University of Ghana http://ugspace.ug.edu.gh EVALUATION OF TWO HERBAL PRODUCTS (FAGARA ZANTHOXYLOIDES FRUIT MEAL AND OCIMUM AMERICANUM LEAF MEAL) AS GROWTH PROMOTERS IN BROILER DIETS BY ELVIS AMANOR (10404632) THIS THESIS IS SUBMITTED TO THE UNIVERSITY OF GHANA, LEGON IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE AWARD OF MASTERS OF PHILOSOPHY IN ANIMAL SCIENCE DEGREE NOVEMBER, 2020 University of Ghana http://ugspace.ug.edu.gh DECLARATION i University of Ghana http://ugspace.ug.edu.gh ABSTRACT This study evaluated Fagara zanthoxyloides fruit meal (FFM) and Ocimum americanum leaf meal (OLM) as growth promoters in broiler diets as replacements for antibiotics. A total of 400 one- day-old Cobb 500 broiler chicks were used for the experiment. The birds were initially raised together on a common starter diet for a week and on day 8, distributed into eight dietary treatment groups in a completely randomized design for six more weeks. The treatment diets were as follows: BD = Basal diet; 0.2FFM = BD + 0.2% FFM; 0.4FFM = BD + 0.4% FFM; 0.2OLM = BD + 0.2% OLM; 0.4OLM = BD + 0.4% OLM; 0.1FFM+0.1OLM = BD + 0.1% FFM + 0.1% OLM; 0.2FFM+0.2OLM = BD + 0.2% FFM + 0.2% OLM; PEN = BD + 0.01% Penicillin V. Each treatment was replicated five times with 10 birds in each replicate. Feed and water were provided ad libitum. The parameters measured include growth performance, carcass characteristics, apparent whole tract nutrient digestibility, nitrogen excretion, serum lipid profile, and faecal microbial count. Data collected were all subjected to analysis of variance using Genstat statistical software (12th edition, 2009), and means with significant differences were separated with Student Newman-Keuls test at a probability of 5%. The results show no significant effects (p>0.05) of FFM, OLM, and penicillin on growth performance, carcass characteristics, digestibility of dry matter, crude protein, ash and crude fibre, nitrogen excretion, serum lipid profile, and counts of faecal pathogenic microbes when compared with birds fed BD. Fat digestibility and faecal microbial load were lowered (p<0.05) by FFM, OLM, and penicillin. Birds fed PEN recorded the least faecal count of yeasts and moulds similar (p>0.05) to that fed 0.4FFM and 0.4OLM. In conclusion, inclusion of FFM and OLM alone or in combination up to 0.4% in diets of broilers did not promote growth performance. ii University of Ghana http://ugspace.ug.edu.gh DEDICATION This work is primarily dedicated to the Almighty God without whose help I could not have come this far, my parents (Mr. Eric Kwao Amanor and Mrs. Sarah Amanor), siblings (Chris Tetteh Kwao Amanor and Marilyn Adamki Amanor), Mr. Gideon Afolayan who encouraged me continually that God will help me to do my project when it looked like all hope was lost and finally, to my very good friend Mr. Daniel Bentum whose contribution towards the success of this work was so wonderful and much appreciated. iii University of Ghana http://ugspace.ug.edu.gh ACKNOWLEDGEMENT My first and foremost gratitude goes to my heavenly Father, The Almighty God for His rich grace and love towards me. He has been my backbone throughout my graduate studies. When the going became tough and I felt like giving up, He strengthened and encouraged me, and here I stand today at the end of the tunnel by His grace. To Him be all the glory, praise, honour, and adoration forever. My next appreciation goes to Dr. Leonard Kofi Adjorlolo, my principal supervisor, and Dr. James Edinam Futse, co-supervisor for their supervision, guidance, counsel, corrections, and instructions throughout the project and compilation of this thesis. I am also indebted to the Livestock and Poultry Research Centre (LIPREC) for providing the research facility, broiler chicks, feed, medication, and water for the project, the Leventis Foundation Scholarship Fellowship for funding the project, and the senior members of the Animal Science Department, University of Ghana for their tutoring, training and contribution towards this work. I also appreciate the wonderful family that God has blessed me with and the efforts of my parents in setting me up on the academic ladder and for their unflinching support throughout my education. I also give God praise for making me meet the late Dr. Thomas Nii Narku Nortey, my former supervisor. Only God will understand how the training I received from him means to me. I learnt so much from him. May His soul rest in perfect peace. Finally, my special gratitude to the following individuals and all who contributed in diverse ways to make this work a success; Prof. Boniface Kayang, Dr. Francis Dogodzi, Mr. Yussif Abdulai, Mr. Gilbert Gbafah, Mr. Christopher Tudeka, Mr. Gideon Afolayan, Mr. Daniel Bentum, Mr. Jonathan Quaye, Mr. Titus Kali, Mr. Solomon Boadu, Mr. Protase Yuorkuu, Mr. Matthew Nyovore, Mr. Benjamin Osei, Mr. Mohammed Bashiru, Mr. Amos Nyarko, Mr. Robert Ntreh, Mrs. Ruth Yeboah, and Mrs. Princess Anane. iv University of Ghana http://ugspace.ug.edu.gh TABLE OF CONTENTS DECLARATION .............................................................................................................................................. i ABSTRACT ..................................................................................................................................................... ii DEDICATION .................................................................................................................................................iii ACKNOWLEDGEMENT ............................................................................................................................... iv TABLE OF CONTENTS ................................................................................................................................. v LIST OF PLATES .......................................................................................................................................... xii LIST OF TABLES ......................................................................................................................................... xiii LIST OF APPENDICES ................................................................................................................................. xv LIST OF ABBREVIATIONS ........................................................................................................................ xvi CHAPTER ONE .............................................................................................................................................. 1 INTRODUCTION ........................................................................................................................................... 1 1.1 Hypothesis.................................................................................................................................................. 3 1.2 Objective of the Study ............................................................................................................................... 3 1.2.1 Specific objectives .............................................................................................................................. 3 CHAPTER TWO ............................................................................................................................................. 4 2.0 LITERATURE REVIEW .......................................................................................................................... 4 2.1 Growth Promoters in Animal Diets ........................................................................................................... 4 2.2 Antibiotics and their Benefits in Poultry Production ................................................................................. 4 2.2.1 Uses of antibiotics in poultry husbandry ............................................................................................. 6 v University of Ghana http://ugspace.ug.edu.gh 2.2.2 Modes of action of antibiotic growth promoters (AGPs) .................................................................... 9 2.2.3 Adverse effects of AGPs on bird and human health ........................................................................... 9 2.2.3.1 Effects of consuming poultry products containing antibiotic residues on human health .......... 10 2.3 Alternatives to Antibiotic Growth Promoters (AGPs) in Poultry Diets ................................................... 11 2.3.1 Probiotics .......................................................................................................................................... 11 2.3.2 Prebiotics........................................................................................................................................... 13 2.3.3 Synbiotics .......................................................................................................................................... 14 2.3.4 Organic acids .................................................................................................................................... 15 2.3.5 Exogenous enzymes .......................................................................................................................... 16 2.3.6 Egg yolk antibodies ........................................................................................................................... 17 2.3.7 Metals ................................................................................................................................................ 18 2.4 Phytogenic Feed Additives in Poultry Nutrition ...................................................................................... 19 2.4.1 Effects of PFAs on feed intake, nutrient digestibility and gut morphology in poultry ..................... 22 2.4.2 Phytogenic feed additives as antimicrobial agents............................................................................ 24 2.4.2.1 Antimicrobial modes of action of phytochemicals ..................................................................... 27 2.4.3 Phytogenic feed additives as growth promoters in poultry diets ...................................................... 27 2.4.3.1 Limitations in the use of phytogenic feed additives ................................................................... 32 2.4.3.2 Benefits of phytogenic feed additives over antibiotic growth promoters ................................... 33 2.4.4 Phytogenic feed additives as antioxidative agents ............................................................................ 33 2.4.5 Phytogenic supplements as immune-stimulatory agents ................................................................... 34 vi University of Ghana http://ugspace.ug.edu.gh 2.4.6 Phytogenic feed additives as coccidiostatic agents ........................................................................... 36 2.4.7 Phytogenic supplements as anthelmintic agents ............................................................................... 37 2.4.8 Effects of phytogenic feed additives on some blood constituents in poultry .................................... 39 2.4.9 Effects of phytogenic feed additives on the quality of poultry products .......................................... 41 2.4.10 Phytogenic supplements as anti-stress agents in poultry ................................................................ 43 2.5 Fagara zanthoxyloides ............................................................................................................................. 47 2.5.1 Biology of Fagara zanthoxyloides .................................................................................................... 47 2.5.2 Chemical composition of Fagara zanthoxyloides............................................................................. 50 2.5.3 Uses of Fagara zanthoxyloides ......................................................................................................... 50 2.5.4 Antimicrobial properties of Fagara zanthoxyloides extracts ............................................................ 52 2.5.5 Anthelmintic properties of Fagara zanthoxyloides extracts ............................................................. 53 2.6 Ocimum americanum ............................................................................................................................... 54 2.6.1 Biology of Ocimum americanum ...................................................................................................... 54 2.6.2 Chemical composition of Ocimum americanum ............................................................................... 55 2.6.3 Uses of Ocimum americanum ........................................................................................................... 56 2.6.4 Antimicrobial activity of Ocimum americanum extracts .................................................................. 56 CHAPTER THREE ....................................................................................................................................... 57 3.0 MATERIALS AND METHODS ............................................................................................................. 57 3.1 Experimental Site and Duration of Study ................................................................................................ 57 3.2 Experimental Birds and Their Management ............................................................................................ 57 vii University of Ghana http://ugspace.ug.edu.gh 3.2.1 Brooding of birds .............................................................................................................................. 57 3.2.3 Vaccination schedule ........................................................................................................................ 59 3.2.4 Lightning ........................................................................................................................................... 59 3.2.5 Litter management ............................................................................................................................ 59 3.3 Preparation of Experimental Diets ........................................................................................................... 59 3.4 Growth Performance Determination ........................................................................................................ 63 3.5 Mortalities ................................................................................................................................................ 63 3.6 Serum Lipid Profile Test .......................................................................................................................... 64 3.7 Carcass Analysis ...................................................................................................................................... 64 3.8 Digestibility Study ................................................................................................................................... 64 3.9 Nitrogen Excretion Determination ........................................................................................................... 65 3.10 Faecal Microbial Analysis ..................................................................................................................... 66 3.11 Chemical Analyses ................................................................................................................................. 66 3.12 Statistical Analysis ................................................................................................................................. 67 CHAPTER FOUR .......................................................................................................................................... 68 4.0 RESULTS ................................................................................................................................................ 68 4.1 Nutrient Composition of FFM and OLM ................................................................................................. 68 4.2 Effects of Dietary Treatments on Growth Performance .......................................................................... 69 4.2.1 Effects of Dietary Treatments on Body Weight ................................................................................ 69 4.2.2 Effects of Dietary Treatments on Average Daily Weight Gain ........................................................ 70 viii University of Ghana http://ugspace.ug.edu.gh 4.2.3 Effects of Dietary Treatments on Average Daily Feed Intake .......................................................... 71 4.2.4 Effects of Dietary Treatments on Feed Conversion Ratio ................................................................ 72 4.3 Effects of Dietary Treatments on Mortality ............................................................................................. 72 4.4 Effects of Dietary Treatments on Carcass and Organ Characteristics ..................................................... 73 4.5 Effects of Dietary Treatments on Apparent Total Tract Nutrient Digestibility and Nitrogen Excretion . 75 4.6 Effects of Dietary Treatments on Serum Lipid Profile ............................................................................ 77 4.7 Effects of Dietary Treatments on Faecal Microbial Composition ........................................................... 78 CHAPTER FIVE ........................................................................................................................................... 81 5.0 DISCUSSION .......................................................................................................................................... 81 5.1 Effects of FFM, OLM and Penicillin Supplementation on Growth Performance.................................... 81 5.1.1 Effects of penicillin supplementation on growth performance ......................................................... 81 5.1.2 Effects of FFM and OLM supplementation on growth performance ................................................ 82 5.1.3 Effects of penicillin, FFM and OLM supplementation on feed intake ............................................. 83 5.2 Effects of Penicillin, FFM and OLM Supplementation on Carcass Parameters ...................................... 84 5.3 Effects of Penicillin, FFM and OLM Supplementation on Nutrient Digestibility ................................... 86 5.4 Effects of Penicillin, FFM and OLM Supplementation on Nitrogen Excretion ...................................... 87 5.5 Effects of Penicillin, FFM and OLM Supplementation on Serum Lipid Profile ..................................... 88 5.6 Effects of FFM, OLM and Penicillin Supplementation on Faecal Microbial Composition .................... 90 5.6.1 Effects of penicillin supplementation on faecal counts of the tested pathogenic microbes .............. 90 5.6.2 Effects of FFM and OLM supplementation on faecal counts of the tested pathogenic microbes .... 91 ix University of Ghana http://ugspace.ug.edu.gh 5.6.3 Effects of penicillin, FFM and OLM supplementation on faecal microbial load ............................. 93 5.6.4 Effects of penicillin, FFM and OLM supplementation on faecal yeasts and moulds count ............. 95 CHAPTER SIX .............................................................................................................................................. 97 6.0 CONCLUSIONS AND RECOMMENDATIONS .................................................................................. 97 6.1 Conclusions .............................................................................................................................................. 97 6.2 Recommendations .................................................................................................................................... 