AKENTEN-APPIAH MENKA UNIVERSITY OF SKILLS TRAINING AND ENTREPRENEURIAL DEVELOPMENT MAMPONG- ASHANTI INFLUENCE OF EXOGENOUS ENZYME (ROVABIOTM) ON THE GROWTH PERFORMANCE, NUTRIENT DIGESTIBILITY, BONE HEALTH AND PROFIT MARGINS IN BROILERS FED DIETARY HIGH LEVELS OF MAIZE BRAN MUSAH ISSAK MASTER OF PHILOSOPHY 2024 AKENTEN-APPIAH MENKA UNIVERSITY OF SKILLS TRAINING AND ENTREPRENEURIAL DEVELOPMENT MAMPONG- ASHANTI INFLUENCE OF EXOGENOUS ENZYME (ROVABIOTM) ON THE GROWTH PERFORMANCE, NUTRIENT DIGESTIBILITY, BONE HEALTH AND PROFIT MARGINS IN BROILERS FED DIETARY HIGH LEVELS OF MAIZE BRAN BY MUSAH ISSAK (8220190004) A thesis in the Department of Animal Science Education, Faculty of Agriculture Education, submitted to the school of Graduate Studies in partial fulfilment of the requirements for the award of the degree of Master of Philosophy (Non-Ruminant Nutrition) In the Akenten-Appiah Menka University of Skills Training and Entrepreneurial Development DECEMBER, 2024 iii DECLARATION STUDENT’S DECLARATION I, Musah Issak, declare that this thesis, with the exception of quotations and references contained in published works which have all been identified and duly acknowledged, is entirely my own original work, and it has not been submitted, either in part or whole, for another degree elsewhere. Musah Issak (Student) Signature……………………………… Date: ………/…………/…………………. SUPERVISOR’S DECLARATION I hereby declare that the preparation and presentation of this work was supervised in accordance with the guidelines for supervision of thesis as laid down by the Akenten-Appiah Menka University of Skills Training and Entrepreneurial Development. DR. Holy Kwabla Zanu (Supervisor) Signature……………………………… Date: ………/…………/………………. iv ACKNOWLEDGMENT My heartfelt appreciation goes to my supervisor, Dr. Holy Kwabla Zanu, for his patience, guidance, and motivation, which were instrumental in the successful completion of this work. I am deeply grateful to Feed2Gain company in the USA for funding this research and to Addisseo in France for providing the enzyme (RovabioTM) used in the study. I would like to acknowledge Mr. Daniel Takah and Mr. Isaac Daitey for their invaluable contributions during data collection and sampling. I also extend my gratitude to Mr. Thomas Quaye and Mr. Larry of AAMUSTED-M for the technical assistance provided in the shed. My sincere thanks go to Mr. Mubarik Iddrisu for proofreading this thesis, as well as to Dr. William Kwenin and Dr. Ismael Coffie of AAMUSTED-M for their advice and support throughout the experimental period. Special recognition goes to my brothers Musah Abdulai and Abdulai ibn Alidu for their unwavering support, as well as to my friend Mr. Kofi Nsiah, whose encouragement and assistance were invaluable throughout the completion of this project. Lastly, I thank everyone who contributed in one way or another to the successful completion of this work. May Almighty Allah richly bless you all. v DEDICATION I dedicate this thesis to my parents, Mr. Abdulai Musah and Mrs. Fatimatu Musah, and my beloved wife Rahima Nuhu. vi TABLE OF CONTENTS DECLARATION ...................................................................................................................... iii ACKNOWLEDGMENT ......................................................................................................... iv DEDICATION ........................................................................................................................... v TABLE OF CONTENTS ......................................................................................................... vi LIST OF TABLES .................................................................................................................... x LIST OF FIGURES ................................................................................................................ xii LIST OF PLATES .................................................................................................................. xiii DEFINITIONS OF ABBREVIATION ................................................................................. xiv ABSTRACT............................................................................................................................. xv CHAPTER ONE ....................................................................................................................... 1 1.0 INTRODUCTION .............................................................................................................. 1 1.1 Background to the Study ...................................................................................................... 1 1.2 Problem Statement ................................................................................................................ 2 1.3 Main Objective ..................................................................................................................... 3 1.4 Specific Objective ................................................................................................................. 3 CHAPTER TWO ...................................................................................................................... 5 2.0 LITERATURE REVIEW .................................................................................................. 5 2.1 Non-Conventional Feed Resources ...................................................................................... 5 2.1.1 The need for non-conventional feed resources .................................................................. 6 2.1.2 Agro-industrial by-products for livestock .......................................................................... 7 2.1.3 Advantages of non-conventional feed resources (NCFR) .................................................. 9 2.2 Maize Bran ............................................................................................................................ 9 2.3 Feed Additives in Poultry Production ................................................................................. 11 2.4 Exogenous Enzyme Activities in Broiler Diets .................................................................. 12 2.4.1 Enzymes that break down non-starch polysaccharides (NSPase) ................................... 14 2.4.2 The enzyme profile of Rovabio ......................................................................................... 16 2.4.3 Phytase enzyme ................................................................................................................ 18 2.5 Exogenous Enzymes' Mode of Action in Broiler Feed ....................................................... 20 vii 2.6 Factors Influencing Enzymes Effectiveness ....................................................................... 23 2.6.1 Impact of dietary nutrient density on enzyme effectiveness ............................................. 23 2.6.2 Effect of dietary ingredients on enzyme efficacy ............................................................. 24 2.6.3 Influence of the birds' age on the enzyme effectiveness ................................................... 26 2.7 The Role of Enzymes in Breaking Down Minerals in Feed Ingredients ............................ 27 2.8 Classification of Non-Starch Polysaccharides .................................................................... 29 2.8.1 Effect of non-starch polysaccharides on nutrient uptake in broilers ............................... 31 2.9 Effect of Phytic Acid on Growth Performance of Broilers ................................................. 33 2.10 Influence of Exogenous Enzyme on Growth Performance of Broilers ............................ 35 2.11 Influence of Exogenous Enzyme on Nutrient Digestibility of Broilers ............................ 38 2.12 Impact of Exogenous Enzyme on Gut pH Of Broilers ..................................................... 40 2.13 Influence of Exogenous Enzyme on Carcass Traits of Broilers ....................................... 41 2.14 Influence of Exogenous Enzyme on Bone Health ............................................................ 42 CHAPTER THREE ................................................................................................................ 44 3.0 MATERIALS AND METHODS...................................................................................... 44 3.1 Study Location and Duration .............................................................................................. 44 3.2 Dietary Treatment and Experimental Design ..................................................................... 44 3.3 Experimental Units and Management ................................................................................ 46 3.4 Parameters Measured .......................................................................................................... 46 3.4.1 Growth Performance ....................................................................................................... 46 3.4.2 Gastrointestinal pH .......................................................................................................... 47 3.4.3 Bone traits ........................................................................................................................ 47 3.4.4 Relative organ weight ...................................................................................................... 48 3.4.5 Protein determination in diets and digesta ...................................................................... 48 3.4.6 Ash determination in diets and digesta ............................................................................ 49 3.4.7 Crude fat determination in diets and digesta .................................................................. 50 3.4.8 Crude fibre determination in diets and digesta ............................................................... 50 3.4.9 Titanium dioxide analysis ................................................................................................ 51 3.4.10 Digestibility calculation ................................................................................................. 52 3.5 Statistical analysis of data ................................................................................................... 52 viii CHAPTER FOUR .................................................................................................................. 53 4.0 RESULTS .......................................................................................................................... 53 4.1 Analyzed Nutrient Composition of Maize Bran and Experimental Diets .......................... 53 4.2 Influence of Exogenous Enzyme and Maize Bran on the Performance of Broilers from d 0 to 14 .............................................................................................................................. 55 4.3 Influence of Exogenous Enzyme and Maize Bran on the Performance of Broilers at d 28 ...................................................................................................................................... 57 4.4 Influence of Exogenous Enzyme and Maize Bran on the Performance of Broilers from d 0 to 42 .............................................................................................................................. 59 4.5 Influence of Exogenous Enzyme and Maize Bran on the Performance of Broilers from d 0 to 56 .............................................................................................................................. 61 4.6 Influence of Exogenous Enzyme and Maize Bran on the Gastrointestinal pH of Broilers, d 28 .................................................................................................................................. 