UNIVERSITY OF MOLISE SPANISH SCIENCE RESEARCH COUNCIL DEPARTIMENT OF AGRICULTURAL, INSTITUTE OF FOOD SCIENCE TECHNOLOGY AND NUTRITION ENVIRONMENTAL AND FOOD SCIENCES INTERNATIONAL Ph.D. in “WELFARE, BIOTECHNOLOGY AND QUALITY OF ANIMAL PRODUCTION” (XXVII CYCLE) Related disciplinary scientific section: 07/G1 (Scienze e Tecnologie Animali) General Coordinator: Prof. Giuseppe Maiorano Doctorate Thesis Title EFFECT OF DIETARY POLYPHENOL-RICH GRAPE BY- PRODUCTS ON GROWTH PERFORMANCE, SOME PHYSIOLOGICAL PARAMETERS, MEAT AND MEAT PRODUCTS QUALITY IN CHICKENS Ph.D. Candidate: Supervisors: Dr. Maria Nardoia Prof. Donato Casamassima Dr. Agustin Brenes Payà Co-supervisor: Dr. Claudia Ruiz-Capillas ACADEMIC YEAR 2014/2015 INTERNATIONAL Ph.D. in “WELFARE, BIOTECHNOLOGY AND QUALITY OF ANIMAL PRODUCTION” (XXVII CYCLE) Related disciplinary scientific section: 07/G1 (Scienze e Tecnologie Animali) General Coordinator: Prof. Giuseppe Maiorano Doctorate Thesis Title EFFECT OF DIETARY POLYPHENOL-RICH GRAPE BY- PRODUCTS ON GROWTH PERFORMANCE, SOME PHYSIOLOGICAL PARAMETERS, MEAT AND MEAT PRODUCTS QUALITY IN CHICKENS Ph.D. Candidate: Supervisors: Dr. Maria Nardoia Prof. Donato Casamassima Dr. Agustin Brenes Payà Co-supervisor: Dr. Claudia Ruiz Capillas ACADEMIC YEAR 2014/2015 2 DECLARATION I hereby declare that the thesis is based on my original work except for citations which have been duly acknowledged. I also declare that this thesis has not been previously or concurrently submitted for any degree or any other institution. Campobasso, 18/02/2016 Dr. Maria Nardoia _____________________________ 3 “Stay hungry. Stay foolish” Steve Jobs (1955-2011) 4 SPANISH SCIENCE RESEARCH COUNCIL INSTITUTE OF FOOD SCIENCE TECHNOLOGY AND NUTRITION The investigation of the present doctoral thesis was carried out at the Departament of Metabolism and Nutrition and the Department of Products at the Institute of Food Science Technology and Nutrition (ICTAN) of the Spanish Science Research Council (CSIC) of Madrid. The experimental trials of this work were part of the projects AGL2012-31355/GAN, S2013/AGR-2913 (MEDGAN) and the Intramural Project 2014470E073, financed by the Spanish Ministry of Economy and Competivitiness (MINECO, Spain), Comunidad Autónoma de Madrid (CAM) and CSIC, respectively, and coordinated by Dr. Agustin Brenes Payà and Dr. Claudia Ruiz-Capillas. 5 ACKNOWLEDGEMENTS I would like to extend my thanks to my Italian supervisor Prof. Donato Casamassima for his support throughout this research. With a very deep sense of gratitude, I would like to express my sincere thanks to my Spanish supervisor Dr. Agustin Brenes Payà and co-supervisor Dr. Claudia Ruiz- Capillas, who gave me the opportunity to work, for 18 months, and to realize this doctoral thesis in their research groups at the Departament of Metabolism and Nutrition and the Department of Products at the Institute of Food Science Technology and Nutrition (ICTAN) of the Spanish Science Research Council (CSIC). To work with you has been a real pleasure to me, with heaps of fun and excitement. I am greatly indebted for your immense help in planning, great valuable suggestions throughout this study and eagerness to lend a hand during the completion of this research and thesis. You have been a steady influence throughout my Ph.D. period in Madrid; you have oriented and supported me with promptness and care, and have always been patient and encouraging in times of new ideas and difficulties. Your ability to select and to approach compelling research problems, your high scientific standards, and your hard work set an example. I admire your ability to balance research interests and personal pursuits. Through your encouragement and teaching, you has made me a more confident scientist. Above all, you made me feel a friend, which I appreciate from my heart. I will always be grateful to Susana Chamorro for her help from the very beginning of my Ph.D. period in Madrid. You supported me and your technical excellence and tremendous grasp of experimental issues had a great impact on me. The experience of life in Spain has been fed day by day with the presence of people in both the Institute and beyond its walls, which have been significant in my way for their joy, enthusiasm, friendship and a lot of good things that I could mention now. I have greatly enjoyed the opportunity to meet and work with the best friend-coworkers that you could ever wont Carlos Cavalleihro and Maria Teresa Echevarria Soto. Thanks for making the job a time of fun and for helping me in times of distress. I wish you all to have the great fortune, like me, to meet along the way of life persons like them. 6 Thanks all the great ICTAN and foregin friends, Carmen de la Mata, Tatiana Pintado (Matilda), Marisabela Palencia Alcantud (Isa), Maria Freire (Goiko), Maria Vazquez (Mocasines), Daniel Marin Peñalver (Dani), Alicia Sànchez Faure (Ali), Nacho Escudero Abad (Rata), Javier Dolz Martin (Jabugo), Victor Serrano (Tuco), Fernando