98 REFERENCES .............................................................................................................................................. 99 APPENDICES ............................................................................................................................................. 148 x University of Ghana http://ugspace.ug.edu.gh LIST OF FIGURES Figure 4. 1: Apparent total tract fat digestibility.............................................................................. 76 Figure 4. 2: Faecal microbial load ................................................................................................... 79 xi University of Ghana http://ugspace.ug.edu.gh LIST OF PLATES Plate 2. 1: Modes of action of phytochemicals against helminths ................................................... 39 Plate 2. 2: Fagara zanthoxyloides plant ........................................................................................... 48 Plate 2. 3: Fruits of Fagara zanthoxyloides ..................................................................................... 49 Plate 2. 4: Ocimum americanum plants ........................................................................................... 54 Plate 3. 1: Birds in a pen after distribution ...................................................................................... 58 xii University of Ghana http://ugspace.ug.edu.gh LIST OF TABLES Table 2. 1: Commonly-used antibiotics in poultry husbandry ........................................................... 5 Table 2. 2: Effects of antibiotic growth promoters (AGPs) in broiler chickens ................................ 8 Table 2. 3: Commonly used botanicals in poultry diets................................................................... 20 Table 2. 4: Effects of phytogenic feed additives on broiler growth indices .................................... 31 Table 2. 6: Effects of phytogenic supplements on Ascaridia galli (A. galli) in chicken ................. 38 Table 2. 7: Effects of PFAs on blood haematology and biochemical indices of broilers ................ 41 Table 2. 8: Effects of phytogenic feed additives on poultry performance, physiology, and productivity under heat stress conditions ......................................................................................... 45 Table 2. 9: Proximate composition of the foliage (at flowering) of Ocimum americanum ............. 55 Table 3. 1: Experimental diets and treatment layout ....................................................................... 60 Table 3. 2: Ingredient composition of experimental starter diets .................................................... 61 Table 3. 3: Ingredient composition of experimental finisher diets .................................................. 62 Table 4. 1: Nutrient composition of FFM and OLM ....................................................................... 68 Table 4. 2: Body weight of broilers fed diets supplemented with FFM, OLM and penicillin ........ 69 Table 4. 3: Average daily weight gain (g) of broilers fed diets supplemented with FFM, OLM and penicillin .......................................................................................................................................... 70 Table 4. 4: Average daily feed intake (g) of broilers fed diets supplemented with FFM, OLM and penicillin .......................................................................................................................................... 71 Table 4. 5: Feed conversion ratio of broilers fed diets supplemented with FFM, OLM and penicillin .......................................................................................................................................... 72 Table 4. 6: Mortality (%) of broilers fed diets supplemented with FFM, OLM and penicillin ....... 72 xiii University of Ghana http://ugspace.ug.edu.gh Table 4. 7: Carcass characteristics of broilers fed diets supplemented with FFM, OLM and penicillin .......................................................................................................................................... 73 Table 4. 8: Internal organ characteristics of broilers fed diets supplemented with FFM, OLM and penicillin .......................................................................................................................................... 74 Table 4. 9: Apparent total tract nutrient digestibility and excreted nitrogen in broilers fed diets supplemented with FFM, OLM and penicillin ................................................................................ 75 Table 4. 10: Serum lipid profile of broilers fed diets supplemented with FFM, OLM and penicillin .......................................................................................................................................................... 77 Table 4. 11: Faecal microbial composition of broilers fed diets supplemented with FFM, OLM and penicillin .......................................................................................................................................... 78 xiv University of Ghana http://ugspace.ug.edu.gh LIST OF APPENDICES Appendix A: Composition of vitamins .......................................................................................... 148 Appendix B: Composition of broiler premix ................................................................................. 149 Appendix C: Agar media used for faecal microbial analysis ........................................................ 150 Appendix D: Analysis of variance (ANOVA) tables .................................................................... 151 xv University of Ghana http://ugspace.ug.edu.gh LIST OF ABBREVIATIONS A. galli Ascaridia galli ADFI Average Daily Feed Intake ADG Average Daily weight Gain AGP Antibiotic Growth Promoter ALT Alanine Aminotransferase AMA American Medical Association ANOVA Analysis of Variance AOAC Association of Official Analytical Chemists APHA American Public Health Association ASM American Society for Microbiology AST Aspartate Aminotransferase BD Basal Diet BMD Bacitracin Methylene Disalicylate BW Body Weight BWG Body Weight Gain CF Crude Fibre CFU Colony-Forming Units xvi University of Ghana http://ugspace.ug.edu.gh CP Crude Protein CRD Completely Randomised Design DCP Dicalcium Phosphate DM Dry Matter E. coli Escherichia Coli EE Ether Extract EO Essential Oil FAO Food and Agricultural Organisation FBW Finishing/ Final Body Weight FCE Feed Conversion Efficiency FCR Feed Conversion Ratio FFM Fagara zanthoxyloides Fruit Meal FI Feed Intake GH Growth Hormone GPX Glutathione Peroxidase H: L Heterophils to Lymphocytes Hb Haemoglobin HDL High-Density Lipoprotein xvii University of Ghana http://ugspace.ug.edu.gh IBD Infectious Bursal Disease LDL Low-Density Lipoprotein LIPREC Livestock and Poultry Research Centre LW Live Weight MDA Malondialdehyde ME Metabolisable Energy MOLM Moringa oleifera Leaf Meal N Nitrogen NaCl Sodium Chloride ND Newcastle Disease OAB Organic Acid Blend OLM Ocimum americanum Leaf Meal PCV Packed Cell Volume PEN Penicillin PFA Phytogenic Feed Additive pH power of Hydrogen ppm parts per million PUFA Polyunsaturated Fatty Acid xviii University of Ghana http://ugspace.ug.edu.gh RBC Red Blood Cell SEM Standard Error of Mean SFA Saturated Fatty Acid SNK Student Newman-Keuls SOD Superoxide Dismutase SS Salmonella and Shigella T3 Tri-iodothyronine T4 Thyroxine VLDL Very-Low-Density Lipoprotein w. weight WBC White Blood Cell WHO World Health Organisation WP Whole Plant Y and M Yeasts and Moulds xix University of Ghana http://ugspace.ug.edu.gh CHAPTER ONE INTRODUCTION Antibiotics have been used since the 1950s in meat poultry production for growth promotion (Thomke and Elwinger, 1998). They were incorporated into diets at sub-therapeutic levels primarily to enhance feed conversion efficiency and concurrently, decrease the population of pathogenic gut bacteria, fight infections, and reduce mortalities (Thomke and Elwinger, 1998; Gauer, 2004; Daramola, 2019). These functions allowed farmers to save on the production cost of chicken. Antibiotics, therefore, contributed significantly to the advancement and prosperity of the poultry industry (Castanon, 2007). Nevertheless, over a decade ago, antibiotics were completely barred from being used for growth promotion by the European Union due to notable adverse effects on animals as well as humans (Dibner and Richards, 2005; Castanon, 2007). Antibiotics destroyed both harmful and beneficial gut microbes (Hernandez et al., 2004; Guban et al., 2006; Henry Ford, 2020). There were also major concerns about residual effects on the consumption of animal food products. Moreover, researchers reported increasing cases of continuous emergence and spread of resistant bacteria strains. The transfer of such germplasm to humans (cross-resistance) increases the risk of multiple drug resistance in humans who consume meat products of antibiotic-fed animals (Klotins, 2005; Owens et al., 2008). The exclusion of antibiotics from feeds however resulted in declines in growth performance, as well as increases in bacterial infections, disease severity, mortalities, and production cost (Castanon, 2007; Allen et al., 2013; Zaunschirm, 2018). As a result, scientists began to diligently search for feed additives that can replace antibiotics. This led to the discovery of antimicrobial peptides, 1 University of Ghana http://ugspace.ug.edu.gh bacteriophages, herbal products, hyper-immune egg yolk antibodies, exogenous enzymes, metals, organic acids, prebiotics, probiotics, and synbiotics (Huyghebaert et al., 2011; Upadhayay and Vishwa, 2014; Gadde et al., 2017). Of these alternatives to AGPs, herbal feed additives appear suitable because they are widely available, cheap and easy to obtain, eco-friendly, safe to use, free from toxins, can easily be planted, harvested and used, and have no residual effects on meat and eggs (Windisch et al., 2008; Wan et al., 2017; Vinus et al., 2018). Numerous herbs and their bioactive components have been reported to have a broad range of antimicrobial activity (Dorman and Deans, 2000; Tucker, 2002). Tucker (2002) indicated that herbs, due to their antimicrobial properties, can replace antibiotic growth promoters. Not only do herbs and their extracts inhibit the activity of pathogenic gut bacteria and promote the growth of beneficial ones (Gill, 1999; Wenk, 2000), they also adapt chicken to environmental stress and improve feed intake, nutrient digestibility, and overall performance (Denli and Demirel, 2018; Vinus et al., 2018). Fagara zanthoxyloides and Ocimum americanum are aromatic herbal plants that are widespread in the tropics, especially in Ghana. They have diverse medicinal properties and are commonly used as condiments (Ngassoum et al., 2004; Rady and Nazif, 2005; Guendéhou et al., 2018). Extracts and metabolites from these plants have antimicrobial, anthelmintic, antioxidative, and anti-inflammatory properties (Adesina, 2005; Nascimento et al., 2011; Adefisoye et al., 2012, Azando et al., 2017; Gberikon et al., 2018). There are however no documented reports on the use of these herbs as additives in poultry feeds, hence this study was designed to evaluate the potential of the fruits of Fagara zanthoxyloides and leaves of Ocimum americanum as growth promoters in broiler diets as replacements for antibiotics. 2 University of Ghana http://ugspace.ug.edu.gh If Fagara zanthoxyloides fruit meal (FFM) and Ocimum americanum leaf meal (OLM) can be shown to promote broiler growth and reduce loads of pathogenic gut microbes and mortalities, they will become suitable feed additives for local poultry farmers. This will save production costs and preclude the negative effects of antibiotics withdrawal from diets on birds. 1.1 Hypothesis Supplementing diets of broiler chickens with Fagara zanthoxyloides fruit meal (FFM) or Ocimum americanum leaf meal (OLM) will promote growth performance and reduce loads of pathogenic gut microbes similar to conventional antibiotics. 1.2 Objective of the Study To assess the growth-promoting effects and antimicrobial efficacy of FFM and OLM in broiler diets as potential alternatives to antibiotic growth promoters. 1.2.1 Specific objectives To evaluate FFM and OLM in broiler diets as growth promoters and antimicrobials in comparison with penicillin on; 1. Growth performance 2. Carcass characteristics 3. Apparent whole tract nutrient digestibility 4. Nitrogen excretion 5. Serum lipid profile 6. Faecal microbial count 3 University of Ghana http://ugspace.ug.edu.gh CHAPTER TWO 2.0 LITERATURE REVIEW 2.1 Growth Promoters in Animal Diets Growth promoters are substances, other than dietary nutrients, that are supplemented into nutritionally balanced feeds to increase the growth rate and feed conversion efficiency of farm animals (Upadhayay and Vishwa, 2014). Growth promoters increase nitrogen retention and utilization to form amino acids for growth enhancement (Wierup, 2001) and may improve the quality of animal products (Al-Dobaib and Mousa, 2009; Herago and Agonafir, 2017). The use of growth promoters is necessary because the traditional methods of raising farm animals allow little opportunity for rapid increases in feed conversion efficiency, weight gain, and profitability (Beermann, 1995). Inclusion of growth promoters in diets helps to produce food animals at cheaper costs to meet the ever-growing demand for animal products (Herago and Agonafir, 2017). 2.2 Antibiotics and their Benefits in Poultry Production Antibiotics are chemical products manufactured synthetically or obtained from some strains of microorganisms to inhibit the growth or cause the death of other microbes (Geidam et al., 2009; Mokhtari et al., 2015). They are among the most commonly-used veterinary drugs in poultry husbandry (Lawal et al., 2015). Antibiotics are used by veterinarians and farmers to treat poultry diseases (particularly, bacterial infections), improve growth performance, feed utilization, health status and egg production (Donoghue, 2003; Chattopadhyay, 2014; Sahu and Saxena, 2014), and reduce mortalities (Schjørring and Krogfelt, 2011). Some examples of antibiotics that are commonly used in the poultry industry are presented in Table 2.1. However, most antibiotic products available on the market for poultry use are combinations of 4 University of Ghana http://ugspace.ug.edu.gh two or more of these antibiotics thus, making them extra potent, whilst other antibiotics are mixed with multivitamins (Lawal et al., 2015). Table 2. 1: Commonly-used antibiotics in poultry husbandry No. Antibiotic Reference 1. Avilamycin (Eslami et al., 2010) 2. BMD (Guban et al., 2006) 3. Bambermycin (Diarra et al., 2007) 4. Chlortetracycline (Proudfoot et al., 1988) 5. Colistin (Jang et al., 2007) 6. Doxycycline (Kana et al., 2017) 7. Flavomycin (Attia et al., 2011) 8. Lincomycin Proudfoot et al., 1990) 9. Monensin (Guban et al., 2006) 10. Oxytetracycline (Alhendi et al., 2000) 11. Penicillin (Onu et al., 2004) 13. Salinomycin (Diarra et al., 2007) 14. Streptomycin (Onifade, 1997) 15. Tylosin (Onifade, 1997) 16. Virginiamycin (Baurhoo et al., 2009) BMD = Bacitracin methylene disalicylate 5 University of Ghana http://ugspace.ug.edu.gh 2.2.1 Uses of antibiotics in poultry husbandry In poultry rearing, antibiotics are used for disease treatment (therapy), prophylaxis, and growth promotion. Prophylactic use of antibiotics involves the use of antibiotics to prevent diseases. With this, birds are given sub-therapeutic doses of the drug either through feed or drinking water when signs and symptoms of infections are absent but suspected. This practice serves as a surety to prevent birds from coming down with diseases (Castanon, 2007; Nisha, 2008; Sahu and Saxena, 2014). For growth promotion, antibiotics are administered in very low doses on regular basis to birds through feed, often over a lifetime, to increase growth rate and productivity. This practice is distinct from the use of antibiotics for therapy and prophylaxis which involve higher doses of the drugs administered generally through drinking water (Castanon, 2007; Geidam et al., 2009; Sahu and Saxena, 2014). The growth-promoting effect of antibiotics was discovered in the United States in the 1940s when birds fed dried Streptomyces aureofaciens broth containing chlortetracycline residues grew at a faster rate (Moore et al., 1946; Al-Dobaib and Mousa, 2009). It was realized that the antibiotic residues in the broth were responsible for the improvement in growth (Sahu and Saxena, 2014). It subsequently became a common practice to supplement poultry diets with antibiotics at sub-therapeutic doses for growth enhancement (Cook, 2004; Sahu and Saxena, 2014). Antibiotics used as growth promoters (AGPs) increase feed utilization by 2 to 5% (Ewing and Cole, 1994) and growth rate by 1 to 10% (Chattopadhyay, 2014). They also promote uniform growth of farm animals and hence, reduce variations in carcass sizes at processing units (Robertsson and Lundeheim, 1994). Also, the quality of meat from animals fed diets containing AGPs is superior (with less fat and high protein content) to those fed diets without AGPs (Hughes and Heritage, 2002; Donoghue, 2003). AGPs also increase egg production and hatchability (Gustafson and Bowen, 6 University of Ghana http://ugspace.ug.edu.gh 1997). Moreover, some antibiotics, especially macrolides, have anti-inflammatory properties. They impede the production of pro-inflammatory cytokines from immune cells that cause anorexia and muscle catabolism (Buret, 2010; Chattopadhyay, 2014). This anti-inflammatory action of AGPs minimises energy wastage and instead, channels dietary energy for production (Niewold, 2007). Additionally, AGPs increase nutrient absorption and the production of growth factors (Prescott and Baggot, 1993). Despite the numerous reported benefits, some researchers observed no effects of AGPs on poultry growth performance (Proudfoot et al., 1990; Baurhoo et al., 2009; Riyazi et al., 2015). This is attributable to factors such as the health status of birds, rearing conditions, management practices, and composition of diets (Klotins, 2005; Sarica et al., 2005). The growth-promoting effects of antibiotics are more noticeable when these factors are suboptimal (Sahu and Saxena, 2014; Biswas et al., 2017). Consequently, when conditions of rearing environments are improved with infection control measures introduced, and overcrowding reduced, there will be no need for AGPs (Prescott and Baggot, 1993). Similarly, Anderson et al. (1999) indicated that animals that are well-nourished and reared in clean environments and at moderate stocking densities do not respond positively to AGPs. The effects of AGPs on broiler growth performance and other parameters observed in some studies are presented in Table 2.2. 7 University of Ghana http://ugspace.ug.edu.gh Table 2. 2: Effects of antibiotic growth promoters (AGPs) in broiler chickens No. AGP Effect(s) References 1. Monensin, BMD  BMD: Increases in FI, BWG, FCE, and (Guban et al., 2006) only or in combination conjugated bile salt concentration (0.50g/kg)  Reduction in population of Lactobacillus salivarius  Monensin: Increase in fat digestibility 2. Neomycin-  Increases in BWG, FBW, and carcass (Onifade, 1997) oxytetracycline, parameters procaine penicillin,  Improvement of FCR streptomycin, tylosin  Increase in FI except for procaine (150ppm each) penicillin 3. Penicillin (50, 100, or  Increases in FI, BWG, and FCE (Onu et al., 2004) 150ppm) 4. Lincomycin (2.2ppm)  No effects on FBW, FCR, mortality, and (Proudfoot et al., monetary indices 1990) 5. Chlortetracycline  No effects on BW and FCE (Proudfoot et al., (5.5mg/kg) 1988) 6. Avilamycin  No effects on FI, FCR, BWG, serum lipid (Riyazi et al. 2015) (150ppm) metabolites, and carcass characteristics 7. Avilamycin  Increase in BW on days 21 and 42 (Bozkurt et al., (10mg/kg)  No effects on FI, FCR, and liveability 2008)  No effect on slaughter characteristics 8. Avilamycin  No effects on concentrations of serum lipid (Eslami et al., (150g/ton) metabolites. 