63 4.7 Influence of Exogenous Enzyme and Maize Bran on the Gastrointestinal pH of Broilers at d 56 ............................................................................................................................... 65 4.8 Influence of Exogenous Enzyme and Maize Bran on the Carcass Traits (% BW) of Broilers from d 0 to 28 ............................................................................................................... 67 4.9 Influence of Exogenous Enzyme and Maize Bran on the Carcass Traits (% BW) of Broilers from d 0 to 56 ............................................................................................................... 69 4.10 Influence of Exogenous Enzyme and Maize Bran on the Femur and Tibial Weight (% BW) of Broilers, d 28 and d 56 ............................................................................................. 71 4.11 Influence of Exogenous Enzyme and Maize Bran on the Tibial and Femur Breaking Strength (N) of Broilers, d 28 and d56 ......................................................................... 73 4.12 Influence of Exogenous Enzyme and Maize Bran on Apparent Ileal Digestibility of Protein, Ash, Fat and Fibre at d 28 ............................................................................... 75 4.13 Influence of Exogenous Enzyme and Maize Bran on Production Economics, d 56 ........ 77 CHAPTER FIVE .................................................................................................................... 79 5.0 DISCUSSION .................................................................................................................... 79 5.1 Analyzed nutrient composition of maize bran .................................................................... 79 5.2 Influence of Exogenous Enzyme and Maize Bran on Growth Performance of Broiler Chickens ....................................................................................................................... 81 ix 5.3 Influence of Exogenous Enzyme and Maize Bran on the Gastrointestinal pH of Broiler Chickens ....................................................................................................................... 84 5.4 Influence of Exogenous Enzyme and Maize Bran on the Carcass Traits (% BW) of Broiler Chickens ....................................................................................................................... 85 5.5 Influence of Exogenous Enzyme and Maize Bran on the Bone Traits of Broiler Chickens ...................................................................................................................................... 86 5.6 Influence of Exogenous Enzyme and Maize Bran on Apparent Ileal Digestibility of Protein, Ash, Fat, and Fibre at d 28 ............................................................................................ 88 5.7 Influence of Exogenous Enzyme and Maize Bran on the Production Economics of Broiler Chickens ....................................................................................................................... 89 CHAPTER SIX ....................................................................................................................... 91 6.0 CONCLUSIONS AND RECOMMENDATIONS .......................................................... 91 6.1 Conclusions......................................................................................................................... 91 6.2 Recommendations ............................................................................................................... 91 REFERENCES ....................................................................................................................... 93 APPENDICES ....................................................................................................................... 112 x LIST OF TABLES Table 2.1: By-Product Feeds from Trees and Crops for Livestocks ........................................... 8 Table 2.2: Proximate composition of maize bran ..................................................................... 11 Table 2.3 Enzyme composition of Rovabio .............................................................................. 17 Table 2.4: Target Substrates, Production Organisms, and Types of Exogenous Enzymes ....... 22 Table 2.5: Content of dry matter (g/100 g, as is basis) and non-starch polysaccharides of the plant-based ingredients ................................................................................................. 33 Table 2.6: Growth performance of broiler chicks fed diets supplemented with or without NSPDE .......................................................................................................................... 38 Table 3.1: Composition and Calculated analysis of experimental diets, % .............................. 45 Table 4.1: Analyzed nutrient composition of maize bran ......................................................... 54 Table 4.2: Analyzed nutrient composition of experimental diets ............................................. 54 Table 4.3: Influence of exogenous enzyme and maize bran on the performance of broilers at d 0 to 14 ........................................................................................................................... 56 Table 4.4: Influence of exogenous enzyme and maize bran on the performance of broilers at d 28 .................................................................................................................................. 58 Table 4.5: Influence of Exogenous Enzyme and Maize Bran on the Performance of Broilers at d 42 ............................................................................................................................... 60 Table 4.6: Influence of exogenous enzyme and maize bran on the performance of broilers at d 56 .................................................................................................................................. 62 Table 4.7: Influence of exogenous enzyme and maize bran on the gastrointestinal pH of broilers, d 28 ............................................................................................................................... 64 Table 4.8 Influence of exogenous enzyme and maize bran on the gastrointestinal pH of broilers, d 56 ............................................................................................................................... 66 Table 4.9: Influence of exogenous enzyme and maize bran on the carcass traits (% BW) of broilers from d 0 to 28 .................................................................................................. 68 Table 4.10: Influence of exogenous enzyme and maize bran on the carcass traits (% BW) of broilers at d 56 .............................................................................................................. 70 Table 4.11: Influence of exogenous enzyme and maize bran on the femur and tibial weight (% BW) of broilers, d 28 and d 56 ..................................................................................... 72 Table 4.12: Influence of exogenous enzyme and maize bran on the tibial and femur breaking strength (N) of broilers, d 28 and 56 ............................................................................. 74 xi Table 4.13: Influence of exogenous enzyme and maize bran on apparent ileal digestibility of protein, ash, fat and fibre at d 28 .................................................................................. 76 Table 4.14: Influence of exogenous enzyme and maize bran on production economics, d56.. 78 xii LIST OF FIGURES Figure 2.1: Classification of non-starch polysaccharides (Choct et al., 2010). ........................ 29 Figure 2.2: The molecular structure of phytic acid (Cherian, 2020) ........................................ 35 xiii LIST OF PLATES Plate 1: Photograph of maize bran ............................................................................................ 11 xiv DEFINITIONS OF ABBREVIATION ABBREVIATION Definition Ca Calcium MB Maize bran Enz Enzyme NSP non-starch polysaccharide NSPase non-starch polysaccharide degrading enzyme MEC Multi enzyme complex AME Apparent metabolizable energy AX Arabinoxylans P Phosphorus FI Feed intake WG Weight Gain BWG Body weight Gain FCR Feed conversion ratio SBM Soyabean meal IDF Insoluble dietary fibre ANF Anti-nutritional factors FTU Phytase unit DCP Dicalcium Phosphate PEI Production Economic Index NRC National Research Council AOAC Association of Official Analytical Chemists. xv ABSTRACT Maize bran (MB) is a potential feed ingredient that can be used to reduce the quantity of maize added to Ghanaian poultry diets. However, MB contains a high level of anti-nutritional factors (ANFs) such as phytic acid and non-starch polysaccharides (NSPs) that negatively affect the utilization of nutrients and allow nutrients to escape enzymatic digestion in the gastrointestinal tract (GIT). Exogenous enzymes have been used over the years to degrade and improve the digestion of nutrients in feedstuffs containing these ANFs. Thus, this study was designed to investigate the hypothesis that exogenous enzyme (RovabioTM) in the presence of high levels of MB could improve the general performance of broilers and increase the profit margins. Three hundred and thirty-six (336) Cobb-500 broiler chicks were allotted to four dietary treatments in a 2 x 2 factorial arrangement in a completely randomized design. The factors were, Enzyme (No vrs yes) and Maize bran MB (No vrs yes) in the starter (d 0 to d 28), grower (d 28 to 42), and finisher diets (d 42 to 56). Weekly intake, body weight (BW), gain, feed conversion ratio, and livability were calculated. The data collected at d 28 and d 56 were nutrient digestibility (crude protein, crude fat, crude fibre and ash), gut pH, carcass traits and bone health (femur and tibial BS), and An Enzyme x MB interaction was detected for FCR (P < 0.05) on d 14 indicating that only in birds fed MB did the enzyme improve feed efficiency. On d 28, No MB as a main effect increased both BW (P < 0.05) and BW gain (P < 0.05) compared to Yes MB diet. The inclusion of enzyme diet increased the gizzard pH of the birds (P < 0.05) at d 28. Maize bran increased gizzard weight and reduced breast weight, % bodyweight (P < 0.05). There was no consistency in the effect of enzyme or maize bran on bone traits. However, the general outcome suggests that the inclusion of enzymes increased feed cost but also increased profitability. In conclusion, the inclusion of the enzyme in the MB-based diet improves broiler performance. 