Diaz Benito (Fer), Diana Ramirez, Deisy, Silvia Diaz Herrero, Ailen Aleman Perez, Isabel Sanchez Alonso, Ruth Nuñez Flores, scientists, technicians and colleagues who provided a very favorable environment and enhancement. When I started this adventure I never imagined and hoped to meet persons like you. I have been very privileged to get to know and to collaborate with you. I learned a lot from you about life, research, how to tackle new problems, how develop techniques to solve them and also…. how to drink beer! The moments spent with you would remain in my mind eternally. In addition, during these months abroad I had the opportunity to meet people outside of the work environment that have been like a family for me, and that made the lighter my moments of melancholy; thanks to Carlos Salas Menarguez and to Dr. Armando Macera for your warmth. I extend my acknowledgements also to my Italian colleagues, that I met during my Ph.D., Michele Di Iorio, Cinzia Abiuso, Domenico Di Memmo, Anna Wilkanowska, Rossella Mucci, Marsia Miranda, Andrea Gentile, Siria Tavaniello, Daniela Cianciullo for being a breath of fresh air in moments of apnea. I kindly thank the technician Dr. Marisa Palazzo for her technical help and for her support. Moreover, there are those whose spiritual support is even more important. Many thanks to Michele Tiano for his constant presence. You have always believed in me and encouraged me during these four years. Finally, my strongest thanks go to my family. I feel a deep sense of gratitude for my mother, father and brother who formed part of my vision and taught me good things that really matter in life. Your infallible love and support has always been my strength. Your patience, sacrifice and humility will remain my inspiration throughout my life. As always it is impossible to mention everybody who direct and indirect supported and helped me completing my thesis in time. I would like to express my gratitude to them all. 7 ABSTRACT Five studies were designed to investigate the potential use grape pomace (GP), grape seeds (GS) and grape skins (SS), the major residues from wine-making industry and a good sources of polyphenols, as a cheaper but functionally equivalent product, with antioxidant activity, that could partially replace vitamin E in broiler chickens diet and be able to improve poultry performance and welfare, besides to warrant high-quality, safe and functional meat products. The studies were carried out at the Department of Metabolism and Nutrition and Department of Products of the Institute of Food Science, Technology and Nutrition (ICTAN-CSIC) of Madrid. The first studies assessed the effect of dietary GS (15 and 30 g/kg), SS (110 g/kg) and GP (37.5 g/kg) and α-tocopheryl acetate (200 mg/kg) inclusion on performance, ileal and excreta content of total polyphenols and tannins, ileal digestibility of protein and ileal and excreta polyphenols digestibility in one hundred and eighty 21-day-old broiler chickens. In addition, plasma and meat α-tocopherol concentration and meat lipid oxidation during refrigerated storage (at 1d and 7d) were evaluated. SS diet reduced daily weight gain (P<0.01) and feed conversion ratio (P<0.001). α-T diet had no effect on ileal and excreta polyphenols content while significantly higher values were observed in GS, SS and GP groups, compared to the control birds. GS, SS and GP diets increased ileal tannins content, with no effect on excreta content. Protein digestibility significantly decreased in birds fed SS diets. Ileal polyphenols digestibility statistically increased in GS, SS and GP groups, while no differences were observed for excreta digestibility. Plasma α-tocopherol increased significantly in birds fed α-T, SS and GP diets, while γ- tocopherol only in birds fed GP diets. Meat α and γ- tocopherol levels were statistically higher in birds fed α-T diet at 1 and 7 days of storage, compared to the other groups. GP and α-T improved the stability of meat to lipid oxidation by reducing MDA values after 1 day and 7 days of storage. The second study aimed to investigate the effect of dietary fermented (FS) and unfermented (UFS) grape skin at different doses (30 g/Kg, FS30 and UFS30, and 60 g/kg, FS60 and UFS60) and of α-tocopheryl acetate (200 mg/kg) in one hundred and fifty 21-day-old broilers. The same parameters of the previous experiment and intestinal microflora were assessed, except for plasma and meat vitamin E concentrations. 8 FS60 and UFS60 negatively affected growth performance and UFS60 significantly decreased protein digestibility. UFS increased ileal and excreta polyphenols content; whereas ileal and excreta tannins content increased in all experimental groups. UFS30 diet statistically increased ileal polyphenols digestibility, while both FS and UFS diets significantly increased the excreta digestibility with higher values in birds fed UFS in comparison to those fed FS. Intestinal microflora was not affected by dietary treatment.