Browsing by Subject "Horse"
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Item Advances in Horse Nutrition and Muscle Protein Synthesis: Grazing Warm Season Annual Forages and Evaluating Muscle Protein Synthesis in Response to Leucine(2016-08) DeBoer, MichelleWarm-season grasses have the potential to provide forage in Upper Midwest pastures. The objectives of this research were to determine yield, forage nutritive value and preference of annual warm-season grasses under horse grazing. Four adult horses (Equus caballus) grazed Japanese millet (Echinochloa esculenta (A. Braun) H. Sholz) Siberian millet (Setaria italica (L.) P. Beauv.), teff (Eragrostis tef (Zucc.) Trotter) sorghum sudan BMR (Sorghum bicolor (L.) Moench), sudangrass (Sorghum sudanense (Piper) Stapf.), and annual ryegrass (Lolium multiflorum L.) at mature and vegetative stages. Forages were seeded in two separate plots utilizing a randomized complete block design with three replicates per plot. Each plot was grazed at a distinct target maturity and each maturity was grazed a minimum of three times each grazing season over a two-year period. Sudangrass was consistently the highest yielding grass (P ≤ 0.0002), producing ≥ 5.5 MT ha-1 at the vegetative stage and ≥ 9.7 MT ha-1 at the mature stage while Japanese and Siberian millet exhibit the lowest yields at ≤ 5.5 MT ha-1 at the vegetative stage and ≤ 6.3 MT ha-1 at the mature stage. Annual ryegrass was the most preferred grass (P ≤ 0.0057) with ≥ 60% removal at the vegetative stage and ≥ 40% removal at the mature stage. Siberian millet was the least preferred grass with ≤ 40% removal at the vegetative stage and ≤ 5% removal at the mature stage. While warm-season grasses meet the nutritional requirements of many classes of horses, Ca:P under 1:1 was observed as well as high NO3-N levels. These conditions could lead to limited mineral availability and possible nitrate toxicity. While teff and sudangrass have potential to be grazed by horses, Ca:P and NO3-N levels should be determined before grazing. Limited research is available regarding the role of leucine in regulating equine skeletal muscle protein synthesis. The objective of this study was to evaluate the effect of leucine on protein synthesis in cultured equine satellite cells by evaluating translation initiation factors in a western blot, the incorporation of puromycin using a nonradioactive surface sensing of translation (SUnSET) method, and measuring the incorporation of [3H]Phenylalanine (3HPhe) in a protein synthesis assay. Satellite cells from equine semimembranosus muscle were grown in cell culture until they developed into myotubes. Treatments used in the western blot consisted of a no leucine control (CON), leucine (LEU), control plus rapamycin (CR), and leucine plus rapamycin (LR). LEU exhibited higher 4E-BP1 and rpS6 phosphorylation when compared to CON with no change observed in mTOR phosphorylation. No increase in phosphorylation was observed in CR or LR treatments. Puromycin incorporation was measured on treatments including a no puromycin control, a no leucine control (CON), and a leucine treatment (LEU). These results showed a 1.6-fold increase in puromycin incorporation between CON and LEU (P = 0.0004). Treatments used in the protein synthesis assay consisted of a leucine titration ranging from 0-nm to 408-nm. These results showed muscle protein synthesis rates increased as a result of leucine treatments with significant differences observed at 204-nM and 408-nM leucine compared to the untreated cells. These treatments exhibited a 1.6-fold increase in protein synthesis rates when compared to the untreated control (P ≤ 0.02). This study demonstrated the phosphorylation of translation initiation factors downstream of mTOR as well as increased 3HPhe and puromycin with leucine treatment. These results suggest leucine can trigger muscle protein synthesis in horses through activation of the mTOR pathway.Item Autologous conditioned serum and subsequent synovial fluid concentrations of cytokines, matrix metalloproteinases, and tissue inhibitors of metalloproteinases after injection into equine osteoarthritic distal interphalangeal joints(2015-07) Tatarniuk, DaneINTRODUCTION: Autologous conditioned serum (ACS) is used for treatment of osteoarthritis in horses. However, there is a lack of critical knowledge of the contents of ACS and any biological effects after injection into osteoarthritic