1 CHAPTER ONE 1.0 INTRODUCTION 1.1 Background to the Study Food safety, environmental impact, and high production costs are just a few of the difficulties the poultry industry faces as it attempts to feed a growing population with high- quality products at reasonably low cost (Pirgozliev et al., 2019). Enhancing the nutritional content of feedstuffs is one strategy to somewhat mitigate the growing cost of feed. Feed additives are chemical and biological supplements, including enzymes, that are used for an added advantage of the feed (Rios et al., 2017). Recent studies have focused on the effects of exogenous enzyme supplementation on nutrient digestibility and performance of chickens. According to Yacoubi et al. (2016), exogenous enzymes are safe to use and enhance feed conversion ratio (FCR) and broiler body weight gain. Globally, maize and soybean meal are primarily used as the main ingredients in chicken feed. Nevertheless, several anti-nutritional factors (ANFs) such as phytic acid, non-starch polysaccharides (NSPs), and anti-trypsin negatively impact their nutritional value (Amerah, 2015; Jlali et al., 2020). Phytate and non-starch polysaccharides (NSP) are examples of these ANFs, which allow essential nutrients to escape digestion in the gastrointestinal tract (GIT) (Rios et al., 2017; Sun et al., 2019). High NSP levels cause digesta viscosity to increase, leading to reduced absorption of important nutrients like proteins, lipids, and starch and also lowering feed efficiency and performance (Amerah, 2015; Jlali et al., 2020; Musigwa et al., 2020). The apparent metabolizable energy (AME) of feedstuffs varies depending on the composition and structure of the NSP (Yacoubi et al., 2017). To improve digestion and optimize energy utilization, non-starch polysaccharide degrading enzymes (NSPase) are 2 added to feedstuffs. This reduces intestinal viscosity and improves performance (Yacoubi et al., 2017; Musigwa et al., 2020). The appropriate addition of NSPase, according to Maharjan et al. (2019), results in an improvement in the metabolizable energy (ME) of the feed ingredients because the breakdown of NSP releases extra energy. NSPase can be used to boost the nutritional value of low-quality maize (Rios et al., 2017). In addition to NSP, phytate also has an adverse effect on feedstuffs' nutritional value. More than 60 % of the total phosphorous (P) in feedstuffs can be bound by phytate, making P inaccessible for absorption (Lawlor et al., 2019). This frequently causes animal feed costs to rise and environmental contamination to increase as the dietary requirements for P inclusion levels are exceeded. To improve growth performance and carcass characteristics of broilers, Jlali et al. (2020) reported that phytase inclusion in broiler diets breaks down phytate and releases P along with other minerals and nutrients that are trapped. 1.2 Problem Statement Maize makes up the majority of the diet of chickens in many African countries whereas, it is used to also prepare the majority of the staple foods for humans, making it a costly commodity. However, Ajila et al. (2012) have identified several locally available agricultural and agro-industrial by-products, including rice bran, wheat bran, and maize bran, as suitable alternatives to maize. Maize bran is a potential feed ingredient that can be used to reduce the quantity of maize grain added to the Ghanaian poultry diet. Moreover, maize bran contains high levels of anti-nutrients such as phytic acid and non-starch polysaccharides (NSPs) that negatively affect the utilization of nutrients and allow nutrients to escape digestion in the gastrointestinal tract (GIT) of monogastric animals (Amoah et al., 3 2018). Several studies have shown that the antinutritional factors (ANFs) in non- conversional feed resources such as rice bran, maize bran and wheat bran could be broken by the activities of exogenous enzymes in the diet. Exogenous enzymes have been used over the years to degrade and improve the digestion of nutrients in feedstuffs containing these ANFs (Alagawany et al., 2018). However, there is a paucity of information on the effect of increasing the inclusion rate of maize bran in broiler diets containing novel exogenous feed enzymes (RovabioTM). The enzyme product is made up of xylanase, phytase, amylase, cellulase, and proteases which helps to improve the utilization of diets suspected to contain high levels of NSPs and phytic acids. Therefore, it was hypothesized that the inclusion of exogenous enzymes would improve the utilization of maize bran or allow their higher inclusion rate in Ghanaian broiler diets while giving better nutrient digestibility, growth performance, bone health, and improving profit margins. 1.3 Main Objective The main objective of this study was to evaluate the influence of exogenous enzyme on nutrient digestibility, growth performance, gut pH, bone health and profit margins in broilers fed diets containing high levels of maize bran. 1.4 Specific Objective Specifically, the study was conducted to evaluate; 1. the influence of exogenous enzyme on broilers fed diets containing high levels of maize bran on the growth performance of broiler chickens 2. the influence of exogenous enzyme on broilers fed diets containing high levels of maize bran on gut pH of broiler chickens 4 3. the influence of exogenous enzyme on broilers fed diets containing high levels of maize bran on carcass characteristics of broiler chickens 4. the influence of exogenous enzyme on broilers fed diets containing high levels of maize bran on bone health of broiler chickens 5. the influence of exogenous enzyme on broilers fed diets containing high levels of maize bran on nutrient digestibility (crude protein, ash, crude fat, and crude fibre) 6. the cost and benefits of feeding broiler chickens with diets containing high levels of maize bran and enzyme supplementation. 5 CHAPTER TWO 2.0 LITERATURE REVIEW 2.1 Non-Conventional Feed Resources The rising cost of conventional feed ingredients, such as maize and soybeans, has made poultry production more expensive, particularly for small and medium-scale farmers. As a result, there is growing interest in utilizing non-conventional feed ingredients that are locally available and often more affordable. Non-conventional feeds refer to alternative ingredients such as agro-industrial by-products, leaves, and other plant materials not traditionally used in poultry diets. These feeds have the potential to reduce feed costs, enhance sustainability, and support the growth of poultry in resource-limited settings (Amata, 2014). However, the use of non-conventional feeds also poses certain challenges regarding nutrient availability, palatability, and potential anti-nutritional factors (ANFs). Studies have shown that non-conventional feed ingredients can have varying effects on the growth performance of poultry, depending on their nutrient composition and inclusion levels. For example, cassava peels, which are high in fibre but low in protein, may reduce growth rates if included at high levels without proper supplementation (Adeyemo et al., 2016). However, when supplemented with protein-rich ingredients such as moringa or soybean meal, cassava peels can be effectively utilized in poultry diets without negatively affecting performance (Ogbuewu & Mbajiorgu, 2023). Rice bran and maize bran, both commonly used in poultry diets, contain relatively high levels of ANFs such as phytates and non-starch polysaccharides (NSPs), which can reduce nutrient digestibility (Ravindran, 2013). The inclusion of enzymes such as phytase or 6 xylanase can improve nutrient availability and enhance the overall performance of birds fed diets containing rice bran or maize bran (Selle et al., 2009). One of the main challenges associated with non-conventional feeds is the presence of ANFs, which can inhibit nutrient absorption and reduce the overall performance of poultry. For example, cassava peels contain cyanogenic glycosides, which can be toxic if not properly processed (Devi & Diarra, 2021). Similarly, Leucaena leaves contain mimosine, an alkaloid that can cause toxicity in poultry if consumed in large quantities (Norfadhilah, 2019). Proper processing methods such as drying, boiling, or fermentation can help reduce the levels of ANFs in non-conventional feed ingredients, making them safer for poultry consumption. Enzyme supplementation has also been widely studied as a strategy to mitigate the negative effects of ANFs in non-conventional feeds. For example, the addition of phytase to diets containing rice bran or maize bran has been shown to increase phosphorus availability and improve growth performance (Selle et al., 2009). Similarly, the use of xylanase or other NSP-degrading enzymes can enhance the digestibility of fibre-rich ingredients, leading to better feed efficiency (Ravindran, 2013). 2.1.1 The need for non-conventional feed resources There are serious shortages in some animal feeds of the conventional type. The grains are required almost exclusively for human consumption. With increasing demand for livestock products as a result of rapid growth in the world population and shrinking land area, future hopes of feeding the animals and safeguarding their food security will depend on the better utilization of non-conventional feed resources that do not compete with human food. The availability of feed resources and their rational utilization for livestock represents possibly 7 the most compelling task facing planners and animal scientists in the world. The situation is acute in numerous developing countries where chronic annual feed deficits and increasing animal populations are common, thus making the problem a continuing saga (Norfadhilah, 2019). Thus, non-conventional feeds could partly fill the gap in the feed supply, decrease competition for food between humans and animals, reduce feed cost, and contribute to self- sufficiency in nutrients from locally available feed sources (Rashid, 2020; Beriso, 2022; Kolawole & Mustapha, 2023). It is therefore imperative to examine for cheaper non- conventional feed resources that can improve intake and digestibility of low-quality forages. Feedstuffs such as fish offal, cassava peel, parm kernel cake, sugarcane bagasse, rice bran, maize bran, cocoa bean waste, coconut meal, corn cob, moringa leaf, leucaena leaf, local brewery and distillery by-products, sisal waste, and coffee pulp are commonly used in Ghana and could be invaluable feed resources for small and medium size holders of livestock (Nortey et al., 2015; Amoah et al., 2017). 2.1.2 Agro-industrial by-products for livestock Appropriate use of relatively inexpensive agricultural and industrial by-products is of paramount importance for profitable livestock production. However, the high cost and low availability of conventional livestock feedstuffs frequently demand consideration of by- products even if the efficiency of utilization is low (Kolawole & Mustapha, 2023). Efficient use of by-products relies on their chemical and physical properties, which influence production system outputs. In developing countries like Ghana, grains, which form the bulk of concentrate feeds for poultry, is both in short supply and expensive due to direct 8 competition with human food uses (Kusi et al., 2015). The increasing human demands for several foods (i.e. olive oil, vegetables, wine, fruit juices, etc.) led to a considerable increase of lands occupied by crops producing these feeds. Consequently, huge amounts of agro- industrial by-products are available in numerous developing countries (e.g. maize bran, rice bran, wheat bran, copra cake etc.), which are still not fully utilized in poultry feeding. Most of these agro-industrial by-products are low in major nutrients. Moreover, the difficulty of the use of these feed sources as fresh material for extended periods and the lack of efficient ways for their integration in feeding calendars may account for their under-utilization (Onte et al., 2019). Table 2.1: By-Product Feeds from Trees and Crops for Livestocks Type Crop BY-PRODUCT FEEDS Tree crop cocoa Cocoa bean waste and cocoa pod husks Coconut Coconut meal Oil palm Oil palm sludge (dry), palm press fibre and palm kernel meal Rubber Rubber seed meal Field crop Castor Castor meal cotton Cotton seed meal Maize Maize bran and maize germ meal Rice Broken rice, rice bran, rice husk and rice straw wheat Wheat bran and wheat straw Cassava Tapioca waste Surgarcane Baggase, green tops, and molasses Source:(Onte et al., 2019). 9 2.1.3 Advantages of non-conventional feed resources (NCFR) The use of NCFR offers several advantages, such as reducing feed costs, minimizing waste, and enhancing sustainability in animal production. Below are some of the advantages of NCFR; a) Concerning the feeds of crop origin, the majority are bulky poor-quality cellulosic roughages with high crude fibre and low nitrogen contents, suitable for feeding to ruminants and poultry (Amoah et al., 2017). b) They are mainly organic and can be in a solid, slurry, or liquid form. Their economic value is often very low (Nortey et al., 2015). c) These are end products of production and consumption that have not been used (Beriso, 2022). d) The feed crops that generate valuable NCFR are excellent sources of fermentable carbohydrates eg. cassava and sweet potato. This is an advantage to ruminants because of their ability to utilize inorganic nitrogen (Amata, 2014). e) Fruit wastes such as banana rejects and pineapple pulp by comparison have sugars that are energetically very beneficial (Amata, 2014; Nortey et al., 2015). f) They have considerable potential as feed materials and their value can be increased if they are converted into some usable products (Ravindra, 2013). 