School of Animal, Rural and Environmental Sciences The efficacy of xylo-oligosaccharides in supporting the gut health, oxidative status and performance of broilers By Alexandra Desbruslais A thesis submitted in partial fulfilment of the requirements of Nottingham Trent University for the degree of Doctor of Philosophy May 2020 i Copyright statement « This work is the intellectual property of the author. You may copy up to 5% of this work for private study, or personal, non-commercial research. Any re-use of the information contained within this document should be fully referenced, quoting the author, title, university, degree level and pagination. Queries or requests for any other use, or if a more substantial copy is required, should be directed to the owner of the intellectual property rights » ii Abstract Population growth and associated food security has led to pressure on the livestock production industries to increase both production output and production sustainability. The 2006 EU ban on the use of non-therapeutic antibiotics had created a need for alternative measures to support the health and growth of livestock. In recent years there has been increasing evidence that broilers may be experience high levels of oxidative stress. This has been linked with an emerging problem with poultry muscle meat myopathies such as woody breast, white stripe and spaghetti meat. Pre-biotics are one area being explored to fill this void. Xylanase is an exogenous enzyme routinely used in broiler production to reduce the anti-nutritional effects of non-starch polysaccharides present in viscous feed grains. It has been speculated that xylanase may also act as a prebiotic by hydrolysing long-chain polysaccharides in the diet into oligosaccharides that can be utilized for fermentation by microbiota in the hind gut. Subsequently, fermentation products can have antimicrobial and energy sparing effects that may be able to enhance broiler performance and gut health. Oligosaccharides have also been shown to have antioxidant properties. A series of bird trials and in vitro studies were conducted to investigate the effect of xylanase, arabinoxylans and xylo-oligosaccharides on the performance, gut health and oxidative status of broilers. Initially a bird trial investigated the effect of an arabinoxylan extract, with and without the addition of xylanase. This study showed performance benefits of supplementing broilers with an arabinoxylan extract with birds consistently gaining more weight than the other treatments (significant during days 21-28) and finally weighing on average over 100g more than the other treatments. Pre-treating the AX extract seemed to negate any performance benefits, possibly due to a mixing or distribution effect of the pre-treatment process. iii A second bird trial investigated the effect of two XOS products derived from different base materials with and without the addition of xylanase in a 3x2 factorial study. This study revealed very few performance effect but did show that xylanase inclusion increased the tibial breaking strength at day 21 and increased calcium digestibility at day 42. An in vitro study investigated novel antioxidant assays as a way to measure the oxidative status of broilers and the antioxidative properties of broiler feed and various commercial oligosaccharides. This study provided initial indications for optimizing the use of this assay in broilers and showed that all of the XOS products investigated had some antioxidative capacity. A final bird trial investigated the dose response of Signis®, a commercial XOS and xylanase supplement. This study compared a commercially recommended dose of 0.1g/kg Signis® with a dose of 0.2g/kg. This study showed significantly improved body weight gain, average bird weight for both doses of Signis® compared to the control and significantly improved feed conversion ratio for the 0.2g/kg dose compared to the control and 0.1g/kg dose. This study did not find any significant difference in digesta viscosity, suggesting that the performance enhancing effects of XOS/xylanase may be due to alternative mechanisms such as the production of xylanase by bacteria in the GIT. This project demonstrated that broilers can gain performance benefits from the supplementation of an AX extract without the addition of xylanase, suggesting that broilers may be able to ferment AX independently and that although broilers may not be able to produce endogenous xylanase, the microbiota within the intestinal tract may be able to and that viscosity and cell wall effects may not be responsible for all beneficial effects of XOS/xylanase supplementation. iv Acknowledgements When I joined NTU as a foundation degree student 5 and a half years ago, I never dreamt I would be where I am today. A number of people have made this incredible journey possible. Without my parents I would not have had the support or capacity to return to education. The endless childcare they have provided and financial help to ensure the needs of my children were met have played a large role in getting me to where I am today. Posthumously, I must that Graham Shelbourne who provided great support during my early years at NTU and suggested that working towards a PhD would be a good aim for me. He was a “once in a lifetime” mentor and is greatly missed by all. During my time with the NTU Poultry Research team I have received immense support, help and friendship from each and every member of the team. Dr Dawn Scholey as my director of studies and Dr Emily Burton have always gone above and beyond the call of duty to ensure both my personal and professional progression. Likewise, I’d like to thank the poultry technical team. They have always helped where they could, and some will remain friends for life. I must say an enormous thank-you to my husband Nick, who joined this journey part-way through, but has always believed in me (often/always), more than I believed in myself. Without his support, childcare and encouragement, I have no doubt that I would not be here today. Finally, my children Gus and Felix need a big thank you for enduring my absence – particularly over Christmas 2019 when I was holed up in the lab ploughing through analysis and during the Corona virus lockdown of 2020 when I was writing up. v List of abbreviations AX – Arabinoxylan AXOS - Arabinoxylan oligosaccharides BWG – Body weight gain BW – Bird weight Ca – Calcium DDGHS – Distillers dried grains with solubles EPEF – European Poultry Efficiency Factor FCR – Feed Conversion Ratio FD – Freeze dried FI – Feed intake ICP-OES – Inductively coupled plasma-optical emission spectrometer KS – Kolmogorov Smirnoff ANOVA – Analysis of variance NSP Non-starch polysaccharide NDO – Non digestible oligosaccharide VFA – Volatile fatty acid XYL – xylanase XOS – xylo-oligosaccharide vi Table of Contents Abstract .................................................................................................................................................. iii Acknowledgements ................................................................................................................................. v List of abbreviations ............................................................................................................................... vi List of Figures ........................................................................................................................................xiv List of Tables .........................................................................................................................................xvi Chapter 1 Review of the literature ......................................................................................................... 1 1.1 Introduction .................................................................................................................................. 1 1.2 Population growth ........................................................................................................................ 3 1.3 Growth of the Poultry Industry ..................................................................................................... 3 1.4 The Avian Digestive Tract .............................................................................................................. 6 1.4.1 The Alimentary canal ............................................................................................................. 6 1.4.2 The Mouth Structure.............................................................................................................. 7 1.4.3 Salivary Glands ....................................................................................................................... 8 1.4.4 Pharynx and Tongue .............................................................................................................. 8 1.4.5 Oesophagus and Crop ............................................................................................................ 8 1.4.6 Proventriculus ........................................................................................................................ 9 1.4.7 Small intestine ........................................................................................................................ 9 1.4.8 Large Intestine – Caeca and Colon ....................................................................................... 11 1.5 Antibiotic growth promotors .....................................................................................................