2010) 9. Bambermycin (2ppm),  No effect on growth performance except (Diarra et al., 2007) bacitracin (55ppm), for penicillin that caused an increase in penicillin (2.2ppm), FCE salinomycin (60ppm),  No effects on intestinal, caecal, and litter [Bacitracin (55ppm) + bacterial counts salinomycin (60ppm)] 8 University of Ghana http://ugspace.ug.edu.gh 10. Virginiamycin  No effects on growth and carcass (Baurhoo et al., (16.5ppm), bacitracin characteristics 2009) (55ppm)  No effects on caecal counts of E. coli and Campylobacter (d 14 and 24)  No effect on caecal count of Lactobacillus (d 14, 24 and 34)  Reduction in caecal count of Bifidobacteria (d 14 and 24) AGP = antibiotic growth promoter; BWG = body weight gain; BW = body weight; BMD = Bacitracin methylene disalicylate; FBW = finishing body weight; FCE = feed conversion efficiency; FI = feed intake; FCR = feed conversion ratio 2.2.2 Modes of action of antibiotic growth promoters (AGPs) The results of several research works show that AGPs mediate growth through their antibacterial effects and hence, do not promote growth in germ-free animals (Feighner and Dashkevicz, 1987; Butaye et al., 2003). AGPs promote growth by reducing intestinal use of nutrients by pathogenic gut microbes (Snyder and Wostmann, 1987), exhibiting antimicrobial activity on enteropathogens, inhibiting the incidence of subclinical infections (Brennan et al., 2003; Humphrey and Klasing, 2004), minimising the production of metabolites that depress growth from Gram-positive bacteria (Knarreborg et al., 2004), and thinning the wall of the small intestine to increase nutrient absorption and utilization (Feighner and Dashkevicz, 1987; Butaye et al., 2003). 2.2.3 Adverse effects of AGPs on bird and human health Despite the well-demonstrated advantages of AGPs, their use is associated with some deleterious effects in both poultry and humans. For instance, AGPs, in addition to destroying pathogenic gut microbes and reducing their populations, also do that to the beneficial ones (Engberg et al., 2000; Knarreborg et al., 2002; Hernandez et al., 2004). For instance, Guban et al. (2006) fed broilers diets containing bacitracin methylene disalicylate and monensin alone or in combination and observed 9 University of Ghana http://ugspace.ug.edu.gh lower ileal count of Lactobacillus salivarius. According to Henry Ford (2020), antibiotics do not differentiate between beneficial and pathogenic bacteria and as such, may destroy beneficial bacteria in the gut. Furthermore, indiscriminate use of AGPs results in the occurrence of drug residues in meat and eggs as well as the emergence and spread of antibiotic-resistant bacteria in birds (Monroe and Polk, 2000). These antibiotic-resistant bacteria strains can be transmitted to humans through food, environment, or direct contact with contaminated meat (Monroe and Polk, 2000). Due to these harmful effects, AGPs have been prohibited in several countries such as Sweden (Cogliani et al., 2011), the United Kingdom, Denmark, Netherlands, and other European Union countries (Dibner and Richards, 2005; Castanon, 2007). Likewise, organizations such as American Public Health Association (APHA), American Medical Association (AMA), and American Society for Microbiology (ASM) have called for restrictions on antibiotic use in animal husbandry and a termination of all non-therapeutic uses of these drugs (Hashemi and Davoodi, 2011). 2.2.3.1 Effects of consuming poultry products containing antibiotic residues on human health The continuous and abusive use of antibiotics results in the incidence of harmful concentrations of antibiotic residues in edible poultry products (Donoghue, 2003; Shareef et al., 2009) which may persist in these foods several days even after cooking (Dipeolu, 2004). The consumption of meat and eggs containing antibiotic residues causes gastrointestinal syndromes (Jing et al., 2009), carcinogenicity, immune-pathological effects, mutagenicity, loss of hearing, nephropathy, hepatotoxicity (Nisha, 2008; Prajwal et al., 2017), imbalance of intestinal microflora (Olatoye and Ehinmowo, 2010), anaphylactic reactions, bone marrow toxicity, and reproductive disorders (Doyle, 2006; Shareef et al., 2009). 10 University of Ghana http://ugspace.ug.edu.gh 2.3 Alternatives to Antibiotic Growth Promoters (AGPs) in Poultry Diets The withdrawal of AGPs from poultry diets resulted in increases in bacterial infections, disease severity, mortalities, and reduction in profitability of poultry enterprise (Castanon, 2007; Allen et al., 2013). Zaunschirm (2018) reported that antibiotic exclusion from diets will create a wide performance gap since sufficient amounts of energy will be channelled away from growth into fighting pathogens. These effects and the increasing awareness of the public about the dangers of AGPs on their health coupled with their quest for meat and eggs free from antibiotic residues encouraged scientists to investigate other products that can replace antibiotics in diets (Gadde et al., 2017). This led to the focus on feed additives such as antimicrobial peptides, bacteriophages, clay, exogenous enzymes, hyper-immune egg yolk antibodies, metals, organic acids, phytogenic products, prebiotics, probiotics, and synbiotics (Huyghebaert et al., 2011; Upadhayay and Vishwa, 2014; Gadde et al., 2017). These products were to enhance growth performance like AGPs but pose no harm to birds and consumers of poultry products (Huyghebaert et al., 2011). The discovery of these products was to an extent guided by the apprehension of the modes of action of AGPs (Gadde et al., 2017). The following section provides an overview of some of the alternatives to AGPs in poultry nutrition with details on their efficacies and mechanisms of action. 2.3.1 Probiotics Probiotics, also called direct-fed microbial supplements, are live microbial organisms that are incorporated into feeds to suppress the growth of harmful microorganisms in the digestive tract and thus, improve the intestinal microbial balance of the animal (AFRC, 1989; Mokhtari et al., 2015). They are administered through drinking water or feed alone or in combination with other additives (Thomke and Elwinger, 1998). Probiotics improve feed intake, enzyme secretion, nutrient digestion 11 University of Ghana http://ugspace.ug.edu.gh and absorption, and health status of farm animals (Wang and Gu, 2010; Ciorba, 2012). Probiotics administered to birds have no negative human health implications. Different species of beneficial bacteria such as Bifidobacterium, Bacillus, Enterococcus, Lactococcus, Lactobacillus, and Streptococcus and sometimes yeast (Saccharomyces) have been used as probiotics in poultry nutrition (Simon et al., 2001; Kabir, 2009; Gadde et al., 2017). Numerous studies have explored and validated the potency of probiotics to improve growth performance of birds. For example, when broiler diets were supplemented with single species of Lactobacillus such as L. bulgaricus, L. casei, L. reuteri, and L. fermentum, live weight and feed conversion efficiency increased (Yeo and Kim, 1997; Apata, 2008). Similar outcomes were also observed when broiler rations were supplemented with multiple Lactobacillus strains (Jin et al., 1998; Mookiah et al., 2014). Furthermore, probiotics prepared with Bacillus species such as B. licheniformis and B. subtilis equally enhanced growth performance (Lee et al., 2011a; Liu et al., 2012). Likewise, inclusion of Rhodopseudomonas palustris (Xu et al., 2014) and Enterococcus faecium (Kabir et al., 2004) in diets significantly increased feed utilization and average daily weight gain of broilers. Karimi Torshizi et al. (2010) supplemented diet or drinking water of broilers with a probiotic mixture made up Aspergillus oryzae, Bifidobacterium bifidum, Candida pintolopesii, Enterococcus faecium, L. acidophilus, L. bulgaricus, L. plantarum, L. rhamnosus, and Streptococcus thermophilus and observed improvements in body weight gain and feed intake. Blajman et al. (2014) performed a meta-analysis of some research trials conducted between 1980 and 2012 to examine the effects of probiotic supplementation on broiler growth performance. These authors reported that incorporating probiotics into rations improved feed conversion ratio and weight gain. Their study also showed that administering probiotics through drinking water is more effective than through feed. Additionally, Blajman et al. (2014) found no significant differences between the 12 University of Ghana http://ugspace.ug.edu.gh effects of single and multiple strain probiotics on broiler growth performance. Probiotic supplementation also improved laying performance and egg sizes of laying chickens (Lei et al., 2013). Also in turkeys, dietary supplementation with probiotics increased daily weight gain and market body weight (Torres-Rodriguez et al., 2007). However, Karaoglu and Durdag (2005) observed no impact of a dietary probiotic (Saccharomyces cerevisiae) on broiler growth and slaughter variables. This may be due to the strain and dosage of the probiotic used, composition of diet, and environmental factors (Gadde et al., 2017). Furthermore, probiotics produce lactic and short-chain fatty acids that lower the gut pH and make the gut microenvironment hostile to pathogenic bacteria. Also, when probiotic bacteria proliferate in the gut, they competitively exclude the pathogenic ones and compete with them for nutrients. Furthermore, some strains of probiotic bacteria (e.g. Bacillus and Lactobacillus spp.) synthesize and release antibacterial substances like bacteriocins that destroy pathogenic bacteria in the gut (Brown, 2011). The bacteria strains that produce bacteriocins have specific immunity to these peptides and hence, are not destroyed by the bacteriocins (Klaenhammer, 1993). 2.3.2 Prebiotics Prebiotics are indigestible complex carbohydrates that selectively enhance the activity and/or growth of beneficial gut bacteria (Patterson and Burkholder, 2003). Some examples of oligosaccharides and non-starch polysaccharides that have been used as prebiotics in poultry diets are fructooligosaccharides, galactooligosaccharides, glucooligosaccharides, inulin, isomaltooligosaccharides, lactitol, lactulose, maltooligosaccharides, mannan oligosaccharides, oligofructose, pyrodextrins, soya-oligosaccharides, and xylooligosaccharides (Patterson and Burkholder, 2003; Steiner, 2006). 13 University of Ghana http://ugspace.ug.edu.gh For instance, dietary supplementation with mannan oligosaccharides improved (p<0.05) feed conversion ratio in broilers (Mohammed et al., 2008). Also, the inclusion of lactulose in broiler diets increased live weight, feed conversion efficiency, Lactobacillus population and concentrations of acetate, butyrate, and propionate in the caecum, goblet cell numbers, and intestinal villi heights (Calik and Ergün, 2015). In another study, isomaltooligosaccharide in broiler ration improved feed conversion ratio and body weight gain (Mookiah et al., 2014). Hooge and Connolly (2011), concluded from a meta-analysis of broiler feeding trials, that prebiotics increase body weights by 5.41% and improve feed conversion ratio by 2.54%. Furthermore, prebiotics stimulate the growth of beneficial gut microorganisms and their production of lactic acid and bacteriocins (Spring et al., 2000). In addition to their effects on probiotics, some prebiotics directly inhibit the growth of certain pathogenic bacteria and hinder them from colonising the gut. For instance, mannan oligosaccharides bind to the type 1 fimbriae of enteropathogens and obstruct them from adhering to enteric epithelial cells (Spring et al., 2000). 2.3.3 Synbiotics Synbiotics are feed supplements that contain mixtures of prebiotics and probiotics such that both constituents act synergistically to improve animal performance (Patterson and Burkholder, 2003). The development of synbiotics was centred on the perception that a blend of prebiotics and probiotics will both implant beneficial microbes in the gut and stimulate their growth (Gibson and Roberfroid, 1995). Several feeding trials were carried out to investigate the efficacy of synbiotics to improve broiler growth performance. For example, Awad et al. (2009) observed that broilers fed a diet containing a synbiotic had significantly higher live weight, weight gain, feed conversion efficiency, and dressing percentage compared to the control group. Mohnl et al. (2007) also found that supplementing broiler diet with a symbiotic increased body weight by 2.04% and decreased 14 University of Ghana http://ugspace.ug.edu.gh mortality by 0.9%. Also, Mookiah et al. (2014) fed broilers a diet admixed with a symbiotic made up of an inulooligosaccharide and a probiotic mixture containing 11 Lactobacillus sp. These researchers observed significant improvements in feed conversion ratio and weight gain (Mookiah et al., 2014). The synbiotic however did not cause a two-fold improvement effect on performance in comparison with birds that fed either a probiotic or prebiotic supplemented diet (Mookiah et al., 2014). Likewise, feeding pullets a diet containing a combination of probiotics and carbohydrates derived from yeast increased body weight gain (Yitbarek et al., 2015). Despite these reported benefits, Jung et al. (2008) supplemented broiler diet with a symbiotic and observed no effects on growth performance which may be due to the inclusion rate of the symbiotic, health status of birds, condition of rearing environment, and dietary factors or composition. The positive effects of synbiotics on poultry growth performance could be due to improvement in digestion and elimination of subclinical infections. The increase in the population of beneficial gut bacteria caused by symbiotics will cause an improvement in digestion and a competitive exclusion of pathogenic bacteria from the gut thus, eliminating the incidence of subclinical infections. These functions will allow birds to grow to their full genetic potential. 2.3.4 Organic acids Dietary supplementation with organic acids (feed acidifiers) such as citric acid (Haque et al., 2010), fumaric acid (Banday et al., 2015), butyric acid, lactic acid (Adil et al., 2010), and formic acid (Hernandez et al., 2006) has been found to promote broiler growth performance. For instance, Banday et al. (2015) supplemented broiler diet with fumaric acid and observed increases in feed conversion efficiency and weight gain. Also, Adil et al. (2010) reported similar growth improvement effects of dietary supplementation with butyric or lactic acid in broilers. Samanta et al. (2010) tested the efficiency of an organic acid blend (OAB) composed of calcium propionate, formic acid, 15 University of Ghana http://ugspace.ug.edu.gh orthophosphoric acid, and propionic acid as growth promoter in broiler diet. These researchers observed increases in feed efficiency and protein accretion among birds that fed the OAB- supplemented diet. Organic acids are administered either as organic acids, their salts or as a blend of several organic acids or their salts (Huyghebaert et al., 2011). Gadde et al. (2017) reported that using a blend of organic acids is more effective and gives better results than using a single organic acid. Organic acids lower the gut pH and make the gut microenvironment unfavourable for pathogenic microbes but allow acid-tolerant beneficial ones like Lactobacilli to thrive. This reduces the load of pathogenic gut microbes and the competition between the beneficial and pathogenic microbes for nutrients (Boroojeni et al., 2014). Organic acids also penetrate cell wall of pathogenic microbes and cause their death (Gadde et al., 2017). Furthermore, organic acids increase protein retention, dry matter digestibility, and absorption and utilization of minerals (Nezhad et al., 2011). Gadde et al. (2017) reported that some organic acids provide energy for the growth of enteric epithelial cells. 2.3.5 Exogenous enzymes Exogenous or in-feed enzymes are protein supplements which when incorporated into feeds bind to feed particles (substrates) and accelerate their breakdown into minute particles for digestion and absorption (Thacker, 2013). Exogenous enzymes perform similar roles as endogenous ones and complement them to improve digestion (Choct, 2006). Some commonly-used exogenous enzymes in poultry nutrition are proteases, phytases, and carbohydrases (e.g. α-galactosidase, α-amylase, β- mannanase, cellulase, pectinase, and xylanase) (Gadde et al., 2017). Exogenous enzymes increase digestibility of nutrients like phytate that are otherwise not digested by endogenous enzymes, eliminate the coating effect of plant cell wall polysaccharides on nutrients to increase nutrient 16 University of Ghana http://ugspace.ug.edu.gh availability, degrade anti-nutritive compounds, reduce digesta viscosity, and increase the solubility and caecal fermentation of non-starch polysaccharides (Choct, 2006; Kiarie et al., 2013). The growth-promoting effects of several types of in-feed enzymes have been reported in poultry (Choct, 2006). Also, several meta-analyses validate the growth-promoting effects of in-feed enzymes. For example, from a meta-analysis, Hooge et al. (2010) found that dietary supplementation with a multi-enzyme complex containing phytase and non‐starch polysaccharide enzymes improved feed conversion ratio and finishing body weights of broilers by 2.64% and 3.73% respectively. From another meta-analysis, it was shown that supplementing male broiler diets with β-mannanase improved feed conversion ratio and weight gain by 4.8 points and 4.2% respectively (Jackson and Hanford, 2014). However, Nortey et al. (2015) supplemented broiler diets containing cocoa pod husk with phytase, an enzyme cocktail or both. The enzyme cocktail contained amylase, β- glucanase, cellulose, pectinase, phytase, protease, and xylanase. These scientists observed no effects of enzyme supplementation on broiler growth performance. The effects of enzyme supplementation on poultry performance is affected by genetic variation among animals, enzyme type, inclusion rate, and composition of diets (Cheng et al., 2014). 2.3.6 Egg yolk antibodies Egg yolk antibodies are equally used as alternatives to antibiotic growth promoters. They are antibodies that are transferred from hens to chicks through egg yolks (Schade et al. 2005). With this, laying hens are inoculated with antigens of interest. The immune systems of the birds respond by producing antibodies that are transported to the yolks. The yolks are then separated from the albumens, and the antibodies are extricated from them, purified, and used as feed supplements (Yegani and Korver, 2010). 17 University of Ghana http://ugspace.ug.edu.gh Cook (2004) reported that dietary supplementation with egg yolk antibodies improves poultry growth. Likewise, Mahdavi et al. (2010) observed that oral administration of an egg yolk antibody to broilers improved feed conversion efficiency and intestinal health. Furthermore, Tamilzarasan et al. (2009) found that some egg yolk antibodies have antibacterial effects on Escherichia coli, and Campylobacter, Clostridium, and Salmonella spp. Egg yolk antibodies bind to bacterial cell structures like the lipoglycans, pili, and flagella, and prevent bacteria from adhering to and colonizing the intestinal epithelium (Suresh et al., 2018). Egg yolk antibodies also cause structural damages to the surfaces of bacterial cells after binding to them and neutralise their toxins (Xu et al., 2011). According to Gadde et al. (2017), egg yolk antibodies are more effective, less toxic, and safer to use than antibiotics, and unlike antibiotics, bacteria cannot develop resistance to them. They are however expensive and susceptible to proteolytic degradation in the digestive tract (Mine and Kovacs-Nolan, 2002). 