2.2 Maize Bran Maize bran is a by-product of dry milling maize, which consists of the bran coating and the maize germ. It is palatable to all classes of farm animals and approaches maize grain in feeding value though it contains more fibre because of the hulls, which are included. 10 Maize bran consists of the outer coating of the kernels, including the hull and tip cap, with little or none of the starchy part of the germ (Saeed et al., 2021). Hussain et al. (2024) defined maize bran as a by-product obtained from the milling of maize, which is the removal of the hull. They added that the hull contains about 15 % crude fibre. Kantanka (2013) reported that maize bran is very much sought after by small and medium scale pig and poultry farmers. It is a very good partial replacement for maize for these species partly because milling machines used in the milling process are not very efficient and the by-product contains most of the germ, bran and some proportions of the endosperm. It is therefore a high-energy source, but unfortunately during the manufacturing process, water is added to the maize and thus the maize bran may be wet. If not dried immediately, it can easily become moldy and may also become rancid. Wherever there are large concentrations of poultry and pigs, the demand is high, and therefore it can be scarce leading to high costs. It is highly fibrous and this limits its utilization because its high fibre cannot be digested by the endogenous enzymes of poultry and can have anti-nutritive effects (Saeed et al., 2021). Maize bran causes an increase in viscosity of intestinal content and entrap large amounts of well digestible nutrients like starch and proteins. This leads to an impaired digestion and digestive problems (Hussain et al., 2024). Table 2.4 shows the proximate composition of maize bran. 11 Table 2.2: Proximate composition of maize bran Composition % Crude protein 9.85 Crude fat 11.66 Moisture content 16.01 Ash content 4.37 Crude fibre 13.29 Total reducing sugar 19.39 Total carbohydrate 58.12 ME (kJ/100g) 1586.83 Source:(Asuk et al., 2016). Plate 1: Photograph of maize bran. Source:(www.alamy.com) 2.3 Feed Additives in Poultry Production Feed additives make up a small percentage of animal feed, yet they can have a significant impact by enhancing feed utilization, increasing growth efficiency, and reducing diseases (Cherian, 2020). The commonly used feed additives in poultry production are pro- and 12 prebiotics, antioxidants, enzymes, and antibiotic growth promoters and each has a unique function. The feed enzyme market has expanded significantly over the last five years, mostly as a result of rising raw material costs (Ravindran, 2013). Even with increased acceptability, there are still many unanswered concerns about how to employ enzymes to provide consistent effects and reactions to enzyme supplementation. However, these inconsistent answers highlight both the existing constraints and the possible ways to improve the advantages of using enzymes. Three essential elements are inescapably linked to limitations in enzyme responses: the substrate, the bird, and the enzyme. Enzymes decrease nutrient loss and lower environmental pollution by enhancing digestion, which increases nutrient availability (Cherian, 2020). According to Pirgozliev (2019), enzymes are proteins that catalyze particular chemical processes and are specific to a particular substrate. The use of exogenous feed enzymes is one of the ways for nutritionists to create diets more economically while enhancing the efficiency of feed and still giving consumers the most economical source of protein because feed costs make up the greater percentage of all input costs (Davids & Meyer, 2017; Boyd et al., 2018). 2.4 Exogenous Enzyme Activities in Broiler Diets According to Zakaria et al. (2010), using exogenous feed enzymes in monogastric diets is a useful strategy for allowing for flexibility in diet composition, as well as for reducing feed costs, improving feed digestibility, and minimizing environmental pollution. According to Doskovic et al. (2013), for exogenous enzymes to be as efficient as possible, they must balance out the function of the animal's endogenous substances. Plant-based raw materials have anti-nutritional factors that limit the availability of nutrients by preventing endogenous 13 enzymes from accessing them, which prevents digestion (Costa et al., 2013). Rios et al. (2017) reported that the digesta transit rate in modern broilers is too quick for optimum digestion, allowing important nutrients to escape digestion in the GIT. In addition to breaking down bound nutrients, exogenous enzymes reduce the cost of producing chicken diets (Boyd et al., 2018). According to Saleh et al. (2019), animal rations contain trace amounts of exogenous enzymes such as phytase, protease, and xylanase which are produced from microbial sources. It has been demonstrated that the use of exogenous enzymes in animal feed improves the body's ability to absorb nutrients that would not otherwise be available (Classen, 1996). The addition of enzymes reduces the adverse effects of ANFs and boosts profitability in poultry production (Costa et al., 2013; Sun et al., 2019). Approximately 1.67- 1.88 MJ of energy per kilogram of feed is not being digested in a conventional corn-soybean diet without enzyme supplementation (Govil et al., 2017). Enzymes can be used to increase protein, fat, and carbohydrate availability as well as to increase more energy being made available for utilization. In poultry feed, enzymes can be added either separately or in the form of multi-enzyme complexes (MEC) (Jlali et al., 2020). Positive outcomes have been reported for both MEC and single. There has been contradictory research on the effectiveness of supplementing with non-starch polysaccharide degrading enzymes in addition to phytase. While NSPase and protease possess distinct target substrates, their actions complement one another because NSPase releases many nutrients and reduces mucus production in the gastrointestinal tract (Rahimi et al., 2020). According to Jlali et al. (2020), the bird's response to MEC use is influenced by its genetic ancestry, age, nutrition, and MEC dose. 14 Adding enzymes to poultry feed, either separately or in combination, has several advantages: i. The release of encapsulated starch in the cell wall minimizes the variation in apparent metabolizable energy (AME) and performance (Amerah, 2015). ii. Less digesta viscosity lowers the amount of wet litter and sticky droppings, which lowers the risk of dermatitis (Wang et al., 1998; Amerah et al., 2017). iii. Due to the immature GITs, young chicks are particularly vulnerable to the negative impact of NSP. Enzymes called carbohydrases help to keep the gut healthy so that an inflammatory gut does not impair performance (Yacoubi et al., 2017). iv. By reducing the digesta viscosity and changing gut microbes by promoting the growth of beneficial microbes, BW, gain, and FCR are improved (Saleh et al., 2019). v. Short-chain fatty acids (SCFA) such as butyrate and acetate are produced by multi- enzyme complexes. Butyrate serves as an energy source for the intestinal epithelial cells in the stomach, promoting both their proliferation and differentiation to improve digestive health (Yacoubi et al., 2016). vi. Phytase inclusion enhances growth performance and carcass characteristics of broilers by releasing trapped P and other nutrients (Jlali et al., 2020). vii. Because nutrients are being utilized, feed costs are decreased (Lawlor et al., 2019). viii. The amount of undigested nutrients excreted into the environment is decreased, which reduces its contribution to environmental contamination (Lawlor et al., 2019). 2.4.1 Enzymes that break down non-starch polysaccharides (NSPase) Enzyme use for commercial applications is a relatively new development, beginning about three decades ago with a focus on addressing the anti-nutritional impact of non-starch polysaccharides (NSP) in cereal-based diets for broiler chickens. NSP, characterized by its 15 high molecular weight, contributes to increased digesta viscosity by forming complex polymers that resist digestion in poultry (Wu et al., 2004). Assessing NSP content in raw materials is crucial for determining the appropriate enzyme levels needed to facilitate energy and nutrient release, thus enhancing diet quality. The specific substrates released by NSPase that contribute to favorable production of short- chain fatty acids (SCFA) in the ceca remain incompletely understood. However, increased fermentation of oligosaccharides and subsequent SCFA production likely play a role in improving apparent metabolizable energy (AME) and influencing gut hormone secretion, which aids in gastric retention and overall gut health. Enhanced growth performance results from improved AME, dry matter retention, and ileal digestible energy. Furthermore, by improving the performance and digestion of nutrients, sticky droppings are reduced in frequency. Furthermore, the proliferation of butyrogenic bacteria in the ceca, facilitated by NSP-degrading enzymes, protects against pathogenic bacteria (Lee et al., 2010). Carbohydrate-degrading enzymes are introduced to high-NSP monogastric feed to break complex carbs down into smaller polymers (Cherian, 2020). Endogenous enzymes are essential for aiding in the breakdown of β 1-3, β 1-4, and β 1-6 links that are present in NSP, but they are absent in monogastric animals. According to Tejede & Kim (2021), the degree of NSP molecule branching affects its solubility, which in turn affects the enzyme's effectiveness (Cherian, 2020). Wu et al. (2004) showed that adding xylanase and phytase to the broiler chicks' diet reduced the digesta viscosity from their duodenum (p < 0.05). Furthermore, Lee et al. (2010) reported that adding phytase and NSPase to the diet decreased the digesta viscosity by 16.8 % and 12.4 %, respectively. These findings imply that NSP 16 degrading enzymes that are capable of dissolving the matrix of the cell wall may make it simpler for phytase to access nutrients that are encapsulated in cell walls by reducing the viscosity of the intestinal contents. 2.4.2 The enzyme profile of Rovabio Rovabio is a commercial multi-enzyme blend manufactured by fermenting the fungus Talaromyces versatilis, which breaks down arabinoxylans (AX) (Bichot et al., 2022). This particular blend consists of 19 enzymatic activities, the most common of which are endo- xylanase and arabinofuranosidase, with a ratio of 3:7, respectively (Table 2.3) (Cozannet et al., 2017; Cozannet et al., 2019; Bichot et al., 2022). Rovabio was evaluated in broilers and was found to improve gut health and growth performance by improving the utilization of energy, fat, fibre, and protein (Cozannet et al., 2019; Saleh et al., 2019). This is because endo-xylanases and arabinofuranosidase work together to increase nutrient digestibility. 