Elisabetta Chiaradia

EFFECT OF EXERCISE TRAINING, SELENIUM AND VITAMIN E ON SOME FREE RADICAL SCAVENGERS IN HORSES (EQUUS CABALLUS)

Physical exercise increases both tissue needs for oxygen and cellular respiration and causes an overproduction of free radicals. When free radical generation exceeds the cells antioxidant capacity tissue-damage develops due to oxidative stress. Therefore, it appears important to increase the scavenger ability of the tissues. Controlled training and dietary supplements may provide ways of doing this. As a model, we used 3-year-old racehorses (Equus caballus) which underwent a series of different physical exercise trials before and after 70 days of daily training and dietary supplements (vitamin E and selenium). The above treatments were able to increase both red blood cell resistance to the peroxidative stress induced in vitro and the glutathione peroxidase activity in lymphocytes. Moreover, they were also able to decrease malondialdehyde (MDA) concentration in the plasma as well as vitamin E consumption and the mobilisation of low molecular weight antioxidants (total peroxyl-radical trapping) following the physical exercise trials. The results obtained indicated that the training and diet supplements we used were able to significantly increase horse antioxidant defences in both the extracellular fluids and blood cells of our horses, thus decreasing peroxidative phenomena following physical exercise.

Physical exercise, oxidative stress and muscle damage in racehorses

Since it has been suggested that lipid peroxidation following free radical overproduction may be one of the causes of physical exercise-induced myopathies and hemolysis in horses, we looked for the possible relationships between these phenomena and muscle fiber damage. We use a homogeneous group of Maremmana stallions which, after a 3-month training period, underwent a series of physical exercises of increasing intensity. We determined the contents of malondialdehyde (MDA), one of the main lipid peroxidation end-products, and glutathione the substrate of one of the most important free radical scavenger enzymes. We also measured creatine phosphokinase and serum lactate dehydrogenase isoenzyme activities whose modification may be indicative of muscle fiber damage. The results obtained indicated that the physical exercise we adopted was able to modify both MDA and glutathione contents in blood. However, its effect on some LDH isoenzyme activities suggested possible damage to tissues other than muscle.

Plasma protein changes in horse after prolonged physical exercise: a proteomic study.

Physical exercise induces various stress responses and metabolic adaptations that have not yet been completely elucidated. Novel biomarkers are needed in sport veterinary medicine to monitor training levels and to detect subclinical conditions that can develop into exercise-related diseases. In this study, protein modifications in horse plasma induced by prolonged, aerobic physical exercise were investigated by using a proteomic approach based on 2-DE and combined mass spectrometry procedures. Thirty-eight protein spots, associated with expression products of 13 genes, showed significant quantitative changes; spots identified as membrane Cu amine oxidase, α-1 antitrypsin, α-1 antitrypsin-related protein, caeruloplasmin, α-2 macroglobulin and complement factor C4 were augmented in relative abundance after the race, while haptoglobin β chain, apolipoprotein A-I, transthyretin, retinol binding protein 4, fibrinogen γ chain, complement factor B and albumin fragments were reduced. These results indicate that prolonged physical exercise affects plasma proteins involved in pathways related to inflammation, coagulation, immune modulation, oxidant/antioxidant activity and cellular and vascular damage, with consequent effects on whole horse metabolism.

Gambling on putative biomarkers of osteoarthritis and osteochondrosis by equine synovial fluid proteomics.

