals with full pedigree information have been genotyped and new data are being generated at an increasing speed. First, we characterized five PRDM9 alleles and generated allele-specific recombination maps using data derived from over 239,000 meioses in Holstein. We found one allele of PRDM9 to be very different from others in both protein composition and recombination landscape. By comparing recombination maps from sperm and pedigree data, we validated the quality of pedigree-based results. Second, we extended our analysis in recombination patterns to four major U.S. dairy cattle breeds, Holstein, Jersey, Ayrshire, and Brown Swiss. We identified over 8.9 million crossover events and constructed eight genome-wide recombination maps for the two sexes in four cattle breeds. We confirmed a longer male genetic map in bovine and found breed-specific recombination hotspots. Our GWAS analyses confirmed seven loci associated with genome-wide recombination rate and the association of the PRDM9 gene with hotspot usage in two sexes and multiple cattle breeds. Third, we explored the plastic nature of recombination in cattle by examining the effect of maternal age and temperature using data derived from 36,999 three-generation families in Holstein for which temperature data were available. We presented a quadratic relationship between recombination frequency and maternal age and a positive correlation between temperature and recombination rate. By analyzing large genomic datasets with pedigree information in cattle, these studies advanced our understanding of meiotic recombination in a domestic livestock species.
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.
CHARACTERIZATION, ENRICHMENT, AND IN VITRO CULTURE OF SPERMATOGONIAL STEM CELLS IN THE DOMESTIC CAT: A MODEL FOR RARE AND ENDANGERED FELIDS
(2014) Vansandt, Lindsey Marie; Keefer, Carol L; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Spermatogenesis is a highly prolific process in which millions of spermatozoa are produced daily. Spermatogonial stem cells (SSCs), the adult stem cell population of the testis, sustain this process by providing a constant source of new progenitor cells. The ability of this stem cell population to self-renew makes it a promising alternative to spermatozoa for genetic preservation of rare and endangered animals. While innovative advances in SSC technologies have been made in the mouse, there is a paucity of information concerning felid SSCs. Therefore, the overall objective of the dissertation was to develop SSC technology in the domestic cat (Felis catus) as a model for rare and endangered felids. In the first study, mRNA transcripts for six SSC marker genes (THY1, GPR125, GFRalpha1, PLZF, UCHL1, and OCT4) were identified in cat testes. Localization within the appropriate in situ niche was confirmed by immunohistochemistry for three of the markers (PLZF, UCHL1, and OCT4). The expression pattern of these markers was conserved in the cheetah (Acinonyx jubatus) and Amur leopard (Panthera pardus orientalis), validating the cat as an appropriate felid model. In Study 2, we explored two techniques to enrich cat testis cells for SSCs. We found that the efficiency of enrichment depends on age of the donor and that prepubertal testes are the preferred source for differential plating. Magnetic-activated cell sorting did not achieve any level of enrichment for cat SSCs, likely due to unsuitability of the antibody. The final study modified the traditional mouse SSC culture system for use in the cat. A clear effect of feeder cell type was demonstrated, with mouse endothelial C166 cells supporting a significantly higher number of germ cell colonies as compared to STO cells or primary cat fetal fibroblasts. Identity of germ cell colonies was confirmed by co-expression of UCHL1, PLZF, and OCT4. During subculture, colonies maintained SSC marker co-expression and displayed alkaline phosphatase activity. At the time of writing, cells had been maintained for 78 days in vitro. Together, these studies provide the groundwork towards application of SSC technology in management of rare and endangered felid populations.