2.3.7 Metals The use of metals like manganese, copper, iron, zinc, and selenium to improve animal growth performance and productivity, even though expensive, has gained acceptance in animal farming (Scott, 2012; Gadde et al., 2017). Metals are supplemented into diets in inorganic or chelated forms or as inorganic salts, e.g sulfates, chlorides, and carbonates (Attia et al., 2012; Gadde et al., 2017). Metals do not only support biosynthetic, digestive, and physiological processes but also promote growth (Richards et al., 2010). For example, copper, in addition to playing important roles in angiogenesis, haemoglobin synthesis, and bone development, acts as a growth promoter in poultry diets (Hoda and Maha, 1995; Vasanth et al., 2015). The inclusion of copper sulphate pentahydrate into diet of broiler chickens at a dose of 250mg/kg improved body weight gain and feed conversion ratio (Pesti and Bakalli, 1996). Likewise, incorporating copper oxychloride, copper sulphate 18 University of Ghana http://ugspace.ug.edu.gh pentahydrate, and copper citrate into diets increased the weight gain of broiler chickens by 4.9, 4.9, and 9.1% respectively (Ewing et al., 1998). Moreover, supplementing basal diet of broilers with zinc sulphate up to 80mg/kg significantly increased body weight gain (Burrell et al., 2004). Also, feeding broilers for four weeks with a diet containing a combination of zinc oxide and sodium selenite resulted in an improvement in growth (Fawzy et al., 2016). Brainer et al. (2003) stated that the growth-promoting effects of metals, when supplemented into diets, is attributable to their antimicrobial properties. Accordingly, Yazdankhah et al. (2014) fed pigs with copper and zinc supplemented diets and observed significantly lower counts of pathogenic intestinal bacteria (Yazdankhah et al., 2014). 2.4 Phytogenic Feed Additives in Poultry Nutrition Phytogenic feed additives (PFAs), also known as botanicals or phytobiotic supplements, are products derived from plants, herbs, and spices that are supplemented into animal diets (Windisch et al., 2008; Grela et al., 2013). PFAs contain several bioactive compounds that affect several physiological functions of farm animals (Paskudska et al., 2018). Some of these compounds improve the taste, flavour and palatability of feeds, thus stimulate the appetite of farm animals (Mirzaei-Aghsaghali, 2012). Others enhance the secretion of digestive juices and activity of endogenous enzymes, promote the growth of beneficial gut microbes, and inhibit the pathogenic ones. These effects culminate in improvements in nutrient absorption and utilization (Dorman and Deans, 2000; Windisch et al., 2008; Mirzaei-Aghsaghali, 2012). Some phytochemicals improve the sensory properties of poultry products (Meineri et al., 2016) and others have anthelmintic, anti-inflammatory, antimicrobial, antioxidative, anti-tumour, anticoccidial, hypoglycemic, and immune-modulatory properties (Grela et al., 2013; Babak and Nahashon, 2014; Suganya et al., 2016). 19 University of Ghana http://ugspace.ug.edu.gh The bioactive compounds of medicinal plants are present in their leaves, fruits, flowers, seeds, stems, roots, and rhizomes (Mirzaei-Aghsaghali, 2012). These plant parts are therefore incorporated into diets often in dried and ground forms, or their extracts are used (Gadde et al., 2017). Some botanicals that are commonly used in poultry diets and the effects of their phytochemicals are presented in Table 2.3. Table 2. 3: Commonly used botanicals in poultry diets Botanical Parts used Phytochemicals Effects of phytochemicals 1. Aloe vera Leaves Anthraquinones Antimicrobial, anti-inflammatory, (Aloe barbadensis) antioxidative, immunomodulatory, and anti- tumour 2. Aniseed Fruit Anethole Digestion stimulation (Pimpinella anisum) 3. Amla Fruit Tannins, ellagic acid, Antioxidative (Emblica officinalis) gallic acid, vitamin C 4. Black cardamom Seeds Cineol Digestion and appetite stimulation (Amomum subulatum) 5. Cinnamon Leaves, Phenolic compounds, Digestion and appetite (Cinnamomum Bark eugenol stimulation, antiseptic, astringent, zeylanicum) carminative, antiviral, antifungal, blood-purifying 6. Cloves Cloves Eugenol Digestion and appetite (Syzygium aromaticum) stimulation, antiseptic 7. Coriander Seeds, Carvone, limonene, Appetite and digestion (Coriandrum sativum) leaves geraniol, linalool, stimulation, carminative flavonoids, elemol, camphor, borneol 8. Cumin Seeds Cuminaldehyde Digestion stimulation, (Cuminum cyminum) carminative 20 University of Ghana http://ugspace.ug.edu.gh 9. Fenugreek Seeds Trigonelline, Appetite stimulation, (Trigonella foenum- trigoneoside, vamogenin antimicrobial, cholesterol- graecum) saponins, protodioscin, reducing diosgenin 10. Garlic Bulb Allicin Digestion stimulation, antiseptic (Allium sativum) 11. Ginger Rhizome Zingerone, ar- Gastric stimulation, methane- (Zingiber officinale) curcumene, β-bisabolene, reducing camphene 12. Horseradish Root Allyl isothiocyanate Appetite stimulation (Armoracia rusticana) 13. Indian ginseng Leaves, Glycine, withanolides, Antistress, analgesic, (Withania somnifera) seeds, root withanine hepatoprotective, immunomodulatory 14. Mint Leaves Menthol Digestion and appetite (Mentha piperita) stimulation, antiseptic 15. Moringa Leaves Ascorbic acid, caffeic Antioxidative, anti-bacterial (Moringa oleifera) acid, carotenoids, chlorogenic acid, flavonoids, phenolics 16. Mustard Seeds Allyl isothiocyanate Digestion stimulation (Brassica Nigra) 17. Neem Leaves Nimbidin, nimbin Antibacterial, antiviral, (Azadirachta indica) antifungal, anthelmintic, stimulation of fibre-degrading enzymes 18. Nutmeg Seeds Sabinene Digestion stimulation (Myristica fragrans) 19. Parsley Leaves Apiol Digestion and appetite (Petroselinum crispum) stimulation, antiseptic 20. Pepper Fruit Piperine Digestion stimulation (Piper nigrum) 21. Rosemary Leaves Cineol, resins, tannins Digestion stimulation, antiseptic, (Rosmarinus officinalis) antioxidative, anti-inflammatory 21 University of Ghana http://ugspace.ug.edu.gh 22. Thyme Whole plant Thymol Digestion stimulation, (Thymus vulgaris) antioxidative, antiseptic 25. Tulsi Leaves Ascorbic acid, beta- Anti-microbial, antioxidative, (Ocimum sanctum) sitosterol, beta-carotene, analgesic, hepatoprotective, eugenol, palmitic acid, anthelmintic, cardioprotective, tannins antispasmodic, antiulcerogenic 26. Turmeric Rhizome Ar-turmerone, Hypocholesterolemic, anti- (Curcuma longa) curcuminoids, curlone, inflammatory, anticarcinogenic, zingiberene antioxidative, antihepatotoxic Sources: Mirzaei-Aghsaghali, 2012; Vinus et al., 2018 2.4.1 Effects of PFAs on feed intake, nutrient digestibility and gut morphology in poultry The digestion process is influenced in several ways by phytogenic feed additives. Some herbal compounds act as sialagogues and hence, stimulate saliva secretion to facilitate swallowing (Suganya et al., 2016). Others improve the palatability and flavour of feeds and excite the taste buds and olfactory nerves to induce feed intake (Al-Kassie, 2009; Muanda et al., 2011). Accordingly, dietary supplementation with Coriandrum sativum (coriander) seeds, Nigella sativa (black seeds) (EL-Shoukary et al., 2014), enzymatically-treated Artemisia annua (sweet annie) whole plant (Wan et al., 2017), Foeniculum vulgare (fennel) seeds (Ragab et al., 2013), Origanum majorana (marjoram) leaf powder (Ali, 2014), and a mixture of Cuminum cyminum (cumin) and Curcuma longa (turmeric) (Al-Kassie et al., 2011a) increased feed intake in broilers. However, increasing the supplementation rate of Allium cepa (onion) extract in broiler diets from 7.5 to 10g/kg significantly reduced feed intake (Aditya et al., 2017). Likewise, a significant decrease in feed intake was observed when the level of Allium sativum (garlic) powder in broiler feed was increased from 3 to 5% (Mulugeta et al., 2019). A similar effect was realized when the amount of Tetrapleura tetraptera (aridan; Prekese) fruit powder was increased from 0.2 to 0.4% in broiler diets 22 University of Ghana http://ugspace.ug.edu.gh (Kana et al., 2017). These depressions in feed intake may be attributed to the consequent increase in the strong odour of these spices which might have negatively affected acceptability. Odoemelam et al. (2013) explained that the beneficial effects of spices are sometimes concealed when they are supplemented in high amounts due to the consequent increase in the levels of anti-nutritional factors like tannins and saponins. These compounds interfere with nutrient utilization and depress growth (Kana et al., 2017). Also, herbs such as aniseed, cumin, fenugreek, ginger, onion, and Capsicum annuum (cayenne pepper) contain compounds that promote the synthesis of bile acid in the liver and its excretion into the bile for lipid digestion and absorption (Suganya et al., 2016). Also, some phytochemicals increase bile flow, as well as the synthesis, secretion, and activity of pancreatic enzymes (Jang et al., 2007; Hashemipour et al., 2014). Others stimulate gastric secretions, activity of digestive enzymes, and digestion, absorption, and assimilation of nutrients (Frankič et al., 2009). These effects lead to improvements in apparent whole tract nutrient digestibility (Hernandez et al., 2004; Wang et al., 2008; Issa and Omar, 2012). Accordingly, Issa and Omar (2012) observed better crude protein, dry matter, and fat digestibility in broilers fed diets containing garlic powder. Similarly, Hernandez et al. (2004) observed increases in apparent total tract and ileal digestibility of dry matter, ether extract, starch, and crude protein in broilers fed diets containing plant extracts. Nonetheless, Jamroz et al. (2003) observed no effects of 150ppm of a standardized mixture containing carvacrol, capsaicin, and cinnamaldehyde in broiler diet on apparent ileal digestibility of dry matter, crude fat, crude ash, and nitrogen. This may be due to the low dietary inclusion rate of the mixture since at a higher inclusion rate of 300ppm, the ileal digestibility of all the fore-mentioned nutrients increased significantly (Jamroz et al., 2003). 23 University of Ghana http://ugspace.ug.edu.gh Furthermore, some phytoconstituents improve gut microanatomy (Murugesan et al., 2015). For example, Ahsan et al. (2018) demonstrated that supplementing broiler diets with a phytogenic product containing cumin, cinnamon, and essential oils of aniseed, garlic, fennel (Foeniculum vulgare), and Mentha balsamea (peppermint) significantly increased the number of goblet cells along with intestinal villus diameter and height. The increased number of goblet cells per villus implies an increase in the production of glycoproteins and mucins that bind to pathogenic microbes and prevent their attachment to the mucosa gut lining (Chacher et al., 2017). Also, the increased villus diameter and height imply an increased intestinal surface area for nutrient digestion and absorption (Murugesan et al., 2015). In addition to increasing goblet cell number and intestinal villus diameter and height, the phytogenic product decreased the thickness of the muscularis (Ahsan et al., 2018). According to Ahsan et al. (2018), the thickness of the muscularis increases with increasing germ load in the gut. The thin muscularis observed among the birds fed the diet containing the phytogenic product, therefore, implies a lower germ load, hence, more dietary nutrients will be channelled for growth (Ahsan et al., 2018). 2.4.2 Phytogenic feed additives as antimicrobial agents Plant extracts in several in vitro and in vivo studies have exhibited strong antimicrobial activity against fungi, bacteria, viruses, and protozoa (Gupta and Charan, 2005; Swiatkiewicz et al., 2009; Sood et al., 2012; Babak and Nahashon, 2014; Suganya et al., 2016). For instance, Junaid et al. (2006) found African basil leaf extract to have antibacterial effects on isolates of Aeromonas hydrophila, Bacillus cereus, Escherichia coli, Salmonella typhimurium, and Yersinia enterocolitica. This antimicrobial effect is attributable to the eugenol content of the plant (Nakamura et al., 1999). Also, cinnamon oil was shown to inhibit the growth of Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus 24 University of Ghana http://ugspace.ug.edu.gh aureus, Staphylococcus epidermis, Salmonella, and Vibrio parahaemolyticus (Montes-Belmont and Carvajal, 1998; Chang et al., 2001). Likewise, Tabak et al. (1999) demonstrated that cinnamon extract at an inclusion rate similar to common antibiotics represses Helicobacter pylori ascribable to the cinnamaldehyde, carvacrol, and eugenol contents of the extract. Furthermore, Goodarzi et al. (2014) studied the effects of onion bulbs on the intestinal microflora composition of broiler chickens. These scientists demonstrated that supplementing diets with 10 and 30g/kg of fresh onion bulb significantly decreased (p<0.05) ileal Escherichia coli population. Besides, the Lactobacillus population in the ilea of the birds that received the diet supplemented with 30g/kg onion bulb significantly increased (Goodarzi et al., 2014). Similarly, Aloe vera gel in feeds decreased the count of Escherichia coli and increased that of Lactobacillus in the ilea of broilers (Darabighane et al., 2012). Kollanoor-Johny et al. (2012) found two plant-derived compounds (i.e. eugenol and trans- cinnamaldehyde) at inclusion rates of 0.75 and 1% to be effective in reducing the colonization of the caeca of broilers by Salmonella enteritis by decreasing their motility and virulence genes. Also, Artemisia sieberi leaf meal in feed significantly reduced coliform and Escherichia coli counts in the caeca of broilers (Khalaji et al., 2011). Similarly, addition of sage, thyme, or rosemary essential oil to diet of laying chickens reduced the counts of coliforms and Escherichia coli in faecal samples (Bölükbaşı et al., 2008). Similarly, a commercial phytogenic product containing extracts of anise, clove, melissa balm, oak, peppermint, and thyme was shown to be as effective as bacitracin methylene disalicylate (BMD) in controlling Escherichia coli, Salmonella, and Clostridium in the caeca of broilers that were orally challenged with Salmonella enteritidis and Escherichia coli (Wati et al., 2015). Moreover, the number of Lactobacillus in the caeca of the birds fed the diet containing the phytogenic product was significantly higher than those fed either the control or BMD- supplemented diet (Wati et al., 2015). Also, Jamroz et al. (2003) reported that dietary 25 University of Ghana http://ugspace.ug.edu.gh supplementation with 300ppm of a standardized mixture containing carvacrol, capsaicin, and cinnamaldehyde significantly reduced Escherichia coli and Clostridium perfringens counts in the recta of broilers to the same extent as an antibiotic (avilamycin) in comparison with the control group. Similarly, Tucker (2002) reported a significant Escherichia coli-inhibition effect of a herbal mixture containing aniseed, cinnamon, garlic, thyme, and rosemary when incorporated into commercial swine diets. Despite the reported proliferation effect of phytogenic feed additives on beneficial gut bacteria, Anugom and Ofongo (2019) administered aqueous African basil leaf extract to broilers and observed significantly lower counts of Lactobacillus in digesta samples collected from the crop, proventriculus, caecum, and ileum on day 35 of the experiment in comparison with the non-treated group. These researchers concluded that the phytochemicals in the herb possibly have bactericidal effects also on beneficial microbes. Likewise, Hernandez et al. (2004) reported that plant extracts, in addition to inhibiting the growth and colonization of pathogenic gut bacteria, may inhibit the beneficial ones. Possibly, similar to antibiotics (Henry Ford, 2020), some phytochemicals do not differentiate between beneficial and pathogenic bacteria and hence, may destroy the beneficial ones. Furthermore, dietary supplementation with Ocimum sanctum (tulsi) leaf powder inhibited in vivo replication of the infectious bursal disease virus in broiler chickens (Gupta and Charan, 2005). Also, extracts of sweet annie inhibited the proliferation of the Newcastle disease virus in chicken embryos (Liu et al., 2009). Likewise, extracts of Eugenia jambolana (jambolan) exhibited very strong virucidal effect on the highly pathogenic avian influenza virus (H5N1) in embryonated eggs inoculated in tissue culture and in ovo (Sood et al., 2012). The anti-viral property of most phytogenic products is attributable to their flavonoid content (Muanda et al., 2011). 26 University of Ghana http://ugspace.ug.edu.gh Furthermore, volatile oils extracted from the leaves of seven Artemisia species namely Artemisia dracunculus, Artemisia cana, Artemisia absinthium, Artemisia biennis, Artemisia ludoviciana, Artemisia frigida, and Artemisia longifolia showed inhibitory effects on the growth of Candida albicans, Cryptococcus neoformans, and Aspergillus niger (Lopes-Lutz et al., 2008). Likewise, essential oil of oregano inhibited the mycelial growth of Aspergillus ochraceus and the production of ochratoxin A from the fungus attributable to its carvacrol and thymol contents (Basilico and Basilico, 1999). 2.4.2.1 Antimicrobial modes of action of phytochemicals In addition to disrupting the membranes of pathogenic microbes and modifying their cell surfaces to reduce their virulence, some phytochemicals activate immune cells like macrophages, monocytes, natural killer cells, and T lymphocytes to destroy the pathogens. Besides, other phytochemicals stimulate the growth and proliferation of beneficial gut bacteria to competitively exclude the pathogenic ones (Diaz-Sanchez et al., 2015; Suganya et al., 2016). When loads of enteric pathogens decrease, nutrient availability increases, sub-clinical infections are eliminated, and mortality decreases (Wilson et al., 2005; Brisbin et al., 2008). 2.4.3 Phytogenic feed additives as growth promoters in poultry diets Currently, herbs and spices are used as natural growth promoters in poultry diets in place of antibiotics (Gadde et al., 2017). Several herbs and spices including Achillea millefolium (yarrow), cinnamon, coriander, ginger, garlic, marjoram, oregano, rosemary, and thyme have been incorporated into poultry diets for growth enhancement (Gadde et al., 2017). Likewise, admixing diets with black cumin seeds (Khalaji et al., 2011), fermented Ginkgo biloba (ginkgo) leaves (Cao et al., 2012), fenugreek seeds (Azouz, 2001), Stevia rebaudiana (stevia) leaf meal (Atteh et al., 2008), and extracts of aniseed (Pimpinella anisum) (Durrani et al., 2007), purslane (Portulaca 27 University of Ghana http://ugspace.ug.edu.gh oleracea) (Zhao et al., 2013), and weeping forsythia (Forsythia suspense) (Wang et al., 2008) improved broiler growth performance. Also, inclusion of 0.2% aridan fruit powder in broiler ration improved feed conversion ratio, live weight, and weight gain (Kana et al., 2017). However, increasing the inclusion rate of the aridan fruit powder from 0.2 to 0.4% depressed feed intake, live weight, weight gain, and carcass yield significantly (Kana et al., 2017). The decline in productive traits could be due to the potential increase in the levels of anti-nutritive factors in the diet which might have negatively imparted nutrient utilization (Kana et al., 2017). Also, Goodarzi et al. (2014) observed that birds fed a diet supplemented with 30g/kg fresh onion bulb had a significantly higher finishing body weight than those fed either a control or virginiamycin-supplemented diet. Incorporating Ocimum basilicum (sweet basil) seeds at a rate of 3g/kg in broiler diet also increased (p<0.05) finishing body weight (Abbas, 2010). Abbas (2010) observed that the broilers fed the sweet basil seed-supplemented diet had the least feed intake but the best feed conversion ratio. Moreover, essential oils of herbs such as basil, clove, garlic, ginger, thyme, caraway, coriander, rosemary, sage, star anise, and turmeric have been used individually or as blends to boost poultry growth (Gadde et al., 2017). For example, a blend of essential oils of cinnamon (ie. cinnamaldehyde) and thyme (ie. thymol), when incorporated into broiler diets improved body weight gain (Tiihonen et al., 2010). Similar findings were made when diets were supplemented with essential oils of coriander (Ghazanfari et al., 2015) and oregano (Basmacioğlu et al., 2010), a blend of essential oils of thyme and star anise (Kim et al., 2016), and a blend of cinnamaldehyde and essential oil of clove (Chalghoumi et al., 2013). Likewise, a blend of cinnamaldehyde, carvacrol, and Capsicum oleoresin in broiler feed improved feed conversion efficiency and weight gain by 9.8% and 14.5% respectively (Bravo et al., 2014). 