Osteoarthritis (OA) and osteochondrosis (OC) are two of the main challenges in orthopedics, whose definitive diagnosis is usually based on radiographic/arthroscopic evidences. Their early diagnosis should allow preventive or timely therapeutic actions, which are generally precluded from the poor relationships occurring between symptomatologic and radiographic evidences. These limitations should be overcome by improving the knowledge on articular tissue metabolism and on molecular factors regulating its normal homeostasis, also identifying novel OA and OC biomarkers suitable for their earlier diagnoses, whenever clinical/pathological inflammatory scenarios between these joint diseases seem somewhat related. To identify proteins involved in their aetiology and progression, we undertook a differential proteomic analysis of equine synovial fluid (SF), which compared the protein pattern of OA or OC patients with that of healthy individuals. Deregulated proteins in OA and OC included components related to inflammatory state, coagulation pathways, oxidative stress and matrix damage, which were suggestive of pathological alterations in articular homeostasis, plasma-SF exchange, joint nutritional status and vessel permeability. Some proteins seemed commonly deregulated in both pathologies indicating that, regardless of the stimulus, common pathways are affected and/or the animal joint uses the same molecular mechanisms to restore its homeostasis. On the other hand, the increased number of deregulated proteins observed in OA with respect to OC, together with their nature, confirmed the high inflammatory character of this disease. Some deregulated proteins in OA found a verification by analyzing the SF of injured arthritic joints following autologous conditioned serum treatment, an emergent therapy that provides positive results for both human and equine OA. Being the horse involved in occupational/sporting activities and considered as an excellent animal model for human joint diseases, our data provide suggestive information for tentative biomedical extrapolations, allowing to overcome the limitations in joint size and workload that are typical of other small animal models.

Proteomic evaluation of sheep serum proteins

BackgroundThe applications of proteomic strategies to ovine medicine remain limited. The definition of serum proteome may be a good tool to identify useful protein biomarkers for recognising sub-clinical conditions and overt disease in sheep. Findings from bovine species are often directly translated for use in ovine medicine. In order to characterize normal protein patterns and improve knowledge of molecular species-specific characteristics, we generated a two-dimensional reference map of sheep serum. The possible application of this approach was tested by analysing serum protein patterns in ewes with mild broncho-pulmonary disease, which is very common in sheep and in the peripartum period which is a stressful time, with a high incidence of infectious and parasitic diseases.ResultsThis study generated the first reference 2-DE maps of sheep serum. Overall, 250 protein spots were analyzed, and 138 identified.Compared with healthy sheep, serum protein profiles of animals with rhino-tracheo-bronchitis showed a significant decrease in protein spots identified as transthyretin, apolipoprotein A1 and a significant increase in spots identified as haptoglobin, endopin 1b and alpha1B glycoprotein.In the peripartum period, haptoglobin, alpha-1-acid glycoprotein, apolipoprotein A1 levels rose, while transthyretin content dropped.ConclusionsThis study describes applications of proteomics in putative biomarker discovery for early diagnosis as well as for monitoring the physiological and metabolic situations critical for ovine welfare.

Ovine subclinical mastitis: proteomic analysis of whey and milk fat globules unveils putative diagnostic biomarkers in milk.

UNLABELLED Subclinical mastitis is one of the main causes of alteration in milk content and has a major impact on both animal welfare and economy in the dairy industry. A better knowledge is needed to understand the ovine mammary gland metabolism and its response to bacterial infection. In this study, the proteomic changes in ovine milk as a result of subclinical mastitis were investigated by comparing both whey and fat globule membrane profiles of samples from Staphylococcus chromogenes-positive individuals, with those from non-infected counterparts having high or low somatic cell count; the latter were used as control. 2-DE and combined MS procedures were utilized for this purpose. Although sample bromatological parameters were very similar, proteomic analysis highlighted significant differences between the three experimental groups. Most relevant changes were observed between samples of infected milk and control. Modifications related to the defense response of the mammary gland to the pathogen were evident, with important consequences on nutritional and technological properties of milk. On the other hand, quantitative protein changes between non-infected samples with low and high levels of somatic cells indicated that the latter may result as a consequence of a probable unpaired cellular metabolism due to cellular stress, hormonal variations or previous infections. Putative biomarkers useful for the monitoring of sheep mammary metabolism and for the careful management of ovine subclinical mastitis to avoid its clinical degeneration are proposed and discussed. BIOLOGICAL SIGNIFICANCE Proteomics has been here applied to the differentiation of healthy and subclinical mastitic sheep milk samples, evidencing the response of the mammary gland to S. chromogenes infection. Presented results propose useful protein biomarkers for the detection of ewe mammary infection at its subclinical stages and, subsequently, mastitis recognition and treatment. Differently from bovine, these data confirm that the increase in somatic cell count in sheep milk is not always associated with protein factors that characterize the mammary gland infection; accordingly, somatic cell count cannot be considered as a useful parameter to certainly diagnose subclinical mastitis in ovine.