17 Table 2.3 Enzyme composition of Rovabio Enzyme Composition Xylanase β-xylosidase and Endo-1,4 β-xylanase β-glucanases Endo-1,3 1,4 β-glucanase, Laminarinase Proteases Metallo protease and Aspartic protease Debranching enzymes α-glucuronidase Ferulic acid esterase, and α- arabinofuranosidase, Cellulases Endo-1,4 β-glucanase, Cellobiohydrolase and β-glucosidase Pectinases Endo-1,5 α-arabinanase, Pectin esterase, Polygalacturonase, α-galactosidase and Rhamnogalacturonase Others Endo-1,4 β-mannanase, β-mannosidase Source: (Plouhinec et al., 2023). Chickens raised on diets high in NSP experience adverse consequences, which can be reduced by adding xylanase as an enzyme supplement (Arczewska-Wlosek et al., 2019). Endo-xylanase facilitates the hydrolysis of the xylan backbone, which releases encapsulated starch and other nutrients and reduces the digesta viscosity caused by soluble non-starch polysaccharides (sNSP). Arabinoxylan is the primary NSP that accounts for at least 50 % of the total carbohydrate fraction (Amerah, 2015; Ward, 2021). It has been documented that certain enzyme, such as β-glucanase, improve the nutritional content of cereal by-products in monogastric animals (Cherian, 2020). According to Jlali et al. (2020), the activity of xylanase causes the release of oligosaccharides, which in turn alters the hindgut microbial population. This improves intestinal health and enhances the capacity for digestion and absorption, ultimately leading to better growth performance. Reducing intestinal viscosity and enhancing the nutritional content of cereal-based diets is achieved 18 by supplementing diets with glucanase and xylanase alone, in combination, or as part of a multi-enzyme complex (Yacoubi et al., 2016). The first (1,3-1,4)-β-glucanase enzyme was isolated from a strain of Bacillus subtilis, which is now known as Bacillus amyloliquefaciens. When introduced to a barley-based diet, broilers responded favourably (Von Wettstein et al., 2000). According to studies conducted by Esteve-Garcia et al. (1997) and Von Wettstein et al. (2000), adding β-glucanase as a supplement to broiler diets has been demonstrated to decrease intestinal viscosity, vent pasting, the frequency of sticky droppings, and increase weight gain. Maize dry matter digestibility has long been enhanced by the use of endo-β-1,4-xylanase and arabinofuranosidase enzyme combinations (Saleh et al., 2019). The avian digestive tract largely passes cellulose and arabinoxylans undigested because no animal enzyme is able to break them down (Cherian, 2020; Ward, 2021). In order to break down the cellulose, which is a contributing factor to the undigested elements in the terminal ileum, microbial cellulase supplements should be given (Khalil et al., 2022). According to Silva et al. (2012) and Zyla et al. (2012), pectinase is an enzyme that hydrolyzes cell walls. When pectin is hydrolyzed, it changes lipid metabolism, the caeca's ability to function, and the GIT's inflammatory response. 2.4.3 Phytase enzyme It is now standard procedure to add phytase to poultry feeds to promote sustainable chicken meat production because phytic acid is considered an anti-nutritional factor (Lui et al., 2014). The first study to add phytase derived from Aspergillus ficuum to a liquid soybean 19 diet was Nelson et al. (2018). The results showed a significant increase in the percentage of bone ash when compared to the control group which did not receive any inorganic P. Ever since, phytase has been a reasonably priced source of inorganic P replacement. According to Outchkourov & Petkov (2019). fungi, bacteria, yeast, and higher plants with different origins can be used to produce phytase at optimal pH and temperature. The phytase enzyme, also known as Myo-inositol hexakisphosphate phosphohydrolase, primarily functions in the upper portion of the gastrointestinal tract (GIT). Its function is to facilitate the stepwise hydrolysis of penta-to monophosphates, which breaks down phytic acid into lower phytate esters and inositol (Feil, 2008; Amerah, 2015; Rahimi et al., 2020; Walk & Roa 2020). It has been reported that inositol release increases broiler growth performance (Bedford & Rousseau, 2017). The animal may now use the phosphorus that was previously bound, and this process also improves the animal's ability to digest and use calcium, amino acids, and energy (Walk & Roa, 2020). According to Rahimi et al. (2020), this minimizes the cost of including inorganic phosphorus and restricts the amount of phosphorus excreted into the environment by enabling a reduced inclusion of the aforementioned nutrients without adversely affecting the animal. There is further evidence that supplementing with phytase improves pre-caecal amino acid digestibility (Siegert et al., 2019). Phytase effectiveness varies depending on the feedstuff and can be attributed to several characteristics of the enzyme, such as ideal pH or temperature, or the feed source (Cherian, 2020; Siegert et al., 2019). 20 2.5 Exogenous Enzymes' Mode of Action in Broiler Feed Cowieson et al. (2010) reported that the mode of action of exogenous enzymes to increase the income of poultry production is improving the apparent digestibility of dietary nutrients and reducing the animal's nutrient requirements. For exogenous enzymes to be applied to dry diets successfully, several requirements are necessary to be met in order for the animal's digestive tract to be active. It must remain active in the physiological conditions of the animal's digestive tract, it must be resistant to proteolysis by the animal's endogenous proteases (Thorpe & Beal, 2001; Cherian, 2020). An enzyme's capacity to break down different substrates is determined by the solubility of non-starch polysaccharides and the intricate structure of the carbohydrate; on the other hand, an enzyme's mode of action is dependent on its efficacy (Cherian, 2020). This means that it should not conflict with the animal's natural digestive enzymes. Variations in the physiology and morphology of the digestive system between different species are likely to alter exogenous enzyme function in this regard. Partridge (1993) and Dierick & Decuypere (2002) demonstrated some of the species’ differences in the utilization of enzymes between poultry and pigs as follows: • Bacterial activity: Compared to pigs, chickens' gut microbiota is far less significant. • Digestive ability: Compared to pigs, poultry has shorter small intestines, which means that there is less chance of enzyme inactivation by microflora. Poultry also have a shorter mean retention time in the small intestine (1 to 2 hours) compared to pigs (4 to 5 hours), and their upper gastrointestinal tracts contain less water. • Fermentation of fibre: Because chickens have significantly smaller hind guts than pigs, birds ferment fibre less than pigs do. 21 • Anatomical: In pigs, feed enters the stomach's acid environment right away after consumption, whereas in poultry, feed enters the crop, where enzymes can function for several hours at a pH of about 6.0 before entering the gizzard. Several commercial enzyme products have been introduced to the feed industry as a result of the efficacy of exogenous feed enzymes in boosting animal performance and raw ingredient utilization, as shown in Table 2.4. 22 Table 2.4: Target Substrates, Production Organisms, and Types of Exogenous Enzymes Enzyme name Classification Production organism Targeted function α – Amylase Carbohydrase Aspergillus ssp, Bacillus spp, Rhizopus Starch hydrolysis β – Amylase Carbohydrase Barley malt Starch hydrolysis and production of Maltose Cellulase Carbohydrase Aspergillus niger Cellulose breakdown α–Galactosidase Carbohydrase Aspergillus niger, Morteirella vinaceae var, Saccharomyces spp Oligosaccharides hydrolysis β – Glucanase Carbohydrase Aspergillus spp, Bacillus spp, β-glucans hydrolysis β – Glucosidase Carbohydrase Aspergillus niger Hydrolyses cellulose degradation products to glucose Hemicellulase Carbohydrase Aspergillus spp, Bacillus spp, Humicola spp, Trichoerma spp Break down hemicellulose Invertase Carbohydrase Aspergillus niger, Sacchatomyces spp Hydrolyse sucrose to glucose and Fructose Lactase Carbohydrase Aspergillus niger, Aspergillus oryzae, Hydrolyse lactose to glucose and galactose β – Mannanase Carbohydrase Aspergillus niger, Bacillus lentus, Trichoderma spp. Trichoderma reeseic Beta-mannans hydrolysis Pectinase Carbohydrase Aspergillus niger, Rhizopus oryzae, Aspergillus aculeatus Pectin hydrolysis Xylanase Carbohydrase spergillus spp, Bacillus spp, Humicola spp., Penicilin spp., Trichoderma spp. Xylan hydrolysis Lipase Lipase Aspergillus niger, Candida spp, Rhizomucor spp, Rhizopus spp. Hydrolyses triglycerides, diglycerides and glycerol monoesters Pepsin Protease Animal stomach Protease hydrolysis Protease Protease Aspergillus niger, Aspergillus spp, Bacillus spp Protease hydrolysis Trypsin Protease Animal pancreas Protease hydrolysis Phytase Phytase Aspergillus niger Phytate hydrolysis Source: (Munir & Maqsood, 2013) 23 2.6 Factors Influencing Enzymes Effectiveness According to Cowieson et al. (2006), one of the main problems with dietary enzyme product is that, adding enzymes may not necessarily result in improved nutrient digestibility or growth performance, and there are a variety of reasons for this. According to Gracia et al. (2003), variations in the types and activities of the microorganisms being used to manufacture the enzyme products as well as their sorts can affect the variation in the results. Additional factors include the degree of inclusion thus single versus mixture (Cowieson & Adeola, 2005; Cowieson et al., 2006). According to Cowieson, (2010), dietary nutritional quality is the most significant factor influencing responses to enzyme products; higher responses are anticipated in diets of lower quality. Ravindran (2013) reported that dietary nutrient density, the type of dietary ingredients, and the age of the birds are some of the major factors contributing to variation in the responses of birds to enzyme inclusion. Ravindran (2013) outlined the key factors necessary for the effective functioning of exogenous enzymes. These include the source of the enzyme, its specific catalytic activity, and its resistance to degradation by pepsin. Additionally, the concentration and accessibility of the substrate, along with the physiological conditions of the digestive tract such as pH, temperature, moisture content, and the duration of digesta in the gut, particularly during the gastric phase where enzyme action is most critical, are essential for optimal enzyme activity. 2.6.1 Impact of dietary nutrient density on enzyme effectiveness According to Moraes et al. (2015), enzyme impacts on performance metrics are typically not noticed when standard diets consisting of nutrients that are highly digested and balanced are fed. 24 Sorbara et al. (2009) reported that adding an enzyme to a broiler's diet that is theoretically perfect is unlikely to yield significant improvement because the birds are already performing to the best of their genetic potential, leaving little possibility for improvement. When improved nutrient utilization is not followed by better growth performance, it is possible that the control diets were not sufficiently limiting in nutrients to reduce growth. A study comparing the effects of xylanases and ß-glucanase with α-galactosidase and ß-mannanase at varying metabolizable energy concentrations discovered that adding these enzymes to the broiler diet increased the digestibility and utilization of energy, which in turn increased the broilers' feed conversion ratio (FCR). Additionally, it was observed that ß-glucanase and xylanases added to a low-energy diet increased feed efficiency (Alqhtani et al., 2014). A study by Gitoee et al. (2015) assessed the efficacy of feeding xylanase, α-amylase, and protease at three distinct metabolizable energy levels. According to the findings, adding enzymes to broiler diets allowed for a reduction in energy content without impairing the performance of the broiler chickens. In an investigation into the impact of several enzyme combinations on apparent metabolizable energy, it was discovered that not a single combination was able to improve the performance of the standard diet. On the other hand, pectinase, protease, and α-amylase greatly enhanced the ME when added to a lower-calorie diet (Kocher et al., 2003). 