28 University of Ghana http://ugspace.ug.edu.gh The improvement in growth of birds fed diets containing phytogenic products is due to improvement in enzymatic function, stimulation of growth and proliferation of beneficial gut microflora, inhibition of pathogenic gut microbes, and decrease in the production of growth-depressing microbial metabolites such as biogenic amines and ammonia. These effects culminate in increases in nutrient absorption and utilisation (Jamroz et al., 2003; Windisch et al., 2008; Frankič et al., 2009) which increases growth performance. Also, some phytogenic feed additives improve protein digestion in the intestine leading to growth improvement (Abbas, 2010). According to Fallah et al. (2013), the growth-promoting effects of phytogenic feed additives arise from the synergistic effect of their bioactive compounds. However, some researchers fed diets admixed with phytogenic products and observed negative or no effects on growth performance. For instance, Goliomytis et al. (2014) supplemented broiler diets with 0.5 and 1g of quercetin (a plant flavonol) per kg of feed and observed no significant effects on cumulative feed intake and body weight. Furthermore, in that study, birds fed the quercetin- supplemented diets had a poorer feed conversion ratio (Goliomytis et al., 2014). Also, other researchers who tested Macleaya cordata (plume poppy) extract (Juskiewicz et al., 2011) and garlic powder (Issa and Omar, 2012) as growth promoters reported no impacts on broiler growth performance. Also, Hernandez et al. (2004) supplemented broiler diets with 5,000ppm Labiatae extract from rosemary, sage, and thyme mixture, and 200ppm essential oil of cinnamon, pepper, and oregano mixture, and observed no effects on feed intake, feed conversion efficiency, live weight, and body weight gain. Similarly, Jang et al. (2007) found no effects of dietary supplementation with a commercial essential oil product containing eugenol, piperine, and thymol on feed intake, body weight gain, and feed conversion ratio of broilers. The lack of response may be due to the inclusion rates of the phytogenic products, health status of the birds, and management practices. These factors 29 University of Ghana http://ugspace.ug.edu.gh influence the effects of phytogenic feed supplements on poultry performance (Yang et al., 2009). Franz et al. (2010) also indicated that the inconsistency in the effects of essential oil on poultry performance could be due to variations in the type and composition of the oils as well as environmental factors. The results of other research trials that examined the effects of phytogenic feed supplements on broiler growth performance are presented in Table 2.4. 30 University of Ghana http://ugspace.ug.edu.gh Table 2. 4: Effects of phytogenic feed additives on broiler growth indices Phytogenic Dietary Effects on growth indices Reference Product inclusion rate 1. Piper guineense 0.2 or 0.4% Increase in BWG (Effiong and Ochagu, (Ashanti pepper) Improvement of FCR 2019) leaf or seed meal 2. Thyme extract 50, 100, 200, Increase in BWG (Alipour et al., 2015) or 400ppm 3. Thyme extract 0.2, 0.4 or No effects on LW and FCR (Pourmahmoud et al., 0.6% 2013) 4. EO from 200ppm Increases in FI and BWG (Al-Kassie, 2009) cinnamon or thyme Improvement of FCR 5. Turmeric powder 0.5% Increase in BWG (Yarru et al., 2009) 6. Turmeric powder 2.5g/kg Increase in BWG up to 21 d of (Akbari, 2014) age 7. Mixture of 0.50, 0.75 or Increases in FI, BWG and FCE (Al-Kassie et al., 2011a) turmeric and cumin 1% 8. Garlic powder 1, 3, or 5% 3%: Increase in FBW and (Mulugeta et al., 2019) improvement of FCR 9. Shatavari 0.5, 1 or 1.5% Increases in BWG and FCE (Rekhate et al., 2004) (Asparagus racemosus) root powder 10. Marjoram leaf 0.5, 1 or 1.5% Increases in FI, BWG and FCE (Ali, 2014) meal 11. Green Tea 0.1 or 0.2g/kg Increases in LW and FCE (Erener et al., 2011) extract 12. Blend of thymol 60, 100 or Increase in BWG (Hashemipour et al., and carvacrol 200mg/kg 2013) 13. Oregano EO 100mg/kg No effects on FI, BWG and (Avila-Ramos et al., FCR 2012) 14. Oregano EO 50 or 100 No effects on LW and FCR (Botsoglou et al., 2002) mg/kg 31 University of Ghana http://ugspace.ug.edu.gh 15. Extract of 1000ppm No effects on LW, FI and FCR (Barreto et al., 2008) cinnamon, clove, oregano, or red pepper 16. Sweet basil EO 200, 400 or No effects on FI, BWG and (Riyazi et al., 2015) 600mg/kg FCR 17. Hesperidin, 0.75 or No effect on BW (Goliomytis et al. 2015) naringin 1.5g/kg 18. Hibiscus 0.25% Hs, No effects on FI, BWG and (Ojelade et al., 2012) sabdariffa (Hs) 0.25% Og, or FCR calyx meal, Ocimum 0.125% Hs + gratissimum (Og) 0.125% Og leaf meal 19. Ginger powder 250, 500 or No effects on FI, BWG and (Mohammed and Yusuf, 750g/100kg FCR 2011) 20. Moringa oleifera 3, 6 or 9% No effects on ADG, FI, FCR (Amevor, 2017) leaf meal and FBW 21. Moringa oleifera 50, 75 or Decreases in FI and LW (Gadzirayi et al., 2012) leaf meal 100% ADG = Average daily gain; BWG = Body weight gain; EO = Essential oil; FCR = Feed conversion ratio; FBW = Final body weight; FCE = Feed conversion efficiency; FI = Feed Intake; LW = Live weight 2.4.3.1 Limitations in the use of phytogenic feed additives The use of phytogenic feed additives is associated with some limitations. For instance, due to their complex compositions, they are not easily quantified and standardized (Suganya et al., 2016). Also, their compositions are affected by soil type, stage of maturity of plants, geographical location, altitude, season of planting or harvesting, method of harvesting, climatic factors, environmental conditions, presence of anti-nutritional compounds, storage conditions, microbial contamination, and processing methods (Windisch et al., 2008; Huyghebaert et al., 2011; Nascimento et al., 2011; 32 University of Ghana http://ugspace.ug.edu.gh Suganya et al., 2016). Also, some phytochemicals are liable to light and temperature variations hence, their availability and concentrations are unstable (Suganya et al., 2016). Some phytogenic feed additives have antagonistic effects on animals when combined (Suganya et al., 2016; Paskudska et al., 2018) whilst others are rather effective when combined than when offered alone (Paskudska et al., 2018). The efficacy of phytogenic feed supplements on poultry performance is also affected by nutritional status of birds as well as composition of diets (Giannenas et al., 2003). 2.4.3.2 Benefits of phytogenic feed additives over antibiotic growth promoters The following characteristics of phytogenic feed additives (PFAs) make them superior to antibiotic growth promoters (AGPs). They are natural, cheap, safe to use, less toxic, eco-friendly, and result in little to no cases of bacteria resistance (Hashemi and Davoodi, 2011; Sethiya, 2016; Nieto, 2017). Suresh et al. (2018) explained that, unlike AGPs, PFAs are composed of a complex blend of phytochemicals with each having a different antimicrobial activity which makes it very challenging for bacteria to gain resistance to them. Furthermore, PFAs have a multifactorial or broad spectrum of action on farm animals unlike AGPs (Steiner, 2006). PFAs also have no residual effects on poultry products and thus, increase the safety of these foods for human consumption (Sanjyal and Sapkota, 2011). 2.4.4 Phytogenic feed additives as antioxidative agents Antioxidants, according to Muanda et al. (2011), are compounds that when added to feeds inhibit or impede lipid oxidation and rancidity thus, maintaining the nutritional quality of feeds. The health- promoting effects of antioxidants from plants possibly arise from their protective role in counteracting reactive oxygen species (Muanda et al., 2011). Some herbs and spices also protect feeds from oxidative deterioration during storage. Owing to this property, essential oils of rosemary, sage, and thyme are commonly used in feed industries for feed preservation (Jacobsen et al., 2008; 33 University of Ghana http://ugspace.ug.edu.gh Nieto, 2017). The antioxidative property of herbs is related to the presence of phenolic compounds such as hydrolysable tannins, flavonoids, phenolic acids, proanthocyanidins, terpenes, and vitamins A, C, and E in them (Muanda et al., 2011). The sulfur-containing molecules in onion and garlic also have antioxidative properties (Higuchi et al., 2003). In an experiment with broiler chickens, Reddy et al. (2009) observed that supplementing diet with tulsi leaf powder increased (p<0.01) the levels of the antioxidative enzymes - superoxide dismutase and catalase and thus, combated oxidative stress in the birds. Also, incorporating tulsi leaf extract into heat-stressed broiler diets significantly increased the activity of plasma glutathione peroxidase; an antioxidative enzyme that scavenges free radicals to prevent lipid peroxidation and maintain intracellular homeostasis (Vasanthakumar et al., 2013). Steiner and Syed (2015) also reported that a blend of volatile oils of caraway, anise, and peppermint activated cellular pathways that protect animal cells from reactive oxygen species. Also, the leaves of Moringa oleifera contain large amounts of Vitamin E; an antioxidant that prevents oxidative spoilage of meat (Moyo et al., 2012). Other herbs that contain antioxidative compounds are Taraxacum officinale (dandelion), Rosmarinus officinalis (rosemary), Salvia officinalis (sage), Matricaria chamomilla (chamomile), Ginkgo biloba (ginkgo), Tagetes erecta (marigold) (Suganya et al., 2016), Capsicum annuum (red pepper), Piper nigrum (black pepper), Capsicum frutescens (chili pepper) (Nakatani, 1997), Prunus dulcis var. amara (bitter almonds), Cinnamomum verum (cinnamon), Mentha piperita (peppermint), Syzygium aromaticum (clove), Laurus nobilis (laurel) (Deans et al., 1993), Carthamus tinctorius (safflower), and Brassica juncea (mustard) (Lee et al., 2007). 2.4.5 Phytogenic supplements as immune-stimulatory agents Some examples of herbs that have immune-stimulatory properties are garlic, Echinacea purpurea (echinacea), Glycyrrhiza glabra (liquorice), and Uncaria tomentosa (cat’s claw). Their 34 University of Ghana http://ugspace.ug.edu.gh phytoconstituents stimulate the synthesis of interferons and improve the activity of macrophages, lymphocytes, and natural killer cells to increase phagocytosis (Frankič et al., 2009). Also, some phytochemicals increase the proliferation of immune cells, antibody titres to antigens, as well as cytokine expressions (Park et al., 2011). Still, others induce the production of heat shock proteins that activate the Toll-like receptors to recognize pathogens and induce immune responses to them (Hashemi and Davoodi, 2012). Supplementing broiler diets with essential oils of Mentha (mint), Satureja hortensis (savory), and Hippophae rhamnoides (sea buckthorn) stimulated the infiltration of leukocytes which suggested an improvement in immune response (Stef et al., 2009). The immune-stimulatory effect of safflower extract on avian lymphocytes and mustard extract on macrophages have been demonstrated (Lee et al., 2007). The safflower extract stimulated the proliferation of avian lymphocytes whilst the mustard extract stimulated the production of nitric oxide by macrophages (Lee et al., 2007). Similarly, cinnamaldehyde, an organic compound derived from cinnamon, stimulated the proliferation of avian spleen lymphocytes and the production of nitric oxide by cultured macrophages (Lee et al., 2011b). Islam et al. (2017) observed a significant improvement of the phagocytic activity of heterophils in broilers that were orally treated with non-dialyzable fraction of Vaccinium macrocarpon (cranberry) extract at a dose of 4mg/ml/bird. Also, oral administration of aqueous and ethanolic extracts of Aloe vera pulp at a rate of 300mg/kg of body weight for three consecutive days resulted in a significant increase in antibody titre to sheep red blood cells in broilers, suggesting an improvement in immune response (Akhtar et al., 2012). Likewise, echinacea extract increased the concentration of immunoglobulins in the blood of laying chickens and improved their health status (Swiatkiewicz and Koreleski, 2007). Furthermore, feeding birds with a diet admixed with Medicago sativa (alfalfa) increased both the masses of the lymphatic organs and the number of lymphocytes produced by these 35 University of Ghana http://ugspace.ug.edu.gh organs (Skomorucha and Sosnówka-Czajka, 2012). Also, Guo et al. (2003) reported that Astragalus membranaceus (astragalus) polysaccharides in feed stimulates the growth of the bursa, spleen, and thymus. 2.4.6 Phytogenic feed additives as coccidiostatic agents Coccidiosis, a very common cause of mortality in poultry, is a disease caused by a protozoa called Eimeria which damages the intestinal epithelium and impairs nutrient absorption (Paskudska et al., 2018). Sweet annie is one of the earliest plants used to combat coccidiosis in poultry. It contains artemisinin; a bioactive compound that inhibits the development of coccidiosis (Świątkiewicz et al., 2009). As such, when dried fruits of sweet annie were incorporated into feed for chickens infected with Eimeria tenella oocysts, intestinal damages were reduced (Swiatkiewicz et al., 2009). Also, Origanum vulgare (oregano) contains compounds that have coccidiostatic properties namely thymol, carvacrol, caffeic acid, rosmarinic acids, flavonoids, ursolic, and phytosterols (Waldenstedt, 2003). Accordingly, the incorporation of oregano extract into diets of broiler chickens infected with coccidia reduced both the counts of Eimeria tenella oocysts in excrement and the extent of intestinal damage (Waldenstedt, 2003). Another herb useful for alleviating the severity of coccidiosis is Prunus salicina (oriental plum) (Swiatkiewicz et al., 2009). It contains phenolic acids, anthocyanins, and flavonoids that have coccidiostatic properties (Swiatkiewicz et al., 2009). Incorporating dried oriental plum into feed for birds infected with Eimeria reduced the number of excreted Eimeria oocytes (Swiatkiewicz et al., 2009). Other herbs that have anticoccidial effects are Aloe vera (Yim et al., 2011), Ocimum gratissimum (Ogunleye, 2019), echinacea (Paskudska et al., 2018), and Nectaroscordum tripedale (honey garlic) (Suganya et al., 2016). 36 University of Ghana http://ugspace.ug.edu.gh 2.4.7 Phytogenic supplements as anthelmintic agents The abusive use of conventional anthelmintic drugs such as piperazine and benzimidazoles (e.g. albendazole and fenbendazole) results in the occurrence of chemical residues in poultry products (Anthony et al., 2005) as well as cases of resistance to these drugs by helminths (Abdelqader et al., 2012). As a result, much attention has currently been placed on the use of botanicals as alternatives to conventional anthelmintic drugs in poultry production (Symeonidou et al., 2018). Some medicinal plants and herbs that have anthelmintic properties are Fagara zanthoxyloides (Athanasiadou et al., 2007), Ocimum gratissimum, black caraway, tulsi, sweet annie, thyme, echinacea, ginkgo, Melia azedarach (chinaberry tree), Carica papaya (pawpaw), Moghania vestita (Sohphlang), Juglan nigra (black walnut), Punica granatum (pomegranate), Embelia ribes (false black pepper), Picrasma excelsa (Jamaican Quassia), Chenopodium ambrosioides (sweet pigweed), Trifolium repens (white clover), Ficus insipida (chalate), Trachyspermum ammi (ajowan caraway), and Cucurbita maxima (pumpkin) (Shifa, 2014). Several of these botanicals have been used as feed additives or water supplements to control helminths in farm animals (Athanasiadou et al., 2007; Symeonidou et al., 2018). Presented in Table 2.6 are the effects of phytogenic supplements on Ascaridia galli; a helminth of poultry known to inflict the most damage (Butcher and Miles, 1995). 37 University of Ghana http://ugspace.ug.edu.gh Table 2. 5: Effects of phytogenic supplements on Ascaridia galli (A. galli) in chicken No. Botanical Part used Dose Effect(s) on A. galli Reference 1. Neem Leaves Aqueous extract at Death of parasite (Khokon et al., (Azadirachta 200mg/kg of BW 2014) indica) 2. Squirrel's claws Seeds Seed powder and Reduction in faecal (Javed et al., (Caesalpinia methanol extract at 30, 40, egg count 1994) crista) and 50mg/kg of BW 3. Chinaberry tree Fruits Fruit powder, aqueous, Inhibition of egg (Akhtar and (Melia ethanol, and methanol development Riffat, 1985) azedarach) extracts all at 20mg/kg of feed 4. Wild bauhinia Bark Ethanol extracts at 100, Reduction in faecal (Asuzu and (Piliostigma 200, and 400mg/kg of egg count Onu, 1994) thonningii) BW 5. Bitter leaf Leaves Aqueous extracts at 1kg/  Reduction in (Siamba et al., (Vernonia litre faecal worm egg 2007) amygdalina), count Fish bean  Reduction in adult (Tephrosia worm population vogelii) BW = Body weight 38 University of Ghana http://ugspace.ug.edu.gh The modes of action of plant secondary metabolites against helminths are shown on Plate 2.1. Plate 2. 1: Modes of action of phytochemicals against helminths (Source: Symeonidou et al., 2018) 2.4.8 Effects of phytogenic feed additives on some blood constituents in poultry Supplementation of broiler diets with 3g/kg each of parsley, fenugreek, and sweet basil seeds reduced blood cholesterol but did not affect total protein, glucose, albumin, and globulin (Abbas, 2010). Fenugreek seeds at 1.5% in feed also reduced blood cholesterol concentration in Muscovi ducklings (El-Ghamry et al., 2004). Also, a blend of cumin and turmeric supplemented at 0.75 and 1% in broiler diets decreased the concentrations of haemoglobin and cholesterol, counts of red and white blood cells, and heterophil to lymphocyte ratio (Al-Kassie et al., 2011a). Moringa leaf powder, when supplemented into broiler diets, equally reduced (p<0.05) serum triglyceride and cholesterol levels (Mandal et al., 2014). Moringa leaves contain large amounts of alkaloids, flavonoids, phenolic 39 University of Ghana http://ugspace.ug.edu.gh compounds, and polyphenols with hypocholesterolemic properties (Moyo et al., 2012; Mandal et al., 2014). Furthermore, supplementing broiler diets with Ocimum gratissimum leaf meal significantly reduced (p<0.05) blood cholesterol but did not affect albumin, creatinine, and total protein (Olumide et al., 2018). Likewise, Prasad et al. (2009) observed that dietary supplementation with garlic powder significantly reduced the concentrations of triglycerides, cholesterol, very-low- density lipoprotein, and low-density lipoprotein, but increased that of high-density lipoprotein in broilers. Qureshi et al. (1983) also supplemented broiler diets with garlic paste and recorded significantly lower serum cholesterol levels. This hypocholesterolaemic effect is caused by the depressing effect of some compounds in garlic namely 3-hydroxyl-3-methyl-glutaryl-CoA reductase, glucose-6-phosphatase dehydrogenase, fatty acid synthase, and malic enzyme on the activities of the cholesterogenic and lipogenic enzymes of the liver (Qureshi et al., 1983; Qureshi et al., 1987). However, dietary inclusion of onion (Allium cepa) extract at the rates of 5, 7.5, and 10g/kg did not affect total cholesterol, high-density lipoprotein, triglycerides, and glucose levels in broilers (Aditya et al., 2017) whilst thyme powder at a concentration of 1g/kg in broiler feed caused increases (p>0.05) in plasma cholesterol and triglycerides (Demir et al., 2005). The effects of phytogenic feed supplements on some blood parameters observed in other studies are presented in Table 2.7. 