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Università Degli Studi Del Molise UNIVERSITÀ DEGLI STUDI DEL MOLISE Department of Agricultural, Environmental and Food Sciences PhD Course in: AGRICULTURE TECHNOLOGY AND BIOTECHNOLOGY (CURRICULUM: Welfare, biotechnology and quality of animal production) (CYCLE XXIX) Related disciplinary scientific section: AGR/19 (Zootecnica Speciale) PhD thesis Effects of different probiotics and synbiotics and mode of their administration on productive performance, carcass traits and meat quality in broiler chickens Coordinator of the PhD Course: Prof. Giuseppe Maiorano Supervisor: Prof. Giuseppe Maiorano PhD Student: Rossella Mucci 151584 ACADEMIC YEAR 2015/2016 To Francesca. It was a honour to be your friend. You will always be with me. ACKNOWLEDGEMENTS I am sincerely and deeply grateful to many people who made possible the realization of this PhD thesis. First and foremost, I would like to thank my supervisor, Professor Giuseppe Maiorano, for his unique guide and support through these three years. His great professionality, precious advices, entusiasm, and unlimited passion for research, were invaluable, and have incomparably contributed both to my personal and professional growth. Special and warm thanks are also given to my Polish supervisor, Professor Marek Bednarczyk, from the Department of Animal Biochemistry and Biotechnology, UTP University of Science and Technology in Bydgoszcz, for giving me the opportunity to work in his laboratory, improving my knowledge and skills. I extend my thanks also to all my Polish collegues for their collaboration and support. Moreover, I would like also to thank all my Italian collegues who shared with me PhD studies. Their support, smiles and advices were very meaningful for me. A special, warm and deserved thank, goes to my coworkers and friends Anna Wilkanowska and Siria Tavaniello. Their friendship and support during these three years, both in good and hard moments, were invaluable. Finally, the most important and deepest thank, goes to my parents Nicola and Anna Maria, for making me who I am, and to my beloved brother Michele. Without their endless love, encouragement and support, especially during hard moments, I never would have realized all my possibilities. Thank you for everything you have done for me. I love you with all my heart. You are my life. ABSTRACT In recent years, the key role of gut microbiota in several aspects of physiology and animal welfare, is increasingly evident. In the poultry industry, antibiotics have been used for a long time, both as therapeutic agents to treat diseases, and also as growth promoters (AGPs) in order to enhance health and performance of birds without therapeutic aims. However, this approach has led to undesired effects, such as the development of antibiotic resistance, and the presence of antibiotic residues in poultry meat and poultry products. In this context the use of AGPs was banned by the European Union from January 1, 2006 (EC regulation No 1831/2003). The ban of AGPs has contributed to increased incidence of enteric diseases (e.g., salmonellosis and campylobacteriosis), resulting in higher mortality rates and lower productivity, as well as increased risks of food-borne diseases in humans, causing serious economic damage to the poultry industry. Since the ban of AGPs in EU, several feed additives have been tested in broiler chickens in order to improve health and performance. Among others, in the post-antibiotics era, probiotics, prebiotics and synbiotics (combination of pro- and prebiotics) offer a natural and safe solution to replace AGPs through the modulation of the activity of the gastrointestinal microbiota, and thus, are considered beneficial to the host animal. The conventional administration of these bioactives in feed and/or water in the first hours/days post-hatching, could lead to conflicting results due to the environmental conditions, such as the individual feed and water intake, the quality of water, and other factors. Moreover, these bioactives should be administered as early in life as possible, minimizing the environmental variables that can compromise their efficacy. To reduce the effect of these factors, in ovo injection technology of probiotics, prebiotics and synbiotics has been developed. The method allows the accurate and precise delivery of the bioactive substance at very low doses to all embryos at early stage of development, minimizing the effect of environmental factors and influencing the microbiome structure in newly hatched chicks. This thesis, which involved two different research works, has aimed to evaluate the effects of different bioactives (probiotics, prebiotics and their combination), administerd in feed (Trial 1) or in ovo (Trial 2) on productive performance and meat quality traits (physico- chemical characteristics, intramuscular collagen properties, total lipids content, cholesterol content, fatty acids composition) in broiler chickens. I The first experiment aimed to assess the effects of a probiotic preparation and a synbiotic combination supplemented in feed on economic impact (European Broiler Index, EBI), performance, carcass traits and meat quality in broiler chickens. 