2.6.2 Effect of dietary ingredients on enzyme efficacy An experiment was conducted by Bhuiyan et al. (2013) to demonstrate the impact of enzyme inclusion on varying diet levels of maize. Enzymes such as xylanase, α-amylase, protease, and phytase were employed in this study. There were three different levels of maize used: 250 g/kg, 500 g/kg, and 750 g/kg. The findings showed that while the FCR remained 25 unchanged, adding the enzymes to the various levels of maize significantly increased the FI and BW. Meng & Slominski (2005) used a multi-carbohydrase cocktail in several diets, the enzyme consists of xylanase, ß-glucanase, pectinase, cellulase, ß-mannanase, and galactanase. The study used four different diets: one that was semi-purified maize and the other three that had 30 % soybean meal, canola meal, or peas in addition to maize. Only when the enzymes were added to the maize and soybean meal diet was an improvement in BWG and FCR seen. According to Walters (2019), there was no discernible difference in broiler BW or FCR when the effects of drought-affected maize and a carbohydrase enzyme mixture including ß-glucanase, cellulase, and xylanase inclusion were assessed on broiler performance and nutrient digestibility. In an experiment, the reaction of broiler chicks to two concentrations of xylanase and ß-glucanase cocktail with one of three digestible lysine levels in the feed was assessed. Between d 1 and 42, the enzyme supplementation reduced the FI by 4.67 % and increased the FCR by 5.53 %, all without affecting the BWG. The inclusion of enzymes made up for a decrease in breast weights at day 42 caused by 300 g of sunflower meal or 8.0 g of digestible lysine/kg of diet. As a result, the relationship between enzyme and sunflower meal was substantial (Woest, 2019). Cowieson & Ravindran (2008b) conducted a study to evaluate the reaction of broilers in the starter phase to three different dosages of an enzyme cocktail that included protease, α- amylase, and xylanase. The outcomes showed that adding the enzyme mixture to the control diet improved performance in a dose-dependent way. The quality of the ingredients, the enzyme combinations in the cocktail, and the concentration of substrates in the diet may all have an impact on the dosage sensitivity. The highest BWG was obtained from the birds 26 with higher doses of the enzyme; however, this may not always be the most cost-effective option. 2.6.3 Influence of the birds' age on the enzyme effectiveness Enzyme inclusion can be beneficial for both young and adult chickens. However, young broiler chickens are generally expected to benefit more from enzyme supplementation because their digestive tracts have limited endogenous enzyme activities, potentially resulting in less efficient feed digestion (Olukosi et al., 2018; Bedford & Apajalahti, 2022). Younger broilers typically have less developed digestive enzyme secretion capacity compared to adult chickens, making the addition of feed enzymes more likely to enhance digestion (Ravindran, 2013). However, the age-dependent effect should be less significant when the supplemented enzyme activities are not naturally present in the chicken’s digestive system and are intended to complement the endogenous digestive enzymes (Aftab et al., 2014). The impact of added enzymes may vary with the bird's age as caecal populations grow in size and variability, leading to more pronounced fermentation responses to cell wall fragments in older birds (Bedford & Cowieson, 2012). As broiler chickens get older, their capacity for digestion and microbiota increases. Feed enzymes may affect broiler performance by interacting with microbial populations, which proliferate with broiler chicken age (Bedford & Apajalahti, 2022). The combined impact of xylanase and arabinofuranosidase debranching enzymes on broiler performance, maize glucuronoarabinoxylan breakdown, and caecal microbial fermentation was recently studied by Ravn et al. (2018). Significant improvements in BW and FCR were seen with the addition of the enzymes; these effects were seen throughout the trial but were 27 especially noticeable on days 21 and 29. The observed improvements in gut morphology and broiler performance were most likely caused by the significantly increased caecal butyrate production. According to a study conducted by Tahir et al. (2012), diets containing phytase along with xylanase or a combination of xylanase, protease, and α-amylase showed significant improvement in the BWG and FCR in broilers at 35 days, but only a partial improvement at 49 days. At seven days of age, Radhi et al. (2023) discovered no discernible variations in the impact of different enzymatic supplements. Nevertheless, regardless of the enzyme utilized, the addition of enzymatic complexes improved the performance of broilers at 21 and 35 days in comparison to the control. Two-enzyme supplementation produced comparable performance to the positive control from days 1–21 in a study where broilers were fed diets with lower levels of energy and minerals, but only modest improvements were seen from days 22–42 (Nunes et al., 2015). 2.7 The Role of Enzymes in Breaking Down Minerals in Feed Ingredients Rahimi et al. (2020) reported that phosphorus is one of the most expensive nutrients in poultry diets, but it is an essential nutrient with multiple important functions in the animal body. According to Xu et al. (2021), insufficient amounts of calcium (Ca) and phosphorus (P) can hinder the growth, mineralization, and strength of bones, respectively. As phytate contains bound P, which accounts for 55 – 85 % of total P in the diet, monogastric animals cannot easily access it (Trayhurn, 2005; Jlali et al., 2020). Because of the way it binds to P and prevents it from being absorbed, this is known as the "phytate effect" (Amerah, 2015; Lawlor et al., 2019). Trayhurn (2005) reported that this binding effect raises the cost of 28 production to supply additional phosphorus that is available to the animal and this contributes to environmental pollution as excess P is excreted into the environment. According to Segobola (2016), the two most prevalent minerals in bone are the macro- minerals, calcium and phosphorus, which account for roughly 37 % and 17 % of bone ash, respectively. To prevent imbalances that might lead to a deficiency of either or both, the animal's intake of Ca and P must be carefully balanced. The primary result of inadequate intake of these minerals is rickets, which can be brought on by either a P or Ca deficiency. This can happen when one nutrient is consumed more than the other or when the dietary intake of one nutrient is excessively high, leading to a deficiency of the other. The availability of P varies greatly depending on the source, but calcium is one of the minerals that is both abundant and highly available from most sources (Whitehead et al., 2004). Because of these differences in nutrient availability and the need to maintain a balanced ratio while avoiding excessive use of P to minimize pollution, dietary levels frequently fall short of requirements. The binding of the nutrient in phytate molecules further complicates the availability of dietary P in cereal grains. Nevertheless, according to Selle & Ravindran (2008), the most widely used standard practice for dephosphorylating phytate and liberating the inherent P component in the diet is the inclusion of exogenous phytases in the diets of pigs and poultry. Naves et al. (2016) found that by supplementing broiler feed with 1500 active units of phytase, it is possible to reduce the level of available phosphorus in broiler feed to 1.0 g/kg. Additionally, the calcium level could be fixed at 6.5 g/kg to maintain performance and optimize the bone mineralization of the birds as well as to improve the retention coefficients of calcium, phytate phosphorus, 29 total phosphorus, and nitrogen, while also decreasing the phosphorus excretion into the environment. 2.8 Classification of Non-Starch Polysaccharides Plant cell walls contain non-starch polysaccharides, which can differ in composition, size, and structure (Maharjan et al., 2019). According to their solubility, they are separated into two factions: soluble NSP (sNSP) and insoluble NSP (iNSP). The NSP classification is shown in Figure 2.1. Figure 2.1: Classification of non-starch polysaccharides (Choct et al., 2010). Since soluble NSP (sNSP) has anti-nutritional effects, its inclusion in broiler diet formulation is limited; as a result, the proportion of water-soluble NSP to total NSP in feed is low (Maharjan et al., 2019; Tejede & Kim, 2021). Yacoubi et al. (2016) reported that the anti-nutritional effects are attributed to arabinoxylans and β-glucans with β-1,4 glycosidic linkage backbones and β-1,3 linkage found in the sNSP fraction. According to Maharaj et al. (2019), it results in a sizable amount of water in the digesta binding, which increases the digesta's viscosity as it passes through the small intestine from the proximal to the distal end. Increased intestinal inflammation, poorer nutrient digestion and absorption, and a decrease 30 in feed AME are all results of this increase in digesta viscosity (Amerah, 2015; Yacoubi et al., 2017; Musigwa et al., 2020). According to Maharjan et al. (2019), valuable nutrients pass through the GIT undigested, resulting in poor feed utilization and potentially poor growth and performance if requirements are not met. The decrease in nutrient digestion is caused by the increase in viscosity, which results in reduced interaction between the intestinal brush border and the digesta, hindering the action of intestinal enzymes. Maharjan et al. (2019) reported that an increase in sticky droppings and consequently wet litter is observed due to the increased water-holding capacity. Tejede & Kim (2021) noted that a key factor in the occurrence of foot pad dermatitis is wet litter, as a result of the increased digesta viscosity and decreased pace of digesta transit, the digestive tract may become hypoxic, which might foster the growth of harmful bacteria. According to Maharjan et al. (2019), the insoluble NSP (iNSP) component is thought to be inert and makes up a higher fraction of the total NSP in broiler diets. According to Musingwa et al. (2020), insoluble NSP has no appreciable impact on digesta viscosity and consequently no negative impact on nutrient digestibility. This component of NSP causes a physical barrier against enzymes which is referred to as a ‘cage effect’ (Rios et al., 2017; Musigwa et al., 2020). According to Rios et al. (2017), nutrients are encapsulated, which may affect energy and nutrient digestibility when iNSP is used in diet formulation. Insoluble NSP has laxative qualities, reduces the bacterial load in the hindgut, and in certain situations, may be beneficial in broiler diets. Since enzymes that can break down the β1-4, β1-3, and β1-6 connections are lacking, insoluble NSP has been utilized as a nutritional diluent (Tejede & Kim, 2021). Due to the slowing down and dilution of nutrient intake, too high inclusions reduce performance (Tejede & Kim, 2021). 