40 University of Ghana http://ugspace.ug.edu.gh Table 2. 6: Effects of PFAs on blood haematology and biochemical indices of broilers PFA Dietary Effects Reference inclusion rate 1. Emblica 0.25, 0.50,  Decreases in serum cholesterol and (Dalal et al., 2018a) officinalis (amla) 0.75 or 1% heterophil count fruit powder  Increase in Hb 2. Garlic bulb, 2 or 4%  Decreases in total cholesterol and (Kim et al., 2009) garlic husk LDL  No effect on HDL 3. Garlic extract 2, 4 or 6%  Decreases in total cholesterol and (Utami et al., 2018) LDL  Increases in HDL and HDL: LDL ratio 4. Ocimum 200, 400 or  Increase in triglycerides (Riyazi et al., 2015) basilicum 600mg/kg  No effects on cholesterol, HDL, and EO LDL 5. Ocimum 0.5 or 1g/kg  0.5 g/kg: Increase in glucose (Osman et al., basilicum  1 g/kg: Increase in lymphocytes 2010) leaf powder  Both: Increase in RBC count but no effects on plasma cholesterol and triglycerides EO = Essential oil; LDL = Low-Density Lipoprotein; Hb = Haemoglobin; H: L = Heterophils to Lymphocytes; HDL = High-Density Lipoprotein; PFA = Phytogenic feed additive; RBC = Red blood cell; WBC = White blood cell 2.4.9 Effects of phytogenic feed additives on the quality of poultry products Lipid oxidation is the primary cause of meat deterioration (Mandal et al., 2014). It is a complex process that occurs in aerobic cells due to the interaction between polyunsaturated fatty acids and molecular oxygen (Verma et al., 2009). Lipid oxidation causes meat spoilage particularly when meat is exposed to oxygen, light, or heat (Mandal et al., 2014). Supplementing animal diets with phytogenic feed additives with antioxidative properties is an effective means to increase the 41 University of Ghana http://ugspace.ug.edu.gh oxidative stability, quality, and shelf life of their meat (Tavárez et al., 2011). Furthermore, Kim et al. (2009) evaluated the sensory and physicochemical characteristics of thigh muscles of broilers fed diets containing different levels of garlic bulb or garlic husk. Garlic supplementation increased the protein content, flavour, and texture of the meat, and reduced the fat content (Kim et al., 2009). Also, increasing the inclusion rate of garlic bulb from 2 to 4% reduced the shear force and thiobarbituric acid reactive substances in the thigh muscles (Kim et al., 2009). Also in that study, the highest level of garlic husk supplementation (4%) decreased (p<0.05) the concentrations of saturated fatty acids and increased (p<0.05) that of unsaturated fatty acids in the meat (Kim et al., 2009). A similar distribution of saturated and unsaturated fatty acids was observed in the meat of broilers fed diets containing peppermint and Viola tricolor var. hortensis (pansy) (Kapica et al., 2006). Furthermore, the flowers of Tagetes erecta (tagetes) and Calendula officinalis (calendula) when added to broiler diets, improved carcass colour (Grela et al., 2013). These flowers added a yellow tint to the skin of the carcasses (Grela et al., 2013). Also, dietary inclusion of Allium sativum and Ocimum gratissimum significantly improved the tenderness and juiciness of the meat of broilers (Odoemelam et al., 2017). Moreover, phytogenic feed additives are used as colouring agents in diets of laying hens to improve egg yolk colour (Nobakht and Moghaddam, 2013). For example, incorporating 2% dried aerial parts powder of Tanacetum balsamita (costmary) into diets of laying chickens did not only lower (p<0.05) blood cholesterol level but also positively imparted egg yolk colour (Nobakht and Moghaddam, 2013). A similar observation was made when diets were supplemented with marigold extract (Sirri et al., 2007). However, Yildirim et al. (2013) and Kopřiva et al. (2014) observed no effects of dietary supplementation with Panax ginseng (Korean ginseng) root extract and dried Beta vulgaris (beetroot) respectively on egg yolk colour. 42 University of Ghana http://ugspace.ug.edu.gh Also, Azeezah et al. (2019) supplemented cockerel diets with Moringa oleifera and Ocimum gratissimum leaf meals and observed increases in meat protein and mineral contents (calcium, magnesium, iron, and phosphorus), and a decrease in cooking loss (Azeezah et al., 2019). Additionally, some phytochemicals have hypocholesterolaemic effects and hence, reduce the levels of cholesterol in meat and eggs. This make these foods safe for human consumption (Grela et al., 2013). For instance, Grela et al. (2013) reported that dietary supplementation with garlic reduces cholesterol levels in meat and eggs. Likewise, adding 1% Ocimum sanctum leaf powder to broiler diet was shown to reduce cholesterol in breast and thigh muscles (Lanjewar et al., 2009). 2.4.10 Phytogenic supplements as anti-stress agents in poultry Heat stress, a commonly-faced problem in the tropics, has been widely reported to deleteriously affect poultry growth performance and productivity (Lara and Rostagno, 2013; Bhadauria et al., 2016). Heat stress occurs when the amount of heat generated from the animal’s body exceeds that released into the environment (Lara and Rostagno, 2013). Of all livestock species, poultry is most negatively affected by heat stress because they lack sweat glands in their skin to allow easy release of internally-generated heat (Bhadauria et al., 2016). Heat stress causes declines in feed consumption, feed utilization, body weight gain, egg production, egg quality, and carcass yield (Whitehead, 1998; Zeferino et al., 2016). The reduction in feed intake leads to reductions in nutrient uptake and utilization and thus, results in poor productivity (Shokryazdan et al., 2017). Chronic heat stress during the growing period of broilers negatively affects the quality of their meat (Lu et al., 2007). Also, heat stress adversely affects the immunity, welfare, and behavior of birds, and ultimately results in mortalities and economic losses to farmers (Lara and Rostagno, 2013; Ranjan et al., 2019). 43 University of Ghana http://ugspace.ug.edu.gh Supplementing drinking water or rations with some phytogenic products is one of the ways to combat heat stress in poultry (Grela et al., 2013, Abd El-Hack et al., 2020). The products of herbs such as black seed, chicory (Cichorium intybus), dill (Anethum graveolens), moringa, ginger, red pepper (Capsicum annuum), thyme, sweet annie, fennel, and rosemary have been successfully used as anti- stress agents in poultry (Abd El-Hack et al., 2020). Herbs with antioxidative properties can equally be used to combat heat stress (Shokryazdan et al., 2017). Heat stress causes oxidative stress in the body, and the oxidation of substrates generates free radicals and other reactive oxygen species that can damage body cells and organs. Hence, antioxidants are neceassary to counteract these effects (Halliwell and Gutteridge, 1984). The effects of some phytogenic feed additives on poultry performance, physiology, and productivity under heat stress conditions are presented in Table 2.8. 44 University of Ghana http://ugspace.ug.edu.gh Table 2. 7: Effects of phytogenic feed additives on poultry performance, physiology, and productivity under heat stress conditions No. PFA Poultry Inclusion rate Effects Reference species 1. Black seed Pigeons 2%  Increases in FI and BWG (Shoukary et (Nigella  Improvement of FCR al., 2018) sativa)  Decrease in aggressive behavior  Decreases in blood cholesterol, catalase, and glucose levels 2. Black seed Broilers 1%  Increases in FI, ADG, and dressing (EL-Shoukary (Nigella percentage et al., 2014) sativa)  Decreases in panting and water/feed ratio  Decreases in T3 and corticosterone concentrations 3. Thyme Broilers 100, 150 or  Improvement of growth performance (Olfati and (Thymus 200mg/kg  Increases in relative weights of bursa of Mojtahedin, vulgaris) Fabricius, spleen, and thymus 2018) EO  Increase in lymphocyte count  Decrease in H: L ratio 4. Ginger Broilers 7.5g or  Increases in BW, BWG, and superoxide (Habibi et al., (Zingiber 150mg/kg dismutase 2014) officinale)  No effects on blood variables and carcass root traits powder  Reduction in MDA level in the liver or EO 5. Sweet Male 1 or 1.25g/kg  Increases in FI, BWG, and carcass traits (Wan et al., annie broilers  Decreases in blood pH and serum levels 2017) (Artemisia of corticosterone, AST, MDA, and ALT annua)  Increases in serum SOD, T3, and T4 Enzymatica levels lly- treated WP 6. Fennel Laying 10 or 20g/kg  Improvement of egg quality traits (Gharaghani et (Foeniculu chickens  Reduction in number of broken eggs al., 2015) m vulgare)  Reductions in carboxyl and MDA in eggs fruits  Reductions in yolk cholesterol and triglyceride levels 45 University of Ghana http://ugspace.ug.edu.gh 7. Fennel Broilers 1 or 2%  Increases in FI, breast meat yield, and (Ragab et al., (Foeniculu circulating leukocytes 2013) m vulgare)  Decreases in body temperature and seeds mortality 8. Sweet basil Broilers 5g/kg  Increases in BWG and dressing (Jahejo et al., (Ocimum percentage 2019) basilicum)  Improvement of FCR seed  Decreases in body temperature, water powder intake, and mortality  Increases in retention of CP, CF, and ME  Increase (p>0.05) in intestinal villi size  No effects on RBC count and PCV  Increases in Hb and WBC count  Increase (p>0.05) in ND antibody titre 9. Coriander Broilers 2%  Increases in FI, ADG, and dressing (EL-Shoukary (Coriandru percentage et al., 2014) m sativum)  Decreases in panting behavior and seeds water/feed ratio  Decreases in T3 and corticosterone concentrations  No effects on drinking behavior, FCR, slaughter weight, and T4 level 10. Javanese Broilers 200 or  Increases in erythrocyte GPX and SOD (Akbarian et turmeric 400mg/kg activity, serum total protein, and plasma al., 2015) (Curcuma GH concentration xanthorrhiza)  Decreases in cholesterol, LDL, EO phosphorus, and chloride concentrations  Increase in bronchitis antibody titre Source: Abd El-Hack et al. (2020) ADG = Average daily gain; AST = Aspartate aminotransferase; ALT = Alanine aminotransferase; BWG = Body weight gain; CP = Crude protein; CF = Crude fibre, EO = Essential oil; FCR = Feed conversion ratio; FI = Feed intake; GH = Growth hormone; GPX = Glutathione peroxidase; Hb = Haemoglobin; H: L = Heterophils to lymphocytes; HDL = High-density lipoproteins; LDL = Low density lipoprotein; MDA = Malondialdehyde; ME = Metabolisable energy; ND = Newcastle disease; PCV = Packed cell volume; PFA = Phytogenic feed additive; PUFA = Polyunsaturated fatty acid; RBC = Red blood cell; SFA = Saturated fatty acids; SOD = Superoxide dismutase; T3 = Tri-iodothyronine; T4 = Thyroxine; WBC = White blood cell; WP = whole plant 46 University of Ghana http://ugspace.ug.edu.gh 2.5 Fagara zanthoxyloides 2.5.1 Biology of Fagara zanthoxyloides Fagara zanthoxyloides is an important medicinal plant in Africa that belongs to the Rutaceae family (Joshua et al., 2016). It is known by other names such as artar root, candlewood, lime-prickly ash, Senegal prickly-ash, wild lime, Zanthoxylum polyganum, Zanthoxylum senegalensis, and Zanthoxylum zanthoxyloides (Ngassoum et al., 2003; Ogwal-Okeng et al., 2003; Joshua et al., 2016; Guendéhou et al., 2018). In Ghana, it is locally called “Haatso”. Fagara zanthoxyloides is woody and grows to a height of about 12m (TPD, 2020a). It has a rough trunk that develops to a diameter of about 0.25m. The bark is gray and has large, sharp, and claw-shaped prickles on it (Plate 2.2). The prickles are widely distributed at regular intervals on the stem and branches. The branches are irregularly shaped with pinnate leaves on them. The leaves are also garnished with thorns. The fruits are capsules with diameter of about 5 to 6mm, as shown on Plate 2.3. Each fruit contains a single seed with a shiny black colour (Guendéhou et al., 2018). 47 University of Ghana http://ugspace.ug.edu.gh Plate 2. 2: Fagara zanthoxyloides plant 48 University of Ghana http://ugspace.ug.edu.gh Plate 2. 3: Fruits of Fagara zanthoxyloides 49 University of Ghana http://ugspace.ug.edu.gh 2.5.2 Chemical composition of Fagara zanthoxyloides Fagara zanthoxyloides is rich in several bioactive compounds such as alkaloids, amides, coumarins, lignins, flavonoids, glycosides, polyphenols, polyterpenes, saponins, sterols, and tannins (Adesina, 2005; Adefisoye et al., 2012; Guendéhou et al., 2018). Enechi et al. (2019) analysed the phytochemical constitution of the leaf extract and observed high levels of alkaloids, flavonoids, phenols, and terpenoids, moderate levels of glycosides and tannins, and low levels of saponins and steroids in the extract. In agreement with this report, Elujoba et al. (2005) reported that Fagara zanthoxyloides contains high amounts of the alkaloids - berberine, canthin-6-one, chelerythrine, fagaronine, and benzoic acid derivatives. Also, Jirovetz et al. (1997) reported the presence of limonene, myrcene, α-phellandrene, α-terpinolene, α-pinene, β-pinene, trans-β-ocimene, sabinene, citronellyl acetate, geraniol, terpinen-4-ol, p-cymene, and methyl citronellate in volatile oil extract of the seeds. 2.5.3 Uses of Fagara zanthoxyloides The bark and leaves are often crushed into powder and used as condiments with smells similar to that of lime (Elbert, 1980). Traditionally, the bark, leaves, stem, and roots are used in medicinal preparations for the treatment of cold, colic, cough, dysentery, digestive disorders, fever, hypertension, dental caries, oral infections, sore gums, toothache, scabies, sickle cell disease, snakebites (Ngassoum et al., 2003; Elujoba et al., 2005; Guendéhou et al., 2018), abdominal pain, dysmenorrhea, elephantiasis, gonorrhoea, lumbago, malaria, sexual impotence, and urinary diseases (Ogwal-Okeng et al., 2003; Adesina, 2005). Also, the root extracts are used in embrocation to treat headaches, and intercostal, lumbar spine, and rheumatic pains (Guendéhou et al., 2018). Moreover, the root powder is sniffed as an emmenagogue likewise the leaf powder to treat migraines 50 University of Ghana http://ugspace.ug.edu.gh (Guendéhou et al., 2018). In West Africa, parts of the plant are used as chewing sticks for tooth cleaning (Ogwal-Okeng et al., 2003). Enechi et al. (2019) demonstrated the antimalarial property of methanolic extract of the leaves. In that study, treatment with the extract normalized the biochemical and haematological anomalies caused by malaria in Plasmodium berghei- parasitised mice. Esan et al. (2014) reported that the root extract is equally effective in controlling malaria due to its inhibitory effects on the in-vitro growth and development of Plasmodium falciparum; a species of plasmodium reported to cause majority of deaths from malaria in Africa (WHO, 2020). The antimalarial property of the extract is attributable to its fagaronine content (Esan et al., 2014). Moreover, the root bark extracts have insecticidal properties (Denloye et al., 2010). They effectively controlled insect pests of cowpea and maize grains such as Callosobruchus maculatus, Sitophilus zeamais, and Tribolium castaneum (Denloye et al., 2010). Similarly, topical application of the root bark powder to cowpea increased (p<0.05) mortality of the seed beetle Callosobruchus maculatus and protected the grains from damage during storage (Musa, 2012). Still, other researchers reported the analgesic, anticonvulsant, antidiabetic, anti-sickling, antioxidative, anthelmintic, antitumor, antitrypanosomal, antiviral, anticancer, hypolipidaemic, hypotensive, and molluscicidal properties of extracts of the plant (Prempeh, 2008; Guendéhou et al., 2018; Dofuor et al., 2019; Enechi et al., 2019). 51 University of Ghana http://ugspace.ug.edu.gh 2.5.4 Antimicrobial properties of Fagara zanthoxyloides extracts The antibacterial and antifungal effects of several extracts of the plant have been reported. For instance, in some in vitro studies, extracts of the fruits exhibited inhibitory effects on Staphylococcus aureus, Salmonella enteritidis, Listeria monocytogenes (Gardini et al., 2009), Bacillus subtilis, Salmonella typhimurium, Streptococcus mutans, Micrococcus luteus, Pseudomonas aeruginosa, and Klebsiella pneumoniae (Misra et al., 2013). This antimicrobial activity is attributable to the high geraniol content of the extracts (Gardini et al., 2009; Sado Kamdem et al., 2015). Another compound isolated from the fruit with antibacterial activity is 3,4,5,7-tetrahydroxy-1-methoxy-10-methyl-9- acridone. This compound inhibited the growth of Pseudomonas aeruginosa and Micrococcus luteus (Misra et al., 2013; Wouatsa et al., 2013). Similarly, Ngassoum et al. (2003) found volatile oil extract of the dried fruits to be effective against Bacillus subtilis, Bacillus cereus, Corynebacterium glutamicum, Enterococcus faecalis, Escherichia coli, Streptococcus faecalis, and Staphylococcus aureus. Also, the crude extract of the powdered stem displayed antibacterial effects on Escherichia coli, Lactobacillus plantarum, Lactobacillus brevis, and Proteus vulgaris (Adefisoye et al., 2012). In an investigation by Ynalvez et al. (2012), the defatted ethanolic extract of the root bark showed great zones of inhibition against Escherichia coli, Staphylococcus aureus, and Enterococcus faecium. Likewise, other researchers reported the antibacterial effects of ethanolic and methanolic extracts of the plant against Bacillus subtilis, Bacillus cereus, Pseudomonas aeruginosa, Enterococcus faecalis, and Staphylococcus aureus (Adebiyi et al., 2009; Adegbolagun and Olukemi, 2010). Similarly, the chemical and chromatographic fractions of the powdered root had effects on Escherichia coli and Staphylococcus aureus which is attributable to the berberine, canthin-6-one, chelerythrine, and flavonoid contents of the extracts (Odebiyi and Sofowora, 1979). 52 University of Ghana http://ugspace.ug.edu.gh Besides, Osho and Adelani (2012) assayed aqueous and ethanolic extracts of chewing sticks made from the plant for their antifungal efficacy against the oral pathogens - Candida tropicalis, Candida albicans, and Candida krusei. Both extracts inhibited the growth of all the tested fungi. 2.5.5 Anthelmintic properties of Fagara zanthoxyloides extracts In vitro studies with crude leaf extracts displayed anthelmintic effects on the sheep parasites Haemonchus contortus and Trichostrongylus colubriformis (Hounzangbe-Adote et al., 2005; Azando et al., 2011). Also, ethanolic and aqueous leaf extracts had effects on Ascaris lumbricoides, Haemonchus contortus, and Trichostrongylus colubriformis (Barnabas et al., 2010; Azando et al., 2011). Similarly, essential oil extract of the seeds affected Strongyloides ratti; a gastrointestinal parasite of rats (Olounladé et al., 2012). Regular consumption of small amounts of the leaves by sheep reduced faecal Haemonchus contortus egg counts (Hounzangbe-Adote et al., 2005). The ethanolic extract of the root bark was found to be effective against Ascaris suum; an endoparasite of pigs (Williams et al., 2016). Owing to this property, extracts of the plant are used to treat ascariasis (Guendéhou et al., 2018). Even in humans, methanolic extract of the root-bark is used to treat worm infections (Ogwal-Okeng, 1990). The flavonoid, terpene, and tannin contents of the extracts are responsible for the anthelmintic effects. These compounds inhibit the development of the larvae of helminths (Prempeh, 2008; Olounladé et al., 2012). Additionally, tannins inhibit egg production from adult helminths (Athanasiadou et al., 2001). 