Quantitation of phospholipids on thin layer chromatographic plates using a desk-top scanner.

Amethod for quantitating phospholipids separated on thin layer chromatographic plates by computer-assisted photodensitometry is described. After development, the plates are stained with molibdic reagent and the image obtained is acquired as TIFF file in the memory of a personal computer. The color intensity of the single spots of the digitalized image is analyzed using a dedicated software.Sensitivity and reproducibility are adequate for most of the needs of lipid chemist. When compared to conventional photodensitometric procedures, the present method offers the advantage of requiring a much cheaper hardware.

Intra-articular administration of lidocaine plus adrenaline in dogs: Pharmacokinetic profile and evaluation of toxicity in vivo and in vitro

The aim of this study was to evaluate the safety of intra-articular (IA) lidocaine plus adrenaline for improving peri-operative analgesia in anaesthetized dogs undergoing arthroscopy of the elbow. A solution of lidocaine (L) 1.98% plus adrenaline 1:100.000 was administered via the IA route and its safety evaluated in terms of cardio-, neuro-, and chondro-toxicity. No bradycardia or hypotension was recorded from induction to the last observational time point. Signs of toxicity of the nervous system could have been masked by the general anaesthesia but lidocaine concentrations detected in the blood were lower than those thought to be capable of producing toxicity. The assessment of in vitro chondrotoxicity showed a dose- and time-dependent effect of lidocaine on the viability of articular cells. Adrenaline appeared to reduce the chondrotoxicity of 1% lidocaine, following an exposure of up to 30 min.

Investigation into omocysteine, vitamin E and malondialdehyde as indicators of successful artificial insemination in synchronized buffalo cows (Bubalus bubalis).

The aim of this study was to describe modifications in plasma homocysteine (Hcy), vitamin E (VitE) and malondialdehyde (MDA) concentrations in the first 56 days after artificial insemination (AI) in buffalo. Thirty-five buffalo cows were divided, ex post, into three groups on the basis of pregnancy diagnosis: pregnant, not pregnant, with embryonic mortality. Pregnancy was diagnosed by ultrasonography and plasma concentrations of pregnancy-associated glycoproteins (PAGs). Our results showed that, in pregnant buffaloes, included those with embryonic mortality, MDA increased progressively while VitE decreased. In non-pregnant buffaloes, MDA and Vit E were unchanged. Hcy concentrations also remained unchanged within each group throughout the study period, but were lower in non-pregnant buffaloes than in the pregnant ones and in those with embryonic mortality. In conclusion, present data suggest that successful pregnancy in buffalo cows might be linked to Hcy metabolism and oxidative stress involvement.

Effect of Physical Exercise on Thiols in the Plasma in the Athletic Horse: Connection with the Immune System

Thiols are a class of organic compounds characterized by the presence of sulphydryl residues (–SH). In biological systems, thiols have a central role in coordinating the intraand extracellular redox balance. In recent years, elevated levels of serum homocysteine (Hcy), a sulphur amino acid, have been recognized as an independent risk factor for cardiovascular disease (House, 1999). Despite the growing evidence of this relationship, the mechanism behind such lesions is still unknown. Scientific debate continues as to whether elevated serum homocysteine is a unique marker for vascular pathology or is associated with pathological processes beyond the vascular system. Indeed, it has recently been reported that Hcy accelerates the rate of endothelial senescence (Xu, 2000) by causing hypomethylation (Hultberg et al., 2000), and by increasing the formation of hydrogen peroxide, especially in association with copper or iron. Considering the involvement of free radicals in the cellular proliferative pathways, it is possible to hypothesize that Hcy plays an important role as mediator of both endothelial cell and immune cell functions (Dudman, 1999).