The objectives of this study were to investigate concentration dependent effects of canine prostatic fluid (PF) on in vitro seminal parameters of cooled canine semen. Sperm motility parameters, plasma membrane integrity and stability, acrosome integrity and DNA fragmentation were measured after the addition of 0%, 10%, 25%, or 50% PF to extended semen of fertile dogs. Assessments were made at 0 h pre-cooling, at 24, and 48 h of cooled storage (4 °C), and after freezing and thawing followed by incubation (37 °C) at 0, 4 and 24 h. Our hypothesis was that lower dilutions of canine semen with PF in an egg yolk-Tris extender would improve plasma membrane stability and acrosome integrity, and preserve sperm kinetics and reduce DNA fragmentation in comparison with higher concentrations of PF in fertile dogs during cooling. Sperm motility parameters were assessed by computer assisted sperm analysis, and plasma membrane integrity by the hypo-osmotic swelling test. Flow cytometry was used after staining with YO-PRO-1/Ethidium Homodimer 1 (EthD-1) to evaluate membrane stability, fluorescent isothiocynate-PNA (Arachis Hypogaea)/propidium iodide to assess acrosome integrity, and sperm chromatin structure assay to assess DNA fragmentation. The data was analyzed using a mixed linear model (ANOVA) and in case of significant effects of time, treatment, or treatment*time interaction (P < 0.05), least square means were used for pairwise comparisons. Acrosome integrity and DNA fragmentation were not affected by treatment with PF. During the cooling period motility parameters were not influenced by PF treatment. A lower proportion of early apoptotic and higher proportion of early necrotic cells was seen during cooling with 50% PF (YO-PRO-1/EthD-1). Although lower concentrations of PF did not improve the evaluated spermatozoal parameters, they did not seem to compromise sperm motility and plasma membrane stability. The presence of 50% PF prior to cryopreservation decreased post thaw motility and produced a shift towards early necrotic cells after thawing. Therefore admixture with more than 10% PF should be avoided prior to cryopreservation of canine semen.

Stem cells exist in various tissues, including dental follicles and dental pulps. Adult stem cells (ASC) can be isolated from patients for autologous transplantation, which eliminates the risk of immune rejection with low or no tumorigenesis. However, one of the challenges is that ASC progressively lose their differentiation ability when cultured in vitro. This prevents expansion of large quantities of high-potential stem cells for therapeutics, especially for stem cells with limited tissue source, such as dental pulp stem cells (DPSC). The goal of this study is to define possible molecular regulation causing loss of differentiation. To achieve this goal, we determined that DPSC at passages 3 and 5 (early passage) possessed strong differentiation capability, and such differentiation capability is completely lost at passage 11 (late passage). Using whole-genome microarray to compare the transcriptomes, we found that the expression of 34 genes were decreased for more than 10-fold in p11 DPSC when compared to p3. After confirming gene expression with RT-PCR, heat shock protein B8 (HspB8) and the GIPC PDZ domain-containing family (Gipc2) were selected for siRNA knockdown study. Knockdown of HspB8 in early-passage DPSC resulted in the cells losing differentiation, but knockdown of Gipc2 had no effect, suggesting that HspB8 plays an important role in maintaining DPSC differentiation. To further study HspB8, we constructed 2 vectors, one containing the coding sequence (CDS) and 3 untranslated region (3UTR) and another containing only the CDS. Transfection of the vectors into early passage DPSC dramatically increased both HspB8 mRNA and protein. However, transfection of the vectors into the late passage DPSC resulted in overexpression of HspB8 mRNA, but increase of HspB8 protein was seen only in CDS transfection. Given that 3UTR of mRNA is the major target region for microRNAs (miRNAs), the results indicate that miRNAs are responsible for down-regulation of HspB8 in long-term culture of DPSCs. We conclude that high-level HspB8 expression is essential for differentiation of DPSC, and down-regulation of HspB8 in cultured DPSC is likely due to increased expression of miRNAs. These are novel findings regarding HspB8 and miRNAs on the regulation of stem cell fate.

