DIETARY INTERVENTIONS TO IMPROVE HEALTHY STATUS IN PIG INTENSIVE FARM CONDITIONS: A MORPHO-FUNCTIONAL STUDY OF TARGET ORGANS.

The aim of my PhD thesis was to give a contribution in the field of improving pig welfare in different crucial phases of the rearing period. This on the basis that: i) keeping farm animals healthy is highly recommended for obtaining animal products that are safe for human consumers, and ii) pathologies and difficulties in food animal species growth result in severe economic losses in farming; really, a correct nutritional management may play a key role in animal production, above all when performed utilizing a multidisciplinary approach. We already know that the appropriate dietary composition and amount of nutrients are determinant for an optimal growth of animals. After the banning (2006) from EU countries of the use of chemotherapeutics as growing substances, the interest of scientific researchers for alternative nutritional strategies appears more and more increasing. Various nutritional approaches with different dietary integrations have been tested in the recent years for finding alternatives to the limited use of chemotherapeutics in animal rearing, as well as for enhancing the intrinsic quality of a food product of animal origin. In the last year most literature focus on the so called nutraceuticals. Here I present three assays aimed at evaluating the effects of different dietary supplementations in pigs in two different phases of the intensive farm industries: weaning, which is most often accompanied by acute stress, and growth-finishing period, which is characterized by a long term stress. In all these studies my approach was micro-anatomical, consistent with morpho-functional analyses of the pig alimentary tract. In addition with the description of important structural details, sometimes differently quantified (histometry) in treated animals in comparison with control ones, the used micro-anatomical approach let me to hypothesize in some instances mechanisms of action of the studied dietary interventions. Moreover I have worked to correlate the results obtained with the used micro-anatomical methods with those aimed at evaluating growth performance, thus respecting a multidisciplinary approach, which is useful when approaching the swine that is important both as a food animal species, and a largely used animal model for approaching biomedical themes important for human medicine. In the first assay I studied the way to manage oxidative stress in the growth-finishing phase. The aim of the study was to investigate whether the use of verbascoside (VB), a polyphenolic plant compound, could modulate pig feeding oxidative and/or nitrosative stress in the gut. Eighteen male piglets were assigned to two groups, which were fed with either a control diet (CON) or a diet supplemented with 5 mg/kg of verbascoside (VB) for 166 days. At slaughter, duodenum and jejunum specimens were collected. Immunohistochemistry and Western blot analyses were performed on the samples to evaluate free radical adducts, including acrolein (ACR), 8-hydroxydeoxyguanosine (8-OHdg) and nitrotyrosine (NT). A KRL test was also used to assess the total blood antioxidant activity. Immunohistochemistry and Western blot showed that dietary treatment decreased the levels of nitrotyrosine in enteroendocrine cell populations (P<0.05). Characterization of the enteroendocrine cell typology was then performed, and serotonin immunoreactive cells were revealed to be directly involved in decreasing nitrosative stress status. This preliminary study demonstrates the important role of dietary VB in decreasing stress biomarkers in swine gut, thus highlighting a possible intervention aimed at building a large prospective for antioxidant dietary supplementation in food animal species. In the second and third assays I studied how to manage the weaning period stressful condition focusing my attention on the intestine in the first work and on the liver in the second one. The effects of dietary supplementation of different copper sulphate forms (CuSO4) in post weaning piglets on growth performance, small intestinal and liver morphology and large intestine microbiota, as well as faecal and liver copper content were studied. At weaning 90 piglets were allotted to three dietary treatments for 18 d: control diet (with no copper sulphate) and two diets supplemented with 150 mg/kg of copper sulphate in protected and unprotected form. Unprotected copper sulphate showed a numerically higher feed:gain ratio. Moreover, duodenum villi length and crypts depth revealed to be higher in the treated animals (unprotected form). Furthermore, the two copper diets showed a decrease in Streptococci in the colon content, and an higher fecal copper content when compared to control animals (P<0.001). The obtained results revealed a modulation in the intestinal environment caused by addition of unprotected copper sulphate, as well as positive structural changes in the duodenum. In the third and last work the aim of the study was to investigate whether the use of verbascoside in weaning pig feeding could modulate oxidative stress in the liver. Twenty four weaned female piglets were assigned to three experimental groups: the first group were fed a diet with 9% of sunflower oil (T1), the second one were fed with the same diet of the T1 but with an integration of antioxidant mix (T2), in particular with a 5 mg/kg of verbascoside (VB), and the third group was fed a control diet (C), where the oil was substituted by starch. The trial went on twenty nine days, and at the end the animals were slaughtered and the liver specimens were collected. Immunohistochemistry and Western blot analyses were performed on the samples to evaluate free radical adducts, including Heat Shock Protein (Hsp) 70, Hsp 90, lysozyme and desmin. These analyses showed an increase of the level of Hsp 70 in the liver treated with sunflower oil. A KRL test was also used to assess the total blood and plasma antioxidant activity. We evaluated also the impact of the use of antioxidant integrations with analyses on some plasma and liver enzymes related to oxidative stress: superoxide dismutase (SOD),catalase (CAT) and glutathione peroxidase (GPX). In the plasma compartment all enzymes were lower in the treated animals in comparison with control group. In the liver only SOD had significant results: T1 group presented a higher level of this enzyme related to other groups. In conclusion, these three works support in my opinion the hypothesis that selected dietary integrations are possibly able to improve pig welfare in intensive farm, directing their actions, among other targets, upon the gut, and it is well known that a healthy gut may be responsible for a healthy individual. This in turn may be of a fundamental importance in the environment of the rearing farm that is so frequently rich of stressors, which may be with efficacy counteracted by nutraceuticals.