joints. OBJECTIVE: The specific aims of this study were to (1) evaluate content of cytokine, matrix metalloproteinases (MMP), and tissue inhibitors of matrix metalloproteinases (TIMP) in ACS, (2) validate/refute that ACS increases anti-inflammatory or pro- inflammatory biomarkers in synovial fluid from naturally occurring osteoarthritic joints, (3) demonstrate whether freeze/thaw effects IL-1ra concentrations in ACS, and (4) evaluate for clinical improvement in lameness following ACS therapy. METHODS: Eleven horses with naturally occurring osteoarthritis in a forelimb distal inter-phalangeal joint were given 3 consecutive intra-articular doses of ACS separated by 7-day intervals. Synovial fluid was collected before administration of each dose at day 0, 7, 14, and 21. Concentrations of cytokines (IL-1ra, IL-1Beta, TNF�, IL-4, IL-6, IL-8, IL-10), MMPs (-1, -3, -9, -13), and TIMPs (-1, -2, -3, -4) were quantified using ELISA and multiplex assays. Serum was compared to unincubated and incubated controls and synovial fluid was compared to baseline (day 0). Horses were videotaped trotting (at day 0, 7, 14, and 21) and graded blindly for degree of lameness. RESULTS: Concentrations of IL-1ra, as well as MMP-1 to TIMP (-1, -2, -3, -4) and MMP-9 to TIMP (-1, -2, -4) ratios were increased in ACS (vs. unincubated control), and IL-4, IL-6, and IL-8 were decreased in ACS (vs. incubated control). Freeze/thaw did not affect biomarker concentrations in ACS. Although ACS contained high concentrations of IL-1ra, no changes in IL-1ra concentrations were observed in synovial fluid 7 days after each injection. No change in median lameness grade was noted throughout study duration (median grade of 1/5 at all time points).Item Proliferative Enteropathy: Pathogenesis and Host Adaptation(2013-03) Vannucci, Fabio AugustoProliferative enteropathy (PE) is an infectious disease caused by an obligate intracellular bacterium, Lawsonia intracellularis, and characterized by thickening of the intestinal epithelium due to enterocyte proliferation. The overall goals of this thesis were to improve the understanding of the pathogenesis of PE by evaluating phenotypic traits, genome variations and transcriptome patterns of L. intracellularis infection and to evaluate the adaptation of the bacterium to porcine and equine hosts. In the first section, the susceptibility of pigs to a homologous porcine L. intracellularis isolate continuously grown in vitro was assessed. A loss of virulence after 40 passages of the bacteria in culture was established. The comparative whole genome analysis of the pathogenic (low passage) and non-pathogenic (high passage) isolates identified the loss of a prophage-associated genomic island in the non-pathogenic variant. This chromosomal island proved not to be essential for the virulent phenotype, since it was not identified in horses clinically affected with PE. However, this genetic element was associated with host-adapted L. intracellularis variants. While pathogenic porcine isolates harbor this genetic element, it was absent in equine isolates and PE-affected horses. Gene expression profiling of a porcine pathogenic isolate showed a wider transcriptional landscape compared with the non-pathogenic variant. In addition, genes highly activated in vitro by the pathogenic variant also were significantly expressed in vivo. However, genes identified in the genomic island were not expressed by intracellular bacteria either in vitro or in vivo. The proliferative changes exhibited by the infected enterocytes in vivo were associated with deregulation of the G1 phase of the host cell cycle and repression of membrane transporters related to nutrient acquisition, characterizing a malabsorptive syndrome as the major mechanism involved in the poor performance and growth of affected animals. Finally, an alternative method for cultivation of L. intracellularis was developed in order to perform a cross-species experimental study evaluating the susceptibility of pigs and horses to a porcine and an equine L. intracellularis isolate. Evident clinical signs, longer periods of bacterial shedding and stronger serologic immune responses were observed in animals infected with species-specific isolates indicating that host susceptibility is driven by the origin of the L. intracellularis strain.Item Tools For Precision Medicine In The Horse(2020-03) Durward-Akhurst, SianOf the 239 recognized genetic