Utilization of patient-specific stem cells in regenerative medicine provides a novel treatment approach for diseases and disorders. Embryonic stem cells and induced pluripotent stem cells can differentiate into any cells found within the body; however, ethical, technical and safety concerns have to be overcome before they can be used in clinics. Patient-specific stem cells can be isolated from adult tissues with no ethical, fewer technical, and safety concerns. Obtaining tissues for stem cell isolation usually requires invasive procedures, but impacted teeth are often extracted in the clinics and can be used for isolation of dental follicle stem cells (DFSCs). The overall goal of this dissertation is to characterize the osteogenic potential of DFSCs and to explore the possibility of using DFSCs for the treatment of craniofacial defects. In this regard, we first showed that DFSCs can be induced to differentiate primarily toward the osteoblast lineage. Our experiments showed that DFSCs at passages 3 to 5 have a strong osteogenic capability that is reduced during in vitro expansion. Comparing DFSCs with non-stem cell dental follicle cells (DFCs), we determined that dentin matrix protein 1 (DMP1) is highly expressed in DFSCs. Further study suggests that DMP1 is likely necessary to maintain the osteogenic differentiation capability of DFSCs via regulating expression of osteogenic genes. Given that adult stem cells exist in a quiescent state under normal physiological conditions, we attempted to activate DFSCs with heat-stress. Culturing DFSCs under mild heat-stress (39ºC-40ºC) could effectively promote their proliferation and osteogenic differentiation. In the final part of this project, in vivo transplantation experiments were conducted to evaluate the osteogenic potential of DFSCs for treatment of calvarial critical-size defects using a rat model. Bone regeneration was assessed by micro-computed tomography (micro-CT) and histological analysis at 4 and 8 weeks post-transplantation. The results showed that transplantation of DFSCs seeded into PCL scaffold significantly improved bone regeneration. An average of 50% bone recovery was observed with treatment of PCL-DFSC transplantation at 8 weeks. In conclusion, this study found that DFSCs are valuable tissue stem cells possessing strong osteogenic potential that can be used for repairing craniofacial defects.

The objective of the survey study was to measure the level of knowledge and attitudes of the study groups with respect to the field of veterinary prosthetics. The objective of the implant study was to validate the biomechanical feasibility of two basic prosthetic implants for horses. The objective of the foot study was to virtually design a simple, inexpensive and accessible prosthetic foot for amputee horses, as well as biomechanically investigate it using finite element analysis (FEA) to define the optimal materials and dimensions. The survey study showed that there is not enough information available about veterinary prosthetics and student populations are more amicable to the field. The implant study showed that both implant designs were adequate in their abilities to withstand equine specific loads in axial compression, cycling and 4-point bending; the implant with and abutment performed statistically significantly better for axial loads and the interlocking screw implant was practically significantly better for bending loads. Using FEA, the most ideal dimensions, out of 0.5, 0.75 or 1 inch heel fillets and toe lengths, for an average-sized equine prosthetic foot were a 0.5 inch diameter heel fillet and 0.75 inch toe length at breakover for both midstance and heel strike. The most ideal material was found to be acrylonitrile butadiene styrene polycarbonate (ABSPC). The survey study indicates that collaborative efforts of different professionals is required to successfully provide veterinary prosthetics and produce more relevant information for advancement of the field. The implant study indicated that the implants, as they are, would best biomechanically resist the forces of small horses or ponies and that more research is required to assess the biomechanical potential of both implant designs together for average or large-sized horses. The prosthetic foot would likely perform better with a rubber heel and rubber lining the sole to absorb shock. It is entirely customizable to any amputee horses specific needs, 3D printable or machinable, inexpensive and accessible as an open source design with free permission to build on and modify as necessary.

The medial geniculate body (MGB) is the target of excitatory and inhibitory inputs from several neural sources. Among these, the inferior colliculus (IC) is an important nucleus in the midbrain that acts as a nexus for many auditory pathways and projections, ascending and descending, throughout the rest of the central auditory system and provides both excitatory and inhibitory projections to the MGB. In addition, the thalamic reticular nucleus (TRN) is a major source of inhibition to the MGB, particularly in rodents. Finally, the auditory cortex (AC) is a major source of descending input to the MGB, providing direct excitation and indirect inhibition via the TRN. In our study, we assessed the relative contribution from these excitatory and inhibitory projection sources to the MGB of the auditory system in mice. Using retrograde tract tracing with CTβ -Alexa Fluor 594 injected into the MGB of the mouse, we quantitatively mapped the projections from both the ipsilateral and contralateral IC, the TRN, and the AC to the ipsilateral MGB. Our results indicate significant GABAergic projections from the IC and TRN to the MGB and excitation from the AC that play an overlooked role in shaping auditory processing. These results complement prior studies in other species, which suggests that these pathways are important factors in the regulation of neuronal activity in the auditory forebrain.