31 2.8.1 Effect of non-starch polysaccharides on nutrient uptake in broilers The costliest raw materials are cereal grains and their by-products, which make up the majority of broiler diets (Cerrate et al., 2019). These cereal grains coupled with protein crops contain the anti-nutritional component NSP that causes variance in the ME of broiler diets (Yacoubi et al., 2016; Musigwa et al., 2020). The NSP concentration in cereal grains varies between 83 and 98 g/kg, according to Sun et al. (2019). Maharjan et al. (2019) found that the presence of NSP, a collection of molecules with varying sizes, structures, and water solubilities, is negatively correlated with the digestion of carbohydrates. According to Rios et al. (2017), this decrease in digestion is caused by increased viscosity of the digesta and intestinal inflammation, which allow important nutrients to escape digestion in the GIT and result in losses of important nutrients. Increased digesta viscosity causes a bacterial overgrowth and delays transit through the gastrointestinal tract (Cherian, 2020; Tejeda & Kim, 2021). Due to the digesta's high viscosity, the intestinal brush border and digesta do not interact well, resulting in limited contact between the digesta and substrates. This prevents breakdown products from being absorbed (Maharjan et al., 2019). The author also emphasizes that other important nutrients are included in addition to the carbohydrate portion. In line with these claims, Rios et al. (2017) describe the "cage" effect of encapsulation, which lowers digestibility and, as a result, the absorption of nutrients like lipids and amino acids because it creates a physical barrier that inhibits enzyme activity. Non-starch polysaccharides make up a large portion of fibre because fibre is the sum of lignin and NSP, and monogastric animals do not secrete the enzymes required to break down NSP (Cherian, 2020). 32 According to Rios et al. (2017), corn and soybean diets are easier to digest than diets formulated with other cereals, such as wheat and barley, which are known to have higher amounts of NSP. According to Changa et al. (2020), broilers obtain their energy and protein from highly digestible raw materials. In a typical broiler corn and soybean diet, maize supplies around 65 % of the total apparent metabolizable energy while soybean meal offers 80 % of the total crude protein (CP) (Rios et al., 2017). Because soybean meal is abundant in protein and satisfies poultry's need for certain amino acids, it offers the highest feeding value among all plant-based protein sources (Frempong et al., 2019). A feedstuff's energy value is determined by how well the starch is absorbed, which is rarely an issue in maize-based diet because broilers completely digest the starch component of maize (Zaefarian et al., 2015). On the other hand, soybean meal has additional ANFs like phytic acid and trypsin inhibitors in addition to NSP (Frempong et al., 2019). The NSP levels in soybean meal present a digestibility issue even though they are lower than those of other vegetable ingredients like wheat and barley (Jamroz et al., 2002; Musigwa et al., 2020). According to Nguyen et al. (2022), diets containing maize and soybeans typically have a total NSP of 10 – 12 % DM, with a water-soluble portion of 1-2.5 % DM. According to Rios et al. (2017) and Saleh et al. (2019), the contents of NSP in maize and soybean meal are around 9 % (97 g/kg) and 29 % (217 g/kg), respectively. However, according to Zaefarian et al. (2015), genetics and environment determine how much NSP is in maize and soybean meal. Frempong et al. (2019) claim that appropriate thermal processing of raw materials can reduce the issues caused by ANFs to some degree. Table 2.5 shows how the NSP contents of several cereal grains differ from one another. 33 Table 2.5: Content of dry matter (g/100 g, as is basis) and non-starch polysaccharides of the plant-based ingredients Ingredients DM 1 sNSP 2 iNSP 3 tNSP 4 Wheat 90.53 14.23 83.08 97.31 Corn 88.97 2.86 64.56 67.42 Barley 90.96 42.36 137.4 179.7 Sorghum 88.45 1.65 53.54 55.2 Soybean meal 90.05 11.22 132.2 143.4 Canola meal 92.16 15.35 146.8 162.1 Wheat bran 91.21 23.16 385.1 408.2 Oat bran 92.40 52.24 65.02 117.3 Soy protein concentrate 93.05 14.69 157.4 172.1 Source: (Nguyen et al., 2022). DM- 1 Dry matter; sNSP- 2 Soluble non-starch polysaccharides; iNSP- 3 Insoluble non-starch polysaccharides; tNSP- 4 Total non-starch polysaccharides 2.9 Effect of Phytic Acid on Growth Performance of Broilers The phosphorylated cyclic sugar alcohol is known as phytic acid (myo-inositol 1, 2, 3, 4, 5, 6-hexakis dihydrogen phosphate). Phytate, the anion form of phytic acid, is found in all plants. Phytin is the chelated form of phytate that is often found in plants when it is combined with cations, proteins, and/or starches (Wang & Guo, 2021). Angel et al. (2015) and Ravindran et al. (2000) reported that in addition to limiting the availability of P, phytate also functions as an anti-nutrient in the diet, affecting the metabolizable energy (ME) and overall digestibility of dietary cations and amino acids. Additionally, it was noted that increased losses of endogenous amino acids were correlated with the amount of phytate included in 34 the diet and had a negative impact on amino acid utilization (Ravindran et al., 1999; Cowieson et al., 2004a; Cowieson & Ravindran, 2007). It has been suggested that the low levels of endogenous phytase are the reason why over two-thirds of the P in plant-based feedstuffs is not easily accessible in poultry because it is bonded to phytic acid (PA) (Bedford, 2000; Woyengo & Nyachoti, 2011). According to recent research, this is untrue because chickens' intestinal mucosa has enough phytase activity. Poor substrate solubility in the small intestine as a result of cation interactions with Ca is the fundamental problem with phytate digestion in poultry (Maenz & Classen, 1998; Cowieson et al. 2011). Phytate phosphorus (PP) and calcium ions combine to create insoluble complexes that inhibit phytase action (Angel et al., 2015). Consequently, reducing the amount of calcium in the diet can enhance the impact of exogenous phytase on the breakdown of PP. Nowadays, it's standard practice to provide exogenous phytase to help birds break down PP. Previous research has demonstrated that the performance of young birds was unaffected by the addition of phytase (500 FTU/kg) and the simultaneous reduction of Ca in starter diets from 1.0 % to 0.67 % in conjunction with decreased non-PP (nPP) levels (Létourneau-Montminy et al., 2010; Powell et al., 2011). According to Lui et al. (2014), there are variations in the concentrations of phytate within various raw materials, and it may be found in the aleurone layers of sorghum and wheat while in maize the germ cell. The charge on the molecule makes it a great chelator. At low pH levels, below the isoelectric point of proteins, proteins have a positive charge, which causes insoluble complexes to form with the negatively charged phytic acid (Feil, 2008). Phytic acid directly affects starch digestion and suppresses amylase activity in broilers due to its capacity to form complexes with other nutrients, including calcium, iron (Fe), zinc (Zn), and manganese (Mn) (Lui et al., 2014). 35 According to Bedford & Rousseau (2017), phytic acid also decreases the rate at which pepsinogen is converted to pepsin and raises endogenous losses linked to the breakdown of gastric mucus. These actions effectively lower the quantity of pepsin available in the stomach. Phytic acid is an ANF that has been shown to affect the effectiveness of digestion and, in turn, the performance of chickens (Bedford & Rousseau, 2017). Because chickens cannot obtain P from their diets, particularly when fed maize and soybeans, costly inorganic sources of P must be added to the diets. This frequently results in dietary P levels exceeding the minimum requirements, which increases feed costs and pollution to the environment (Feil, 2008; Lawlor et al., 2019; Poernama et al., 2021). Figure 2.2: The molecular structure of phytic acid (Cherian, 2020) 2.10 Influence of Exogenous Enzyme on Growth Performance of Broilers The poultry industry has witnessed significant advancements in feed additives aimed at improving the growth performance and overall health of broilers. One such additive gaining attention is the exogenous enzyme which is designed to enhance nutrient utilization in poultry diets, thereby positively influencing broiler growth and performance. Studies by Choct et al. (2010) have shown that the inclusion of exogenous enzymes in broiler diets leads to increased enzymatic activity, particularly in the hydrolysis of NSPs and proteins. A 36 study by Bedford and Cowieson (2012) demonstrated that the addition of exogenous enzyme to broiler diets resulted in improved feed conversion ratios (FCR), indicating enhanced utilization of nutrients for growth. The increased efficiency in converting feed into body mass suggests the economic feasibility of incorporating exogenous enzymes in broiler production systems. When a multi-enzyme complex was introduced to the diets of broiler chickens fed maize and soybean meal, Rios et al. (2017) assessed its effects on the growth performance, energy, and amino acid consumption of the birds. According to the authors, feed conversion ratio, digestible energy, and digestible amino acid levels all improved with enzyme addition. Nadeem et al. (2005) found that adding feed enzyme to a diet containing 50 Kcal/kg less ME than the control diet at a rate of 0.05 g/kg did not significantly affect weight gain. However, it did significantly increase feed intake and decrease feed conversion rate (FCR) during the initial (1-28 days) and overall (1-42 days) growing periods (Table 2.6). However, during the finisher phase (29–42 days), these authors did not find any appreciable variations in these parameters. The research conducted by Khan et al. (2006) also demonstrated that adding an exogenous enzyme to chicken feed (0.05 g/kg) increased weight gain and feed conversion ratio (FCR) by 8 % when the diet was corn-based and sunflower meal; feed intake was unaffected. Nutrient digestibility was improved in the diet that included enzyme supplements. These results support the hypothesis that, in comparison to high-digestible feedstuffs, the positive effects of NSP-degrading enzymes may be slightly greater with low- digestible feedstuffs such as sunflower meal (14–18 % crude fibre). A study by Chalghoumi et al. (2020) reported that supplementing Rovabio Excel at 0.05 g/kg improved final live BW, daily BWG, daily FI, FCR, and production index for broilers from (day 7-21) and (day 37 22-37) but was statistically similar to the standard control diet. Broilers fed maize and SBM diets containing NSPase showed improvements in BWG and FCR of 3.9 % and 3.2 %, respectively, according to Slominski (2011). In contrast to the aforementioned findings, research by West et al. (2007) shows that the addition of Rovabio Excel (0.022 %) was without impact on growth, feed conversion, and carcass characteristics but decreased (P = 0.06) mortality at days 1-14 and 1-42 respectively. 38 Table 2.6: Growth performance of broiler chicks fed diets supplemented with or without NSPDE Parameter Diet A B C D Starter phase (0-28 days) Average initial body weight (g/bird) 44.00 ± 0.03 45 ± 0.08 43 ± 0.07 44 ± 0.06 Average body weight (g/bird) 1002 ± 12 1069 ± 11 1063 ± 14 1060 ± 16 Average feed intake (g/bird 2200b ± 5 2457a ± 7 2336b ± 5 2414a ± 4 Feed efficiency 2.20b ± 0.04 2.30a ± 0.02 2.20b ± 0.05 2.28a ± 0.03 Finisher phase (29-42 days) Average body weight (g/bird) 969 ± 21 969 ± 22 966 ± 19 982 ± 15 Average feed intake (g/bird 2355 ± 6 2443 ± 4 2403 ± 8 2491± 9 Feed efficiency 2.43 ± 0.08 2.52 ± 0.07 2.49 ± 0.03 2.54 ± 0.02 Overall (0-42 days) Average body weight (g/bird) 1971 ± 16 2038 ± 9 2029 ± 15 2042 ± 16 Average feed intake (g/bird 4555b ± 5 4900a ± 3 4739b ± 9 4905a ± 8 Feed efficiency 2.31b ± 0.06 2.40a ± 0.00 2.33b ± 0.04 2.40a ± 0.03 Source: (Nadeem et al., 2005). Value (means ± SEM) in rows with different superscripts differ significantly (p<0.05). * Diet A: commercial broiler diet without NSPDE; Diet B: commercial broiler diet without NSPDE; Diet C: commercial broiler diet having 50 Kcal/Kg less ME and without NSPDE and Diet D: commercial broiler diet having 50 Kcal/Kg less ME and with 0.05 g/Kg NSPDE 2.11 Influence of Exogenous Enzyme on Nutrient Digestibility of Broilers The carbohydrases such as xylanases and cellulases, target non-starch polysaccharides (NSPs) present in feed ingredients. Through their enzymatic action, they break down complex carbohydrates into simpler, more digestible forms, potentially improving nutrient availability for absorption in the gastrointestinal tract (Bedford, 2018). 