traits and disorders in the horse, the causative variants have been identified for less than 50 genetic disease phenotypes, leaving a vast deficit in our understanding of genetic disease in this species. Large-scale studies of genetic variation from genome sequencing have been used with great success in humans, dogs, cats, and other species, to improve understanding of genetic variation in the general population (i.e.; not phenotyped for a single disease) and to facilitate identification of causative variants. These steps are two of the first and most important steps towards Precision Medicine, or genome-driven medical decision making. Precision Medicine has been demonstrated to facilitate disease diagnosis, allow for target treatment options addressing the specific disease-causing variant, and provide more accurate prognostic information based on the response of patients with the same disease-causing variants to treatment. At this time, Precision Medicine is in its infancy in the equine field and the largest catalog of genetic variation is from < 100 horses. In this thesis, we develop the first large-scale studies of single nucleotide polymorphism (SNP) and structural (copy number variants [CNVs], insertions, deletions, inversions, and intra- and interchromosomal translocations) variation in the horse. We also calculate the number of variants computationally predicted to have a detrimental effect on phenotype (i.e.; the genetic burden) and the frequencies of 154 previously identified phenotype-causing and -associated variants. Lastly, we demonstrate the utility of these resources for prioritizing putative phenotype-causing variants for 11 equine phenotypes that are analogous to Mendelian phenotypes in humans (alopecia areata, atrial fibrillation, congenital bilateral absence of the vas deferens, eosinophilic myositis, hemochromatosis, hyperkalemic periodic paralysis, skeletal muscle hypertrophy (hypertrophy), idiopathic renal hematuria, malignant hyperthermia, microphthalmia, and myotonia). We identified SNPs and small and large structural variants from whole genome sequence of 534 horses from 46 breeds and discovered 28,273,058 SNPs, 1,609,215 small insertions and deletions, 500,780 copy number variants (CNVs), 16,982,525 structural variants (insertions, deletions, inversions, and intrachromosomal translocations), and 11,149,562 interchromosomal translocations. The genetic burden in this population was 0.02% (5,852 variants), with 5,020 of those variants predicted to lead to complete loss of function (LOF) of the gene carrying the variant. Each individual horse carried 865 genetic burden variants (705 LOF variants). Of the 154 phenotype-causing and -associated variants, 94 were identified in this catalog of genetic variation. Finally, we developed 3 pipelines for prioritizing putative-phenotype-causing variants. A stringent candidate gene approach excluded ~100% of variants for our 11 equine phenotypes, and this pipeline was determined to be too stringent. The other 2 pipelines prioritized variants using the expected allele frequency of the causative variant based on the disease prevalence and Hardy-Weinberg equilibrium, with a measure of gene constraint (A) and without (B). The pipelines led to a decrease in the number of possible variants by 99.99% (A) and 99.96% (B), leaving on average 211 (A) and 554 (B) putative phenotype-causing variants for follow-up. Overall, we have produced the largest catalog of genetic variation in the horse to date. This resource can be used to prioritize phenotype-causing variants for all future genetic investigations in the horse. The genetic burden is higher in horses than in humans, which is expected given the reduced genetic diversity in the horse compared to humans and the reduced quality of the horse reference genome and annotation files compared with humans. This catalog of variation was successfully used to provide additional evidence that all the previously published disease-causing variants are likely the true variants, based on their low frequency in this population. We also demonstrated the utility of this catalog for prioritizing phenotype-causing variants for suspected equine genetic phenotypes that are analogous to Mendelian phenotypes in humans. These putative variants will be followed up using additional computational tools and through genotyping in additional cases to identify the true phenotype-causing variants.