Escherichia coli biofilm formation is believed to be a significant cause of chronic equine endometritis, but limited studies exist to support this theory. In total 130 equine reproductive tract E. coli were collected, with 35 isolates showing strong in vitro biofilm formation (OD570 > 0.4). Changing incubation conditions did not significantly alter the distribution of E. coli between biofilm-forming groups, and strong biofilm formation began as early as 4 hours after initiation of incubation. The collected uterine isolates could be screened for biofilm formation using an adapted crystal violet (CV) assay consisting of chromogenic agar incubation and polyvinyl chloride conical tubes, with results demonstrating an in-house technique for screening isolates for biofilm formation. The strong biofilm-forming uterine E. coli showed a 7- to 11-fold increase over corresponding planktonic state in antibiotic resistance to ampicillin, ticarcillin, ceftiofur, gentamicin, amikacin, tobramycin, polymixin B and enrofloxacin. The exposure of certain biofilm-forming uterine E. coli isolates to 12.5% and 25% sub-MIC ceftiofur concentrations resulted in a significant dose-dependent increase in biofilm biomass after 24 hours incubation. All E. coli isolates evaluated (total = 101) were shown to carry the genes for Type-1 fimbriae, S-fimbriae, curli and the PGA polysaccharide, all of which are associated with biofilm formation. When exposed to endometrial explants E. coli adherence occurred rapidly and early stages of biofilm formation were visible on endometrial surfaces using SEM. Attempts to demonstrate uropathogenic E.coli associated cellular internalization were unsuccessful, but instead showed that adhered E. coli acquire biofilm associated antibiotic resistance rapidly after adhering to endometrial surfaces. In conclusion these experiments have shown that approximately 30% of equine reproductive tract E. coli can form strong in vitro biofilm, and show significant resistance to commonly used equine reproductive antibiotics. Also there is potential for increased biofilm formation to occur after exposure to low-level antibiotic exposure. The CV assay is an important screening assay for biofilm formation and can be adapted for use by clinicians in equine practice. The results of these experiments have shown the need to include biofilm-screening assays in equine practice for effective treatment of chronic endometritis.

The use of advanced imaging techniques has greatly improved orthopedic research and education. Histology of bone is a method of evaluating bone morphology, bony cells, and bone remodeling in two-dimensions, while micro-computed tomography (microCT) is a three-dimensional analysis of bone morphology. Combined, these methods assist in providing a comprehensive analysis of bone. In this dissertation, these techniques were utilized to answer questions currently outstanding in veterinary medicine: the effect of bisphosphonates on equine bone, and variation in murine tarsal anatomy. Bisphosphonates are drugs that reduce osteoclast-mediated bone resorption and have recently been approved for the use in horses. Despite prolific clinical use, there has been little evidence of their effect on bone in horses. Therefore, the goal of these studies was to determine the impact of bisphosphonates on bone in normal, young horses. This was accomplished by evaluating bone biopsies taken before and 60 days after a single bisphosphonate administration. Biopsies were analyzed using microCT and histomorphometry. We found that the bisphosphonates studied have minimal to no effect on bone morphology and remodeling, and therefore conclude that these drugs do not negatively impact bone, and have no effect after 60 days. Mice are the most commonly utilized animal model for orthopedic research, and knowledge of normal anatomy is critical to identify pathologies secondary to disease. We found conflicting evidence regarding the tarsal anatomy in the mouse. While normal tarsal variation exists in other species, this has not been documented in the mouse. Therefore, the purpose of this study was to characterize the tarsal anatomy of the mouse. MicroCT data from muroid tarsi were collected, and representative tarsi were evaluated by histology. Fusion of the central and tarsal bone III was present in all laboratory mice evaluated, but was not present in the laboratory rat or wild white-footed mouse. This fusion was confirmed via histology; however, hyaline cartilage was present, surrounded by mature trabecular bone indicating a joint remnant despite the fused state of the bones. We conclude that in certain laboratory mouse strains, the central and tarsal bone III are fused into a single bone.

The Mississippi Sandhill Crane (Grus canadensis pulla), the most endangered North American crane, is considered critically endangered and is protected by Federal and State law. Substantial funding has established the Mississippi Sandhill Crane National Wildlife Refuge in Southern Mississippi and an artificial insemination / breeding facility at the Audubon Center for Research of Endangered Species in New Orleans, Louisiana to promote species recovery. In spite of extensive time, labor, and money invested in captive propagation, juvenile Mississippi Sandhill Cranes suffer substantial mortality due to predation by bobcats (Lynx rufus), coyotes (Canis latrans) and red tailed hawks (Buteo jamaicensis) upon reintroduction to the refuge. Studies have shown decreased avian mortality in reintroduction programs incorporating antipredator conditioning. Appropriate antipredator behaviors are likely innate in cranes, however the object at which to direct these behaviors may require social learning in lieu of the normally long period of parental care known in this species. An antipredator conditioning program was conducted prior to release for 2 years in juvenile Mississippi Sandhill Cranes using live tame predators and conspecific presence to teach predator recognition and appropriate responses. Death of juvenile cranes upon reintroduction to the refuge due to predation has not occurred since the inception of the program. However, factors such as an increase in predator control or differing weather conditions may have contributed to these results. Behavioral results strongly suggest that the presence of adult cranes during antipredator conditioning of subadult cranes is of benefit. With the presence of an adult pair of cranes (models), subadult cranes show significantly more vigilance in the form of the tall alert behavior. Contact call and guard call occurrence were associated with age, however appropriate vocal response to predator presence occurred regardless of whether a model was present. No cranes were harmed during antipredator conditioning procedures, and time and money expenditures were minimal. Antipredator conditioning programs for cranes can be relatively simple and inexpensive with minimal risk to participants. We strongly recommend similar procedures be incorporated into other avian endangered species reintroduction programs.