53 University of Ghana http://ugspace.ug.edu.gh 2.6 Ocimum americanum 2.6.1 Biology of Ocimum americanum Ocimum americanum is an aromatic herb with lavender or white flowers that grows to a height of about 20 to 30cm (TPD, 2020b). Despite its name, Ocimum americanum is innate to Africa, the Indian subcontinent, Southeast Asia, and China (Junaid et al., 2006). It is variously called American basil, English camphor basil, basilic camphor, hairy or hoary basil, lime or lemon basil, and Ocimum canum (Chagonda et al., 2000; Anuradha, 2014; Gberikon et al., 2018). In Ghana, it is locally known as “akokobesa” or “eme” (Berhow et al., 2012). It belongs to the kingdom Plantae, phylum Tracheophyta, division Magnoliophyta, class Magnoliopsida, order Lamiales, family Lamiaceae, genus Ocimum, and specie americanum (Anuradha, 2014). Plate 2. 4: Ocimum americanum plants 54 University of Ghana http://ugspace.ug.edu.gh 2.6.2 Chemical composition of Ocimum americanum Anuradha (2014) analysed aqueous leaf extract of the plant for its phytochemical composition and found alkaloids, amino acids, carbohydrates, flavonoids, phenolic compounds, steroids, and terpenoids in it. In another study, Gberikon et al. (2018) observed significant concentrations of cardiac glycosides, steroids, and flavonoids in both flower and leaf extracts. The leaf extract additionally contained tannins that were absent in the flower extract (Gberikon et al., 2018). Also, volatile oil extracted from aerial parts and leaves of the plant contained 1,8-cineole, bornyl acetate, bicyclogermacrene, borneol, cadinol, camphor, cedrol, endo-fenchyl acetate, eugenol, germacrene A, germacrene D, linalool, methyl chavicol, terpinen-4-ol, trans-caryophyllene, α-bergamotene, trans-β-terpineol, α-terpineol, β-elemene, and γ-cadinene (Nascimento et al., 2011). Furthermore, Mustafa and El-kamali (2019) performed proximate analyses on dried leaves and flowers of the plant and reported the results shown in Table 2.9. Table 2. 8: Proximate composition of the foliage (at flowering) of Ocimum americanum Nutrient (%) Leaves Flowers Moisture 5.5±0.1 5.53±0.1 Crude protein 18.43±0.31 10.15±0.9 Total ash 19.09±0.1 8.87±0.05 Crude fibre 13.8±0.1 29.23±0.2 Source: Mustafa and El-kamali (2019) 55 University of Ghana http://ugspace.ug.edu.gh 2.6.3 Uses of Ocimum americanum The leaves are used for cooking and in traditional medicines to treat cold, diabetes, fever, headaches, inflammation of joints, malaria, as well as genitourinary and skin diseases (Ngassoum et al., 2004; Asase et al., 2005; Berhow et al., 2012; Gberikon et al., 2018). Also, essential oils extracted from the plant are used to manufacture perfumes (Rady and Nazif, 2005). Furthermore, the phytoconstituents of Ocimum americanum have analgesic, anticancer, antiasthmatic, antistress, antibacterial (Nascimento et al., 2011), antioxidative (Gberikon et al., 2018), antidiabetic (Nyarko et al., 2002), nematicidal, insecticidal, and fungicidal properties (Karuppusamy et al., 2002). 2.6.4 Antimicrobial activity of Ocimum americanum extracts Silver nanoparticles derived from aqueous Ocimum americanum leaf extract exhibited tremendous antibacterial activities against Escherichia coli and Staphylococci aureus (Anuradha, 2014). The antibacterial property of Ocimum americanum extracts is attributable to the cardiac glycosides and tannins in them (Gberikon et al., 2018). The antifungal property of extracts of Ocimum americanum has also been reported (Anuradha, 2014; Gberikon et al., 2018). In a study conducted by Gberikon et al. (2018), the leaf and flower extracts had effects on Trichophyton mentagrophytes; a fungus that causes skin infections in animals and humans. Anuradha (2014) also found silver nanoparticles obtained from the aqueous leaf extract to inhibit the mycelia growth of Aspergillus niger. This antifungal effect is attributable to the methyl chavicol content of the leaves (Anuradha, 2014). 56 University of Ghana http://ugspace.ug.edu.gh CHAPTER THREE 3.0 MATERIALS AND METHODS 3.1 Experimental Site and Duration of Study This research work was carried out at the Livestock and Poultry Research Centre (LIPREC) of the University of Ghana. LIPREC is in the coastal savannah area of Ghana and on latitude 05040’N and longitude 00016’W. The area experiences a bimodal rainfall distribution. The major rainy season begins from March to July whilst the minor rains begin from August to November. The annual mean rainfall of the area ranges from 128 to 1709mm, and the annual mean temperature is 26.9°C. The relative humidity at LIPREC lies between 58 and 83.7% at 1500h. 3.2 Experimental Birds and Their Management 3.2.1 Brooding of birds A total of four hundred unsexed one-day-old Cobb 500 broiler chicks were used for the trial. Before the birds arrived, the brooder house was cleaned and fumigated to eradicate pests in the building. Clay pots containing live charcoal and electric lamps were used to provide warmth for the birds. The floor of the brooder house was covered with wood shavings. The chicks on arrival were first introduced to clean drinking water mixed with glucose D and vitamins (see appendix A for composition). The glucose and vitamin mixture was given to relieve the birds of the stress they might have suffered during transportation. The glucose further supplied them with energy. The birds were allowed to drink for about two hours before feed was offered. They were brooded for seven days together on a common starter diet and fed ad libitum throughout this period. Not knowing how birds will react or respond to FFM and OLM since they are novel feed additives, it was planned for trial 57 University of Ghana http://ugspace.ug.edu.gh to start in week two about which time their body systems will be quite developed to handle such additives. 3.2.2 Distribution of birds after brooding On the 8th day, the birds were transferred into an open-sided poultry house and kept there until the end of the feeding trial. A total of forty deep litter pens measuring 1.5m x 2.5m were used for raising the birds. Wood shavings served as bedding materials in the pens. Ten birds were randomly selected from the lot, weighed, and distributed into each of these pens in a completely randomised design. There were eight dietary treatment groups with five replicates each, and each replicate had 10 birds. Plate 3.1 is an image of the birds in one of the pens after distribution. Plate 3. 1: Birds in a pen after distribution 58 University of Ghana http://ugspace.ug.edu.gh 3.2.3 Vaccination schedule The birds were orally vaccinated against infectious bursal disease (IBD) on days 7, 21, and 35, and Newcastle disease on days 14 and 28. 3.2.4 Lightning The birds were provided with lights in the evenings such that they had 24 hours of continuous light throughout the experiment. 3.2.5 Litter management Wood shavings were used as litter materials throughout the experiment. They covered the floor to a depth of about 4cm during the brooding stage and between 6 and 8cm as the birds grew. The wood shavings were treated with insecticide, disinfected, and allowed to dry before use. The litter was turned every two to three days to avoid litter compaction and allow the escape of ammonia gas from it. Occasionally, wet and caked portions of the litter were scooped out and replaced with fresh ones. Wood shavings were changed every two weeks. 3.3 Preparation of Experimental Diets The Fagara zanthoxyloides fruits and Ocimum americanum leaves were harvested from plants growing at LIPREC. They were air-dried under shade for five to seven days, ground in a Retsch mill through a one-mm-sieve into meals, stored, and used to prepare the experimental diets. Obtaining Ocimum americanum leaves was fairly easy. However, it was quite difficult to obtain Fagara zanthoxyloides fruits due to the thorns on the stems and leaves of the plant. Eight experimental diets were formulated and fed to the birds. Diet BD was the basal diet and served as the negative control diet. The remaining diets were supplemented with FFM or OLM alone or in 59 University of Ghana http://ugspace.ug.edu.gh combination at different inclusion rates, or antibiotic (Penicillin V), as illustrated in Table 3.1. The basal diet (BD) served as the negative control diet whilst that supplemented with Penicillin V (PEN) was the positive control diet. The inclusion rates of FFM, OLM, and Penicillin V, shown in Table 3.1, were maintained for both the experimental starter and finisher diets. Table 3. 1: Experimental diets and treatment layout Dietary Composition No. of No. of treatment Replicates birds/replicate BD Basal Diet (BD) 5 10 0.2FFM BD + 0.2% FFM 5 10 0.4FFM BD + 0.4% FFM 5 10 0.2OLM BD + 0.2% OLM 5 10 0.4OLM BD + 0.4% OLM 5 10 0.1FFM+0.1OLM BD + 0.1% FFM + 0.1% OLM 5 10 0.2FFM+0.2OLM BD + 0.2% FFM + 0.2% OLM 5 10 PEN BD + 0.01% Penicillin V 5 10 The experimental starter diets were fed from day 8 to 28 and finisher diets from day 29 to 49. The ingredient compositions of the experimental starter and finisher diets are presented in Tables 3.2 and 3.3 respectively. The diets were supplied ad libitum throughout the trial. 60 University of Ghana http://ugspace.ug.edu.gh Table 3. 2: Ingredient composition of experimental starter diets Percentage (%) inclusion of ingredients Ingredient BD* 0.2FF 0.4FF 0.2OL 0.4OL 0.1FFM+ 0.2FFM+ PEN M M M M 0.1OLM 0.2OLM FFM 0.00 0.20 0.40 0.00 0.00 0.10 0.20 0.00 OLM 0.00 0.00 0.00 0.20 0.40 0.10 0.20 0.00 Penicillin V 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.01 Maize 50.00 49.90 49.80 49.90 49.80 49.90 49.80 50.00 Soya bean meal 35.55 35.48 35.41 35.48 35.41 35.48 35.41 35.55 Wheat bran 12.00 11.98 11.95 11.98 11.95 11.98 11.95 12.00 Limestone 1.20 1.20 1.20 1.20 1.20 1.20 1.20 1.20 Methionine 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 Lysine 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 DCP 0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.20 Broiler premix+ 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 NaCl 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 Total 100 100 100 100 100 100 100 100 Calculated proximate composition (% DM) Dry matter 90.79 91.22 91.14 90.78 90.96 91.38 91.60 91.02 Ash 6.55 7.55 7.03 7.10 7.20 6.86 8.07 7.33 Ether extract 3.39 3.44 3.84 4.05 3.84 3.69 3.55 3.37 Crude protein 23.45 23.40 23.36 23.40 23.36 23.40 23.36 23.45 Crude fibre 3.90 4.04 4.12 4.32 4.59 3.93 4.51 3.89 ME (kcal/kg) 3048 3043 3037 3042 3037 3042 3037 3049 Cost (GH¢ 199.22 200.04 200.86 199.84 200.46 199.94 200.66 207.22 100kg) FFM = Fagara zanthoxyloides fruit meal; OLM = Ocimum americanum leaf meal; DCP = Dicalcium phosphate; NaCl = Sodium chloride; ME= Metabolisable energy. Toxin binder (Biomin®) was added to diets at the manufacturer's recommended rate. +Broiler premix composition is shown in Appendix B. BD* served as the common starter diet fed to the birds during the brooding stage (from day 1 to 7). 61 University of Ghana http://ugspace.ug.edu.gh Table 3. 3: Ingredient composition of experimental finisher diets Percentage (%) inclusion of ingredients Ingredient BD 0.2FF 0.4FF 0.2OL 0.4OL 0.1FFM+ 0.2FFM+ PEN M M M M 0.1OLM 0.2OLM FFM 0.00 0.20 0.40 0.00 0.00 0.10 0.20 0.00 OLM 0.00 0.00 0.00 0.20 0.40 0.10 0.20 0.00 Penicillin V 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.01 Maize 56.61 56.50 56.39 56.50 56.39 56.50 56.39 56.61 Soya bean meal 23.55 23.50 23.45 23.50 23.45 23.50 23.45 23.54 Wheat bran 17.03 17.00 16.97 17.00 16.97 17.00 16.97 17.03 Limestone 1.50 1.50 1.50 1.50 1.50 1.50 1.50 1.50 Methionine 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 Lysine 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 DCP 0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.20 Broiler premix+ 0.30 0.30 0.30 0.30 0.30 0.30 0.30 0.30 NaCl 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 Total 100 100 100 100 100 100 100 100 Calculated proximate composition (% DM) Dry matter 87.74 87.96 88.03 87.55 87.40 87.53 87.92 88.00 Ash 5.45 5.46 5.71 5.51 5.37 5.57 5.22 5.49 Ether extract 3.09 3.27 3.85 3.26 3.48 3.47 3.40 3.08 Crude protein 19.08 19.04 19.00 19.04 19.00 19.04 19.00 19.07 Crude fibre 5.43 5.99 5.82 5.51 5.89 6.01 6.39 5.42 ME (kcal/kg) 2966 2960 2954 2960 2954 2956 2954 2965 Cost (GH¢/ 191.83 192.67 193.51 192.47 193.11 192.57 193.31 199.81 100kg) FFM = Fagara zanthoxyloides fruit meal; OLM = Ocimum americanum leaf meal; DCP = Dicalcium phosphate; NaCl = Sodium chloride; ME= Metabolisable energy. Toxin binder (Biomin®) was added to all the diets at the manufacturer's recommended rate. +Broiler premix composition is shown in Appendix B. 62 University of Ghana http://ugspace.ug.edu.gh 3.4 Growth Performance Determination The birds were weighed at the start of the growth trial and subsequently at weekly intervals. Also, the amount of feed offered each time was recorded and that refused weighed weekly throughout the experiment. These values were used to estimate average daily feed intake (ADFI), average daily weight gain (ADG), and feed conversion ratio (FCR) using the formulas listed below. Feed offered – feed refused ADFI (g) = Number of days Difference in body weights between two successive weeks ADG (g) = Number of days Average daily feed intake (ADFI) FCR = Average daily weight gain (ADG) Mortalities were accounted for in determining ADFI. 3.5 Mortalities Mortalities were recorded each time they occurred throughout the experiment. Mortality percentage was estimated as the ratio of the number of dead birds to the total number of birds per treatment multiplied by 100, as represented below. Number of dead birds Mortality (%) = x 100 Total number of birds per treatment 63 University of Ghana http://ugspace.ug.edu.gh 3.6 Serum Lipid Profile Test After the feeding trial, five birds per treatment (a bird from each treatment replicate) were randomly selected, wing tagged, and starved overnight (about 9 hours). Thereafter, 2ml of blood was collected from their medial wing (brachial) veins by venipuncture into serum separator tubes (SSTs) containing gel and clot activator. The blood samples were allowed to stand for about 30 minutes to clot and then centrifuged at 3000 revolutions per minute (rpm) for 3 minutes. The sera were extracted and analysed for the concentrations of triglycerides, cholesterol, high-density lipoprotein (HDL), and low-density lipoprotein (LDL) using the fully automatic veterinary chemistry analyzer URIT- 8021AVet (URIT Medical Electronic Co., Ltd.). 3.7 Carcass Analysis The birds from which blood samples were taken for the serum lipid profile test were slaughtered and examined for weights of carcass parts, internal organs, and abdominal fat. Live weight before slaughter as well as de-feathered and dressed (carcass) weights were taken. The carcasses were cut into commercial parts consisting of necks, thighs, drumsticks, breasts, and wings. These parts as well as internal organs including the proventriculus, gizzard, liver, heart, and intestine were also weighed. 3.8 Digestibility Study Three birds from each treatment were randomly selected and transferred into newly-prepared pens for a digestibility trial that lasted four days. The procedure followed that indicated by Aditya et al. (2017) however, slight modifications were made. The floors of the pens were covered with polythene sheets for easy collection of excreta. The birds were housed individually and allowed two days of acclimatisation to the new pens before data collection began. Afterwards, they were fasted overnight 64 University of Ghana http://ugspace.ug.edu.gh for about 11 hours to get rid of almost every digesta from their digestive tracts. The birds were then fed 120g of their respective finisher diets daily for three days. On the fourth day, feeds were withdrawn and leftover feeds weighed to determine total feed intake per bird. Excreta from the birds were collected regularly each day into containers. At the end of each day, the hoarded excreta from each bird was transferred from the collection container into a plastic bag and frozen at –18oC. Excreta were collected also on the fourth day for 24 hours even though the birds were not fed this day to obtain the remaining faeces from the feeds they consumed. The birds had access to water throughout the digestibility trial. Later, the excreta were removed from the freezer and allowed to thaw. The excreta collected from each bird over the four days were bulked and oven-dried until constant weight at 55oC. The oven- dried excreta were weighed, ground in a one-mm-sieve Retsch mill, and analysed for dry matter, ash, crude fibre, crude protein, and fat. Also, samples of the finisher diets served to the birds during the digestibility trial were analysed for the same nutrients. The results were used to estimate the apparent total tract digestibility for each nutrient using the formulae below stated by Liu et al. (2020). Nutrient intake − Nutrient voided Apparent total tract nutrient digestibility (%) = X 100 Nutrient intake Nutrient intake = Nutrient in diet x Feed intake Nutrient voided = Nutrient in faeces x Amount of faeces voided 3.9 Nitrogen Excretion Determination The nitrogen (N) contents of the finisher diets and excreta of the birds used for the digestibility trial were used to estimate the relative amount of N excreted from each bird as 100% minus N digestibility. 65 University of Ghana http://ugspace.ug.edu.gh 3.10 Faecal Microbial Analysis Eight new pens were prepared and their floors were covered with polythene sheets. Each day for five days, four birds from each treatment replicate were randomly selected from their original pens and transferred into the new pens. Faecal samples were collected into sterile falcon tubes and stored at 4°C for analysis. Faecal collection was done immediately after they were voided to avoid contamination. The faecal samples were analysed for total plate count (microbial load), and counts of coliforms, Escherichia coli, Salmonella and Shigella, as well as yeasts and moulds. Faecal microbial analysis followed the procedure indicated by Murugesan et al. (2015) but slight modifications were made. 1g of each sample was taken, and 1000μl of sterile saline solution was added to it and thoroughly mixed. An aliquot of the resulting homogenous solution was taken and serially diluted in sterile saline solution from 10-2 to 10-12, cultured with the appropriate agar media (see appendix C), and incubated. The agar plates for microbial load, coliforms, and Salmonella and Shigella were incubated at 37°C for 24 hours, Escherichia coli at 44°C for 24 hours, and yeasts and moulds at 37°C for 72 hours. Thereafter, visible microbial colonies that appeared on the culture plates were counted, and the results were expressed as log10 CFU (colony-forming units) per gram of the samples before statistical analysis. 3.11 Chemical Analyses Samples of the experimental diets, FFM, OLM, and excreta were analysed for dry matter (DM), ash, crude fibre (CF), crude protein (CP), and ether extract (EE) following the methods described by the Association of Official Analytical Chemists (AOAC, 2000). The results were all expressed on percentage dry matter basis. 66 University of Ghana http://ugspace.ug.edu.gh 3.12 Statistical Analysis The data collected were all subjected to analysis of variance (ANOVA) and analysed as a completely randomised design (CRD) using the Genstat statistical software (12th edition, 2009). Mean values with probability less than 5% (p<0.05) were considered significant and separated using the Student Newman-Keuls (SNK) test. The weight of birds at the onset of the trial (d 8) were not statistically different (p>0.05). 