39 The impact of exogenous enzyme on the nutrient digestibility of broilers has been the subject of several investigations. Choct et al. (2017) reported increased ileal digestibility of nutrients, including proteins and amino acids, when broilers were fed diets supplemented with feed enzyme. The enhanced digestibility of nutrients suggests that feed enzyme may play a crucial role in breaking down dietary components, making them more accessible for absorption in the small intestine. One aspect of nutrient digestibility influenced by feed enzymes is the degradation of dietary fibres. Carbohydrases target fibrous components, such as arabinoxylans and cellulose, leading to their breakdown. Bedford (2018) highlighted the importance of this enzymatic action in reducing the anti-nutritional effects associated with fibre, ultimately contributing to improved nutrient utilization by broilers. A study by Choct et al. (2017), demonstrated that an exogenous enzyme (0.05 %) contributes to the breakdown of complex carbohydrates into fermentable substrates, potentially increasing the production of short-chain fatty acids in the caeca. This fermentation process can enhance energy availability for the bird, influencing overall nutrient digestibility and utilization. Bedford (2018) proposed that the enzymatic breakdown of NSPs by exogenous enzyme reduces the viscosity of the digesta, promoting nutrient absorption. Additionally, the liberation of oligosaccharides during NSP degradation may serve as prebiotics, fostering a healthier gut environment for improved nutrient assimilation (Choct et al., 2017). It has also been demonstrated by Wiseman et al. (2000) that there is a strong correlation between the AME values of several wheat varieties and the rate of starch digestion in vitro. The findings indicate that the degree of amylolytic enzyme accessibility to starch granules varies among wheat varieties, with the cell wall architecture of the grain potentially serving as a major determining factor. In the small intestine of chickens, Bedford (2002) reported that a 40 significant quantity of nutrients, including starch, are retained inside the cell walls and are eliminated when xylanase is added. According to D'Alfonso & McCracken (2003), there is a notable fluctuation in the nutritional content of maize (93 samples analyzed) for hens, with starch digestibility ranging from 84 % to 90 % and ileal digestible energy value varying by 2.04 MJ/kg DM. This variance was decreased using an enzyme product including xylanase, protease, and amylase. According to Cowieson (2005), rather than viscosity reduction, as is frequently the case for viscous grains, the capacity of enzymes, in particular, glycanases, to boost the nutritional content of corn-soy diets is likely mediated by changes in cell wall architecture of the gain. 2.12 Impact of Exogenous Enzyme on Gut pH Of Broilers Research has shown that exogenous enzyme supplementation influences nutrient utilization in broilers by breaking down NSPs and other complex substrates. Exogenous enzyme facilitates the release of fermentable substrates in the gastrointestinal tract. Choct et al. (2017) reported that this enzymatic action may contribute to an increase in short-chain fatty acid production in the ceca, potentially influencing gut pH through the fermentation process. Digesta viscosity is a critical factor affecting nutrient absorption in the gastrointestinal tract. Bedford (2018) proposed that the enzymatic degradation of NSPs by exogenous enzymes reduce the viscosity of the digesta, creating an environment conducive to nutrient absorption. This reduction in viscosity may contribute to changes in gut pH, as the availability and absorption of nutrients are closely linked to the physical properties of the digesta. The gut microbiota plays a crucial role in maintaining gut health and influencing pH. The breakdown of NSPs by exogenous enzymes generates oligosaccharides, which may act as prebiotics, 41 promoting the growth of beneficial microorganisms. The modulation of the microbial population in the gut can have downstream effects on fermentation processes and, consequently, on gut pH regulation (Choct et al., 2017; Bedford, 2018). Morgan et al. (2022) observed a trend towards a more acidic pH in the caeca of broilers receiving exogenous enzyme supplementation. This observation aligns with the notion that enhanced microbial fermentation, facilitated by exogenous enzyme, can lead to the production of organic acids, influencing gut pH. 2.13 Influence of Exogenous Enzyme on Carcass Traits of Broilers The composition of broiler carcasses, including the distribution of muscle and fat, is critical for meat quality. Bedford (2018) suggested that the enzymatic action of exogenous enzyme on lipids might impact fat deposition and, subsequently, carcass composition. Additionally, changes in nutrient availability and utilization may influence muscle development, potentially influencing the yield and quality of broiler meat. Morgan et al. (2022) reported improved carcass yield and breast meat percentage in broilers receiving exogenous enzyme supplementation. The authors attributed these effects to enhanced nutrient utilization and the promotion of a healthier gut environment. While Zanella et al. (1999) demonstrated that the inclusion of exogenous enzyme had no significant influence on the relative weight of leg, breast muscle, and wings, Selle et al. (2003a) found that supplementing wheat-based diets with xylanase plus phytase increased breast weight by 5.8 %. Wu et al. (2004) reported that the addition of phytase and xylanase separately resulted in a significant decrease in the small intestine's relative length and weight (p<0.05). In contrast, Brenes et al. (1993) showed that adding xylanase to wheat-based diets had no effect 42 on the relative weights of the pancreas, liver, proventriculus, or small intestine in broiler chicks. Similarly, Lee et al. (2010) found no significant differences in the relative weights of the liver, abdominal fat, right leg, or right breast muscle between treatment groups when using Rovabio® Max (0.02%). Chicks fed diets containing Rovabio® Max had slightly larger relative weights of the right leg and right breast muscle than chicks in the negative control groups lacking Rovabio® Max, but these changes were not statistically significant. 2.14 Influence of Exogenous Enzyme on Bone Health Bone mineralization is a key determinant of bone strength and overall skeletal integrity. Research by Morgan et al. (2022) demonstrated that broilers receiving exogenous enzyme supplementation exhibited improved bone mineralization. The authors attributed this effect to the enhanced digestion of phytate-bound minerals, releasing phosphorus for better utilization in bone formation. Improved bone mineralization is indicative of enhanced bone strength, which is essential for the overall well-being of broilers. When comparing the tibia- breaking strength and ash of chicks fed diets containing exogenous enzyme to those of the negative control groups that did not contain exogenous enzyme, Lee et al. (2010) found no significant differences in the relative weight and length of the tibia among the treatments. Chicks fed diets with exogenous enzyme had tibia-breaking strength and ash considerably higher than those of the negative control groups that did not receive enzyme (p < 0.05). The phytase action may have increased the retention of calcium and phosphorus from the phytate- mineral complex, which would have improved the ash percentage. These findings are thought to be a strong indicator of the correlation between enhanced tibia mineralization and the addition of phytase to the diet, as several publications have found comparable results (Martínez-Vallespín et al., 2022). 43 The breakdown of NSPs and phytate by exogenous enzyme may reduce the anti-nutritional effects associated with these compounds, leading to increased mineral availability for bone development (Bedford, 2018). Additionally, the potential prebiotic effects of exogenous enzyme on the gut microbiota may indirectly influence mineral absorption and utilization, contributing to improved bone health in broilers. In an experiment to determine the effects of multiple enzymes composed of phytase plus carbohydrolases found that the supplementation of the multiple enzyme improved the growth performance and bone mineralization in broiler chicks (Alagawany et al., 2018). Another study by Javaid et al. (2022) evaluated the impact of an indigenously produced multi-enzyme complex on broiler growth and found that the supplementation led to significantly higher tibia ash values and phosphorus content in the bones of the birds. https://pubmed.ncbi.nlm.nih.gov/?term=Alagawany%20M%5BAuthor%5D 44 CHAPTER THREE 3.0 MATERIALS AND METHODS 3.1 Study Location and Duration The research was carried out within the poultry unit of the Department of Animal Science at Akenten-Appiah Menka University of Skills Training and Entrepreneurial Development, situated on the Mampong- Ashanti campus. Mampong is positioned in the intermediate region linking the Guinea savannah to the north and the tropical rainforest to the south of Ghana, along the Kumasi-Ejura Road. The research spanned from February 2024 to June 2024. 3.2 Dietary Treatment and Experimental Design The proximate composition of the primary ingredients (maize, maize bran, soybean meal, and fishmeal) was Analyzed and used to formulate four experimental diets (Table 3). These diets were structured based on a 2 × 2 factorial design, with treatments randomized in a completely randomized design (CRD). The factors include Enzyme (Enz) levels (No vrs Yes) and Maize Bran levels (No vrs Yes) across starter, grower, and finisher diets. The Concept 4 feed formulation program from Creative Formulation Concepts, LLC, Annapolis, MD, was employed for diet formulation. The MB-based diets were made to be a bit deficient in crude protein, energy, lysine and methionine. The addition of enzyme on top of the diets, without using the manufacturer's matrix values as is the practice in Ghana was expected to improve nutrient retention and compensate for the deficiency of the dietary nutrient. Subsequently, the diets were subjected to proximate and chemical analyses following the procedures of AOAC (1990). Throughout the starter (d 0 to 28) and grower-finisher (d 28 to 56) phases, the diets were provided ad libitum in mash form. 45 Table 3.1: Composition and Calculated analysis of experimental diets, % Vitamin A, 8,000,000 IU; Vitamin B1, 1300 mg, Vitamin B2, 2500 mg, Vitamin D3, 3000 IU; Vitamin E, 10,000 IU; Vitamin K3, 1,500 mg; Vitamin B6, 1,000 mg; Vitamin B12, 6 mg, Nicotinic Acid, 5,000 mg,Pantothenic Acid, 4000 mg; Choline Chloride, 8000 mg; Copper, 2,500 mg; Cobalt, 700 mg; Iron, 4,500 mg; Zinc, 55, 000 mg; Methionine, 50,000 mg; Lysine, 200,000 mg; Selenium (1%), 1,300 mg; Iodine, 2,000 mg; Manganese, 60, 000 mg; Antioxidant, 625 mg. Starter Grower /Finisher Ingredients T1 T2 T3 T4 T1 T2 T3 T4 Maize grain Soybean meal Maize bran Fishmeal Dicalcium Phosphate Salt Oyster shell Mineral premix TiO2 DL-Methionine Enzyme (RovabioTM) Total Calculated Nutrient Crude Protein Fibre Calcium Avail Phosphorus M.E. (Kcal/Kg) Methionine, total Lysine, total Sodium Chloride 63 19.5 0 15.18 0.5 0.4 0.32 0.5 0.5 0.1 0 100 22.43 2.84 0.9 0.34 2998 0.59 1.43 0.29 0.24 63 19.5 0 15.18 0.5 0.4 0.32 0.5 0.5 0.1 0.03 100 22.43 2.84 0.9 0.34 2998 0.59 1.43 0.29 0.24 50 15.5 19.76 12 0.99 0.4 0.25 0.5 0.5 0.1 0 100 20.15 4.68 0.94 0.45 2795 0.53 1.22 0.28 0.24 50 15.5 19.76 12