Hearing loss in dogs and cats following dental or ear procedures performed under anesthesia has recently been reported. The most likely causes for this acute-onset deafness were considered to be mechanical or vascular. Jaw manipulation as a possible cause of acute-onset deafness in the dog was investigated in the current study. Structures adjacent to the temporomandibular joint were of interest because changes in jaw orientation could disrupt vessels and nerves in this area. Current descriptions of the anatomy of the vasculature supplying the canine ear are either incomplete or inconsistent. Another considered cause was a jaw-ear connection via a ligamentous remnant of Meckels cartilage. A ligament from the jaw to the ear, while recognized in humans, has not been described in the dog. The goal of this research was to provide more accurate anatomical descriptions of relevant canine juxta-articular structures to support future physiological studies. The occurrence of hearing loss in dogs and cats following procedures performed under anesthesia was documented. Occurrence was low, with only 62 identified cases between 2002 and 2009, but the true occurrence may be greater. No relationship was observed between hearing impairment following these procedures and breed, gender, size of dogs, or anesthetic drug used; however, older animals may be more vulnerable. New anatomical variations of three arteries are described in this study. (1) The rostral tympanic artery is a branch of the mandibular ramus and is accommodated by a small foramen located within a depression medial to the temporomandibular joint. (2) A rostral auditory tube branch of the caudal deep temporal artery was identified. (3) The origin of the caudal auricular artery occurred opposite the lingual artery in 25% of dissected specimens, contrary to published descriptions. The rostral tympanic artery and the rostral auditory tube branch may be susceptible to damage during jaw manipulation due to their locations. Variability of the caudal auricular artery can create problems during surgical procedures. The tympanomandibular ligament, a remnant of Meckels cartilage and equivalent to the human sphenomandibular ligament, was established as a jaw-ear connection in the dog. Tension on this ligament did not produce malleus movement.

Ayahuasca denotes an Amazonian psychotropic plant tea obtained from Banisteriopsis caapi, which contains beta-carboline (b-carboline) alkaloids, chiefly harmine, harmaline and tetrahydroharmine. The tea usually incorporates the leaves of Psychotria viridis, which are rich in N,N-dimethyltryptamine (DMT), a psychoactive 5-HT2A agonist. The beta-carbolines reversibly inhibit monoamine-oxidase (MAO), effectively preventing oxidative deamination of the orally inactive DMT and allowing its absorption and access to the central nervous system. Despite increased use of the tea worldwide, easy to perform and validated methods for its characterization do not exist and the metabolism and excretion of DMT and the b-carbolines has not been studied systematically in humans following ayahuasca consumption. Thus, we developed a liquid chromatographyelectrospray ionization-tandem mass spectrometry procedure for the simultaneous quantification of the major alkaloid components of ayahuasca, including several known and potential metabolites. The assay was applied to a variety of ayahuasca samples and modified to be applicable to human blood and urine samples before and after consumption of ayahuasca. The major components present in ayahuasca samples were tetrahydroharmine and harmine, followed by DMT and harmaline. The major metabolite of DMT was the corresponding N-oxide, DMT-N-oxide which was found in both blood plasma and urine, although not detectable in ayahuasca samples. Less than 1% of the administered DMT dose was detected in urine or blood plasma, despite the inhibition of monoamine oxidase afforded by the presence of the harmala alkaloids in ayahuasca. The major harmala alkaloid excreted was tetrahydroharmine. The methods developed would be suitable for the study of ayahuasca in human and ethnobotanical research, as well as in forensic examinations of ayahuasca preparations. The characteristics of the methods suggest that their sensitivity, selectivity and reproducibility are adequate for use in further toxicological and clinical research on ayahuasca as well as functioning as an assay to screen biological samples for endogenous hallucinogens. Based on the results of these studies we also present a critical review of 69 published studies reporting the detection in human body fluids of three indole alkaloids that possess differing degrees of psychedelic activity. Suggestions for the future directions of ayahuasca and endogenous psychedelics research are offered.