The role of vitamin A during bio-mineral tissue development in pigs

PhD ThesisSkeletal health is a critical determinant of animal health and welfare. Kyphosis is one such idiopathic skeletal disease that compromises the welfare of commercial pigs. Vitamin A regulates the expression of genes that define bone growth and development, and has been suggested to associate with kyphosis. This thesis aimed to establish the molecular basis of kyphosis, clarify vitamin A’s role on the expression of genes that regulate skeletal development, and to ascertain vitamin A’s role on gene expression in kyphotic pigs. The TGF-β signalling pathway was associated with kyphotic bone and cartilage tissues. This was due to bone and cartilage tissues showing associations with small-leucine rich proteoglycans, ASPN and DCN respectively, which regulate TGF- β signalling. Potential effects as a result of differential gene expression include reduced endochondral bone growth and deterioration of articular cartilage. The dose of vitamin A was observed to be a critical factor in the regulation of expression of skeletal genes within bone tissue. Genes related to the family of Rho-GTPases, which control cytoskeletal dynamics, were observed to be differentially regulated within the trabecular bone in response to vitamin A. In addition, vitamin A dose was observed to initially antagonise serum 25(OH) D, and upon full saturation of the liver with vitamin A, serum 25(OH) D was restored through, as of yet, unknown mechanisms. The gene GIT2, which associates with Rho-GTPases, was observed to be differentially downregulated within kyphotic trabecular bone, and showed a dose-response relationship with vitamin A supplementation. Furthermore, kyphotic pigs were indicated to have reduced vitamin D status. This research has outlined the molecular basis of kyphosis in pigs, and has indicated vitamin A and vitamin D drive the disease. The research also outlines the role of how excessive vitamin A controls the expression of genes that regulate bio-mineralisation in trabecular bone. The thesis has also offered novel insights into vitamin A’s potential role in regulating gene expression during kyphotic development

Differential responses by aortic valve interstitial cells using in vitro models mimicking metastatic and dystrophic calcification: critical role of inorganic phosphate and additional effects of other pro-calcific agents including LDLs

Calcific aortic valve stenosis (CAVS) is the most severe valvulopathy affecting western world population. In the present thesis, in vitro models stimulating metastatic or dystrophic calcification in cultured bovine aortic valve interstitial cells (AVICs) were studied. Multivaried stimulations of primary cultures of AVICs were performed using inorganic phosphate (Pi) at critical concentrations, bacterial endotoxin lipopolysaccharide (LPS), and conditioned medium (CM) from cultures of allogeneic LPS-stimulated macrophages. Spectrophotometric estimations revealed calcification primarily to depend on elevated Pi levels ( 65 2.0 mM), which were comparable with the most elevated normophosphatemic levels and hyperphosphatemic ones. Pi-dependent effects resulted to be enhanced by adding LPS and CM together but not alone. Ultrastructurally, the calcific process consisted in a distinct AVIC degeneration as found for actual CAVS, in which the crucial event was a progressive plasmamembrane and organelle membrane colliquation that culminated in the generation of an acid-phospholipid-rich material outlining dying cells and their remnants and acting as major hydroxyapatite (HA) nucleator. AVIC remnants included rounded paracrystalline calcospherulae mirroring those described for CAVS. Consistently, Raman micro-spectroscopy applied to calcifying cultured AVICs revealed peripheral localization of HA and acidic phospholipids. Formation of calcific nodules also in the presence of apoptosis inhibitors and negative immunoreactivity to cleaved caspase 3 and annexin V led to exclude apoptosis to occur for calcifying AVICs. Conversely, surviving AVICs were found to undergo initial autophagocytosis, as revealed by immunopositivity to marker MAP1-LC3A, and subsequent derangement as revealed by ultrastructural detection of unusual hypertrophy of endoplasmic reticulum correlating with incubation time course and Pi concentration. In CAVS-affected valves atherosclerosis-like features were detected, with (i) Raman analysis showing colocalization between HA, phospholipids, cholesterol and carotenoids, and (ii) parallel electron microscopy revealing the presence of typical intra- and extracellular cholesterol crystals. Thus, the effects exerted by native low density lipoproteins in both native (nLDL) and aggregated (agLDL) form were explored on cultured AVICs using thin layer chromatography (TLC), spectrophotometry, histochemistry and electron microscopy. The chromatographically detected amounts of internalized esterified cholesterol differently correlated with the spectrophotometrically calcium estimations. Concerning pro-calcific effects, nLDLs and agLDLs alone induced mild calcification, whereas their combination with pro-calcific Pi+LPS+CM mixture unexpectedly provoked negligible calcification for nLDLs but prominent calcification for agLDLs. In conclusion, LDLs are further potential candidates for conditioning AVIC-dependent calcification in addition to Pi and other pro-calcific factors

UWOMJ Volume 34

Schulich School of Medicine & Dentistryhttps://ir.lib.uwo.ca/uwomj/1008/thumbnail.jp

Biomechanical studies of locomotion in pigs

PhD ThesisLameness is a major cause of lost productivity for the pig industry. The objective of this PhD was to develop an objective motion capture method for growing pigs and assess (1) the repeatability and sensitivity of the method (2) the gait characteristics of pigs housed on different floor types and (3) gait differences in pigs with conformational deficiencies, joint disease and/or clinical lameness. Infrared camera-based motion capture was applied to three different cohorts of pigs in three experiments, including an observational study following 84 gilts from grower- to second-parity stage. 3D coordinate data of reflective skin markers attached to head, neck, trunk and leg anatomical landmarks were collected. Temporal (time), linear (displacement) and angular (joint angles) kinematic gait parameters were calculated. Repeatability of the method varied with amount of overlying tissue and/or prominence of anatomical landmarks used for marker placement, but not necessarily with walking speed. Gait development of pigs reared on fully-slatted, partly-slatted or deep straw-bedded floors was not different. Lameness detection and evaluation was possible using relative linear and temporal kinematics. The within-stride trajectory of head and pelvic regions during walking differentiated pigs with front and multi-leg lameness from normal pigs, respectively. The ipsilateral swing-to-stance time ratio detected lameness in hind legs, but was not affected during multi-leg lameness. The frequency and magnitude of irregular steps was increased in lame pigs and in pigs with subclinical joint lesions of osteochondrosis diagnosed post slaughter. Step irregularity (as reflected in the step-to-stride length ratio) was also predictive of impending lameness. The step-to-stride length ratio is a dimensionless and ideal parameter to monitor pigs of different age and size, moving at a self-chosen walking speed. Flexion asymmetry and joint flexion patterns were indicative of locomotor problems in some cases. Gait analysis therefore offers potential for automated prediction and early detection of lameness.British Pig Executive (BPEX

An Introduction to Zooarchaeology

zooarchaeology is a self-reproducing field taught in many university departments of anthropology or archaeology. As archaeologists have literally taken faunal analysis into their own hands, they have debated how best to use animal remains to study everything from early hominin hunting or scavenging to animal production in ancient market economies. Animal remains from archaeological sites have been used to infer three kinds of information: the age of deposits (chronology); paleoenvironment and paleoecological relations among humans and other species; human choices and actions related to use of animals as food and raw materials. Methods for reconstructing human diet and behavior have undergone the greatest growth over the last four decades, and most of this book addresses the second and third areas. This book deals with what I know best: vertebrate zooarchaeology, and within that, analysis of mammalian bones and teeth