360 one-day-old female chicks (Ross 308) were randomly allotted to 3 dietary treatments: basal diet (Control, C); basal diet with 1% of Lavipan® (JHJ Sp. z o. o., Gizałki, Poland) (L) consisting of L. lactis IBB500, C. divergens S1, L. casei ŁOCK 0915, L. plantarum ŁOCK 0862 and S. cerevisiae ŁOCK 0141; basal diet with a combination of Lavipan® (1%) with RFO (0.8%) (LR). Both formulations were supplemented for the first 7 days of chick’s life. Chickens were reared in 30 floor pens (10 replicate pens/treatment, 12 chicks/pen). To provide commercial conditions, the poultry house was filled with 9000 as-hatched chicks. Animals were fed ad libitum with commercial diets and had free access to water. Mortality for the overall experimental period was calculated for each pen replicate. Body weight gain (BWG), feed intake (FI) and feed conversion ratio (FCR), at 10, 21 and 40 days of age, were calculated on pen basis. At 41 days of age, 10 randomly chosen birds per treatment, between more big, were weighed and slaughtered. The yields of carcass, breast and legs were calculated. pH, color and water holding capacity (WHC) were measured at 24 hours post mortem on right pectoral muscle (PM). Total lipids and fatty acids analyses on left PM were carried out. Data were evaluated by one way ANOVA. Scheffé’s test was applied to compare the differences among means. The mortality was lower in groups fed with supplementation of probiotic (1 %) and synbiotic (2.27 %) compared with C group (5 %), however, the differences were not significant (P > 0.05). The BWG within the first 10 days of life was affected by dietary probiotic and synbiotic formulation (P < 0.05). In general, both L and LR groups were characterized by better BWG values in comparison with C (207.89, 208.99, and 197.63 g/bird, respectively; P = 0.037). No significant differences in the BWG were found for the rest of the rearing period even if the treated groups showed a slightly higher (P > 0.05) BWG compared to C. FI within the first 10 days of life was affected by the treatment; it was higher (P < 0.05) for chickens of L group compared to C. The total amount of FI was slightly higher (P > 0.05) both in L and LR groups in comparison with C. In the present research, FCR was similar between experimental groups for the whole rearing period (P > 0.05). EBI was better both in L and LR groups compared with C; however treatment with L was associated with a higher (P < 0.05) value (+2.6%) in comparison with C. The final body weight was similar (P > 0.05) between experimental groups. The dietary probiotic and synbiotic supplementation had no effects (P > 0.05) on carcass weight and carcass yield, as well as on PM weight, PM yield, and legs weight and legs yield, as well as physico-chemical traits (pH, color and WHC). The total content of lipids, SFA, MUFA and PUFA, as well as the individual fatty acids were not II affected (P > 0.05) by the treatment. Dietary probiotic and synbiotic supplementation had no effects (P > 0.05) on all calculated nutritional ratios (n-6/n-3, PUFA/SFA). Atherogenic and thrombogenic indices were similar (P > 0.05) between experimental groups as well. The results indicate that, taking into account the effects on growth performance, the economic impact could be relevant if considered to feed a high number of animals reared in commercial conditions with basal diet with 1% of Lavipan® and basal diet with a combination of Lavipan® (1%) with 0.8% RFO. The aim of the second trial was to evaluate the effect of two different synbiotics administered in ovo on performance, carcass traits and meat quality in broiler chickens. On day 12 of incubation, 5850 eggs (Cobb 500FF) were randomly divided into 3 experimental groups and were in ovo injected with either: 0.2 ml of a physiological saline solution (Control, C); 0.2 ml of a synbiotic formulation containing 2 mg/embryo of Bi2tos (Clasado BioSciences Ltd., Sliema, Malta), trans-galactooligosaccharides enriched with 105cfu/embryo of Lactobacillus salivarius IBB3154 (SYN1) or 0.2 ml of a synbiotic formulation containing 2 mg/embryo of raffinose family oligosaccharides (RFO) enriched with 105 cfu/embryo of Lactobacillus plantarum IBB3036 (SYN2). After the injection, the injection hole was covered with a drop of organic glue and the incubation was continued until hatching. Among the hatched chickens, 2040 males (680 per each group) were randomly chosen and reared in a commercial poultry house. Chickens were raised in pens (n = 75/pen) with 8 pen replicates per treatment for effect on performance. Moreover, separate pens for sampling (n = 10 birds per pen: 8 replications per each experimental group) were included in the experimental design. Animals were fed ad libitum with commercial diets according to their age and had free access to water.
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