67 University of Ghana http://ugspace.ug.edu.gh CHAPTER FOUR 4.0 RESULTS 4.1 Nutrient Composition of FFM and OLM The results of the proximate analysis performed on samples of FFM and OLM to ascertain their nutritive value are presented in Table 4.1. Table 4. 1: Nutrient composition of FFM and OLM Nutrient (% DM) FFM OLM Dry matter 89.40 92.22 Crude protein 16.13 15.03 Ash 6.95 8.75 Fat 23.76 6.25 Crude fibre 25.23 31.98 ME (Kcal/kg) 2924 1306 FFM = Fagara zanthoxyloides fruit meal; OLM = Ocimum americanum leaf meal; ME= Metabolisable Energy 68 University of Ghana http://ugspace.ug.edu.gh 4.2 Effects of Dietary Treatments on Growth Performance 4.2.1 Effects of Dietary Treatments on Body Weight Shown in Table 4.2 are the effects of the dietary treatments on body weight. Compared with the negative control group (BD), there were no significant differences in body weight throughout the experiment. However, at the end of the first week, birds fed 0.2FFM were heavier (p<0.05) than that fed PEN. Also, in week 3, the weight of birds fed 0.2FFM was higher than those fed 0.2OLM and 0.1FFM+0.1OLM. Birds in the latter groups however recorded similar (p>0.05) weight. Table 4. 2: Body weight of broilers fed diets supplemented with FFM, OLM and penicillin Experi Dietary treatment means mental BD 0.2FF 0.4FFM 0.2OL 0.4OL 0.1FFM+ 0.2FFM+ PEN SEM p- week M M M 0.1OLM 0.2OLM value 0 156.7 162.3 155.7 153.3 154.8 155.9 156.4 152.2 4.25 0.448 1 409.2ab 427.8a 397.8ab 394.2ab 409.8ab 391.4ab 393.2ab 382.5b 11.99 0.024 2 838.6 864.6 814.5 801.1 826.3 795.4 828.2 811.9 21.45 0.072 3 1272.4 ab 1316.4a 1243.3ab 1193.8b 1245.8ab 1194.6b 1265.8ab 1252.8ab 37.09 0.049 4 1722 1772 1680 1662 1693 1649 1709 1705 53.0 0.426 5 2220 2219 2152 2079 2203 2136 2181 2130 71.3 0.472 6 2597 2516 2543 2500 2633 2510 2553 2434 100.1 0.633 a,b Means on the same row bearing similar superscripts are not significantly different (p>0.05) SEM = Standard error of means 69 University of Ghana http://ugspace.ug.edu.gh 4.2.2 Effects of Dietary Treatments on Average Daily Weight Gain Dietary supplementation with FFM, OLM, and penicillin did not affect (p>0.05) average daily weight gain (ADG) throughout the trial in comparison with birds fed the basal diet (BD). However, only in week 1, birds fed 0.2FFM gained more weight on daily basis than those fed 0.1FFM+0.1OLM, 0.2FFM+0.2OLM and PEN. Thereafter, ADG remained uniform (p>0.05) among the eight dietary treatment groups, as depicted in Table 4.3. Table 4. 3: Average daily weight gain (g) of broilers fed diets supplemented with FFM, OLM and penicillin Experi Dietary treatment means mental BD 0.2FFM 0.4FF 0.2OL 0.4OL 0.1FFM+ 0.2FFM+ PEN SEM p- week M M M 0.1OLM 0.2OLM value 1 36.07ab 37.93a 34.59ab 34.41ab 36.43ab 33.64b 33.83b 32.90b 1.320 0.012 2 61.34 62.40 59.52 58.13 59.50 57.71 62.14 61.34 1.927 0.140 3 61.97 64.54 61.26 56.09 59.93 57.03 62.51 62.99 3.082 0.123 4 64.3 65.1 62.3 66.9 64.0 64.9 63.3 64.6 4.01 0.976 5 65.7 63.9 67.5 59.6 72.8 69.5 67.4 60.8 5.07 0.207 6 53.8 42.4 55.8 60.1 61.5 53.5 53.1 46.5 7.69 0.252 a,b Means on the same row bearing similar superscripts are not significantly different (p>0.05). SEM = Standard error of means 70 University of Ghana http://ugspace.ug.edu.gh 4.2.3 Effects of Dietary Treatments on Average Daily Feed Intake The effects of dietary treatments on average daily feed intake are presented in Table 4.4. In comparison with the negative control group (BD), there were no differences (p>0.05) in average daily feed intake among the treatment groups. However, birds in the 0.2FFM group consumed more (p<0.05) feed than those in PEN in week 1. Table 4. 4: Average daily feed intake (g) of broilers fed diets supplemented with FFM, OLM and penicillin Experi Dietary treatment means mental BD 0.2FF 0.4FF 0.2OL 0.4OL 0.1FFM+ 0.2FFM+ PEN SEM p- week M M M M 0.1OLM 0.2OLM value 1 60.63ab 63.37a 61.60 ab 60.17 ab 59.66 ab 56.94 ab 56.11 ab 54.86b 2.384 0.017 2 123.5 131.6 125.3 124.9 122.6 120.2 119.4 124.3 4.57 0.271 3 133.0 133.1 137.5 128.2 128.4 135.4 128.3 132.9 5.72 0.632 4 148.4 153.8 150.2 146.4 145.5 144.1 145.3 144.3 7.35 0.882 5 160.5 169.6 159.4 160.2 167.0 155.3 167.3 156.3 7.34 0.410 6 148.3 160.5 160.4 159.6 162.0 158.3 162.9 152.8 9.92 0.819 a,b Means on the same row bearing similar superscripts are not significantly different (p>0.05). SEM = Standard error of means 71 University of Ghana http://ugspace.ug.edu.gh 4.2.4 Effects of Dietary Treatments on Feed Conversion Ratio Throughout the six-week experimental period, feed conversion ratio was unaffected (p>0.05) by treatment diets, as displayed in Table 4.5. Table 4. 5: Feed conversion ratio of broilers fed diets supplemented with FFM, OLM and penicillin Experi Dietary treatment means mental BD 0.2F 0.4FF 0.2O 0.4OL 0.1FFM+ 0.2FFM+ PEN SEM p-value week FM M LM M 0.1OLM 0.2OLM 1 1.68 1.68 1.78 1.75 1.64 1.70 1.66 1.67 0.069 0.468 2 2.02 2.11 2.11 2.16 2.06 2.09 1.92 2.04 0.106 0.492 3 2.16 2.06 2.25 2.31 2.15 2.38 2.07 2.11 0.143 0.293 4 2.32 2.41 2.42 2.22 2.28 2.23 2.31 2.24 0.171 0.883 5 2.31 2.75 2.38 2.74 2.31 2.26 2.51 2.58 0.249 0.314 6 2.83 4.7 2.98 2.70 2.64 3.11 3.13 3.68 0.708 0.128 All the means in this table are not significantly different (p>0.05). SEM = Standard error of means 4.3 Effects of Dietary Treatments on Mortality As presented in Table 4.6, dietary treatments did not affect (p<0.05) mortality throughout the study. Table 4. 6: Mortality (%) of broilers fed diets supplemented with FFM, OLM and penicillin Experi Dietary treatment means mental BD 0.2F 0.4FF 0.2O 0.4OL 0.1FFM+ 0.2FFM+ PEN SEM p-value week FM M LM M 0.1OLM 0.2OLM 1-6 2.00 4.00 10.00 8.00 2.00 4.00 2.00 8.00 3.761 0.224 The means in this table are not significantly different (p>0.05). SEM = Standard error of means 72 University of Ghana http://ugspace.ug.edu.gh 4.4 Effects of Dietary Treatments on Carcass and Organ Characteristics There were no significant differences (p>0.05) among the eight dietary treatment groups in terms of live weight before slaughter, as well as de-feathered, dressed, carcass part, abdominal fat (Table 4.7), and internal organ (Tables 4.8) weights. Table 4. 7: Carcass characteristics of broilers fed diets supplemented with FFM, OLM and penicillin Dietary treatment means Parameter (g) BD 0.2FF 0.4FF 0.2O 0.4O 0.1FFM+ 0.2FFM+ PEN SEM p-value M M LM LM 0.1OLM 0.2OLM Live w. 2652 2798 2616 2541 2768 2606 3012 2662 162.2 0.150 Defeathered w. 2470 2591 2439 2378 2547 2419 2778 2473 146.4 0.203 Dressed w. 2056 2127 2019 1960 2112 2002 2328 2081 135.8 0.252 Neck 99.4 98.4 92.6 94.6 99.0 110.6 113.0 103.4 7.33 0.100 Breast 791 863 775 758 804 703 881 749 73.7 0.296 Drumsticks 280.6 298.2 267.8 265.8 270.6 264.8 291.0 274.0 23.66 0.794 Thighs 307.0 312.0 299.2 308.0 314.6 316.0 344.2 328.0 24.07 0.686 Wings 225.0 215.6 242.0 199.6 221.4 237.8 247.0 216.6 16.07 0.101 Abdominal fat 11.6 17.8 20.0 17.6 14.2 14.2 18.8 17.8 6.49 0.903 All the means in this table are not significantly different (p>0.05). SEM = Standard error of means; w. = weight 73 University of Ghana http://ugspace.ug.edu.gh Table 4. 8: Internal organ characteristics of broilers fed diets supplemented with FFM, OLM and penicillin Dietary treatment means Parameter BD 0.2FF 0.4FF 0.2OL 0.4OL 0.1FFM+ 0.2FFM+ PEN SEM p- (g) M M M M 0.1OLM 0.2OLM value Proventriculus 8.40 10.00 9.40 9.40 10.40 10.20 11.40 9.40 1.24 0.428 F. gizzard 64.0 63.6 55.6 55.8 67.0 63.0 62.6 53.6 6.20 0.310 E. gizzard 43.0 45.8 40.4 40.6 42.2 43.2 44.4 36.8 4.31 0.568 Liver 36.0 39.6 36.8 34.0 41.8 36.4 42.2 35.8 3.35 0.173 Heart 8.80 9.00 8.40 8.60 10.80 9.80 9.20 9.40 1.02 0.362 F. intestine 75.4 89.6 75.6 76.8 85.2 89.0 91.8 69.2 8.48 0.101 E. intestine 53.8 66.2 55.6 54.4 60.4 65.0 66.8 52.2 5.91 0.078 All the means in this table are not statistically different (p>0.05) E. = Empty; F. = Full; SEM = Standard error of means 74 University of Ghana http://ugspace.ug.edu.gh 4.5 Effects of Dietary Treatments on Apparent Total Tract Nutrient Digestibility and Nitrogen Excretion The results for apparent total tract nutrient digestibility and nitrogen excretion are both presented in Table 4.9. Dry matter, crude protein, ash, and crude fibre digestibility, as well as nitrogen excretion, were not influenced (p>0.05) by the treatment diets. However, in terms of fat digestibility, there was a significant difference between the treatment groups and the negative control group. Table 4. 9: Apparent total tract nutrient digestibility and excreted nitrogen in broilers fed diets supplemented with FFM, OLM and penicillin Dietary treatment means Nutrient (%) BD 0.2FF 0.4FF 0.2OL 0.4OL 0.1FFM+ 0.2FFM+ PEN SEM p-value M M M M 0.1OLM 0.2OLM Dry matter 74.40 71.08 74.38 73.69 74.96 72.54 75.72 75.98 2.218 0.418 Crude protein 68.6 59.9 70.1 67.0 69.5 65.5 72.1 67.8 4.99 0.420 Fat 97.75a 62.59b 70.62b 67.99b 66.74b 64.12b 64.50b 72.25b 3.376 <.001 Ash 28.4 25.7 38.7 24.4 24.2 25.7 30.4 37.1 8.93 0.592 Crude fibre 38.3 47.8 49.8 43.4 48.0 39.2 50.2 51.3 5.10 0.133 Excreted N 31.4 40.1 29.9 33.0 30.5 34.5 27.9 32.2 4.99 0.420 a,b Means on the same row bearing different superscripts are significantly different (p<0.05). N = Nitrogen; SEM = Standard error of means 75 University of Ghana http://ugspace.ug.edu.gh The supplements (FFM, OLM and penicillin) caused a significant decrease (p<0.05) in fat digestibility to the same extent as shown in Figure 4.1. 120 a 100 80 b bb b b b b 60 40 20 0 Dietary treatments Figure 4. 1: Apparent total tract fat digestibility a,b Bars with similar letters are not statistically different (p>0.05) 76 Fat digestibility (%) BD 0.2FFM 0.4FFM 0.2OLM 0.4OLM 0.1FFM +0.1OLM 0.2FFM +0.2OLM PEN University of Ghana http://ugspace.ug.edu.gh 4.6 Effects of Dietary Treatments on Serum Lipid Profile The results of the serum lipid analysis in Table 4.10 reveal no effects (p>0.05) of the dietary treatments on the concentrations of cholesterol, low-density lipoprotein (LDL), high-density lipoprotein (HDL), and triglycerides. Table 4. 10: Serum lipid profile of broilers fed diets supplemented with FFM, OLM and penicillin Dietary treatment means Variable BD 0.2FF 0.4FF 0.2O 0.4O 0.1FFM+ 0.2FFM+ PEN SEM p- (mmol/L) M M LM LM 0.1OLM 0.2OLM value Cholesterol 3.82 4.13 2.47 3.75 3.75 3.75 3.65 3.97 0.500 0.086 HDL 1.102 1.170 0.652 1.206 1.104 1.114 1.042 1.156 0.1737 0.085 LDL 2.456 2.692 1.632 2.224 2.384 2.308 2.348 2.504 0.3174 0.103 Triglycerides 0.568 0.600 0.418 0.708 0.580 0.728 0.580 0.684 0.1613 0.622 All the means in this table are not significantly different (p>0.05). SEM = Standard error of means 77 University of Ghana http://ugspace.ug.edu.gh 4.7 Effects of Dietary Treatments on Faecal Microbial Composition The results for the faecal microbial analysis are presented in Table 4.11. Except for the counts of total microbes (microbial load) and yeasts and moulds, the counts of coliforms, Escherichia coli, as well as Salmonella and Shigella in faecal samples were not affected (p>0.05) by the dietary treatments. Table 4. 11: Faecal microbial composition of broilers fed diets supplemented with FFM, OLM and penicillin Dietary treatment means Parameter BD 0.2FF 0.4FF 0.2OL 0.4OL 0.1FFM+ 0.2FFM+ PEN SEM P-value (log10 M M M M 0.1OLM 0.2OLM CFU/g) Microbial 13.23a 10.65b 9.46b 10.24b 10.08b 9.46b 9.53b 9.11b 0.605 <.001 load Coliform 5.01 5.21 4.51 2.39 5.11 3.99 4.65 4.16 0.875 0.066 E. coli 5.07 1.59 1.35 1.74 1.34 1.70 1.64 2.12 1.221 0.083 SS 4.73 5.48 4.91 1.85 4.90 4.29 3.92 4.72 1.071 0.071 Y and M 8.28a 6.91a 4.79ab 7.10a 4.91ab 6.31a 5.86a 2.36b 1.184 0.001 a,b Means on the same row bearing different superscripts are significantly different (p<0.05). CFU = Colony-forming units; E. coli = Escherichia coli; SEM = Standard error of means; SS = Salmonella and Shigella; Y and M = Yeasts and Moulds 78 University of Ghana http://ugspace.ug.edu.gh FFM, OLM, and penicillin reduced (p<0.05) faecal microbial load to the same extent when compared with birds that received the basal diet (BD), as depicted in Figure 4.2. 14 a 12 b b b 10 b b b b 8 6 4 2 0 Dietary treatments Figure 4. 2: Faecal microbial load a,b Bars with similar letters are not statistically different (p>0.05) 79 Microbial load (CFU/g) BD 0.2FFM 0.4FFM 0.2OLM 0.4OLM 0.1FFM +0.1OLM 0.2FFM +0.2OLM PEN University of Ghana http://ugspace.ug.edu.gh Furthermore, birds fed the diet supplemented with penicillin (PEN) recorded the least faecal count of yeasts and moulds which was not different (p>0.05) from that of birds fed 0.4FFM and 0.4OLM. However, the counts of yeasts and moulds in faecal samples of the birds fed the remaining diets (BD, 0.2FFM, 0.2OLM, 0.1FFM+0.1OLM, and 0.2FFM+0.2OLM) was similar (p>0.05), as shown in Figure 4.3. 9 a 8 a a 7 a a 6 ab ab 5 4 3 b 2 1 0 Dietary treatments Figure 4. 3: Faecal count of yeasts and moulds a,b Bars with similar letters are not statistically different (p>0.05) 80 Yeasts and moulds count (log10 CFU) BD 0.2FFM 0.4FFM 0.2OLM 0.4OLM 0.1FFM +0.1OLM 0.2FFM +0.2OLM PEN University of Ghana http://ugspace.ug.edu.gh CHAPTER FIVE 5.0 DISCUSSION 5.1 Effects of FFM, OLM and Penicillin Supplementation on Growth Performance 5.1.1 Effects of penicillin supplementation on growth performance In the present study, supplementing diet with penicillin did not affect feed intake, body weight gain, and feed conversion ratio compared with the negative control group. This result is consistent with those of other researchers who supplemented broiler diets with lincomycin (Proudfoot et al., 1990) and avilamycin (Riyazi et al., 2015). Similarly, Baurhoo et al. (2009) reported no growth-promoting effect of bacitracin or virginiamycin in feed on broilers. In contrast, Onu et al. (2004) fed broilers a diet containing penicillin at the same inclusion rate tested in this study (i.e. 100ppm) and observed significant increases in feed intake, body weight gain, and feed conversion efficiency. Other scientists equally found significant improvement effects of antibiotic supplementation on broiler growth performance (Onifade, 1997; Guban et al., 2006). The absence of growth-promoting effects of penicillin noted in this study could be due to the low stocking density, high biosecurity, and good hygienic conditions under which the birds were raised. It is well established that birds that are well-nourished and reared in clean environments and at moderate stocking densities do not respond positively to antibiotic growth promoters (Prescott and Baggot, 1993; Anderson et al., 1999) and that, the growth-promoting effects of antibiotics are realized only when animals are raised under unhygienic conditions (Sahu and Saxena, 2014; Biswas et al., 2017). This implies that under hygienic rearing conditions, farmers do not need to add antibiotics to diets. 81 University of Ghana http://ugspace.ug.edu.gh Furthermore, antibiotics are well known to promote growth through their antibacterial effects on pathogenic gut microbes (Butaye et al., 2003). However, penicillin in this study did not affect any of the tested pathogenic microbes (coliforms, Escherichia coli, and Salmonella and Shigella). This can further explain why the birds fed the penicillin-supplemented diet did not show any improvement in growth. 5.1.2 Effects of FFM and OLM supplementation on growth performance FFM and OLM at all the inclusion rates tested in this experiment did not affect growth performance in comparison with birds fed the basal diet. This finding concurs with those of other researchers who admixed diets with PFAs (Hernandez et al., 2004; Mohammed and Yusuf, 2011; Issa and Omar, 2012; Ojelade et al., 2012; Riyazi et al., 2015). For example, Ojelade et al. (2012) supplemented broiler diets with Hibiscus sabdariffa (roselle) calyx meal and/or Ocimum gratissimum (African basil) leaf meal and found no effects on feed intake, body weight gain, and feed conversion ratio. Similar observations were made when broiler rations were supplemented with ginger powder (Mohammed and Yusuf, 2011), garlic powder (Issa and Omar, 2012), and sweet basil essential oil (Riyazi et al., 2015). Conversely, inclusion of Shatavari root powder (Rekhate et al., 2004), thyme or cinnamon essential oil (Al-Kassie, 2009), marjoram leaf meal (Ali, 2014), aridan fruit powder (Kana et al., 2017), and Ashanti pepper leaf or seed meal (Effiong and Ochagu, 2019) in broiler diets significantly increased growth performance. The inclusion of FFM and OLM in diets was expected to improve growth performance. The absence of growth-promoting effects could be due to the low dietary inclusion rates tested in this study. Yang et al. (2009) reported that the inclusion rates of phytogenic products determine the concentrations of their active compounds in diets and ultimately their effects on farm animals. Higher inclusion rates of FFM and OLM beyond 0.4% could have increased growth performance. Parallel to this assertion, 82 University of Ghana http://ugspace.ug.edu.gh Al-Kassie et al. (2011a) added a mixture of cumin and turmeric to broiler diet at 0.25% and observed no effect (p>0.05) on body weight gain. However, higher inclusion rates of the mixture (0.5, 0.75, and 1%) increased body weight gain significantly (Al-Kassie et al., 2011a). Similarly, increasing the supplementation rate of garlic powder in ration from 1 to 3% increased (p<0.05) body weight gain and final body weight of broilers (Mulugeta et al., 2019). Phytogenic feed additives also promote growth through their antimicrobial effects on pathogenic gut microbes (Al-Kassie, 2009; Goodarzi et al., 2014; Murugesan et al., 2015). As such, in clean rearing environments where pathogens are absent or at minimal levels, phytogenic feed additives do not influence growth, similar to antibiotics. In this research work, FFM and OLM did not affect the counts of the tested pathogenic microbes and also, the rearing environment was good. This may further explain the non-effect of FFM and OLM on growth performance. Furthermore, the failure of FFM and OLM to improve protein digestibility or reduce nitrogen excretion in this study may also explain their inability to promote growth. This is because phytogenic supplements that promote growth do so by increasing protein digestibility and decreasing nitrogen excretion (Abbas, 2010; Jahejo et al., 2019). Even though FFM and OLM did not promote growth, they had no negative effects on growth performance. Since FFM and OLM did not promote the growth performance of birds, there is no need to include them in diets at the levels tested in this study, especially when the rearing conditions are good. 5.1.3 Effects of penicillin, FFM and OLM supplementation on feed intake In this study, there was no influence of the antibiotic or herbal products on feed intake compared to the control group. Similar to this finding, other researchers observed no effects of dietary 83 University of Ghana http://ugspace.ug.edu.gh supplementation with avilamycin (Hernandez et al., 2004), bacitracin or virginiamycin (Baurhoo et al., 2009) on feed intake by broilers. These results contradict the findings of Onifade (1997) and Guban et al. (2006) who observed increased feed intake among birds fed diets supplemented with antibiotics. Similar to the finding of the present study, Avila-Ramos et al. (2012) and Goliomytis et al. (2014) found no effects of phytogenic feed additives on broiler feed intake. In contrast, some scientists observed increased feed intake among birds fed diets containing phytogenic products (Ragab et al., 2013; EL-Shoukary et al., 2014; Wan et al., 2017). Possibly, FFM and OLM at the tested dietary inclusion rates did not impact the taste, flavour, and palatability of the feeds to influence feed intake. Borneol is a compound present in some medicinal plants that stimulate appetite of farm animals (Mirzaei-Aghsaghali, 2012). It is present in aniseed (Al-Kassie, 2008), hot red pepper (Al-Kassie et al., 2011b), coriander (EL-Shoukary et al., 2014), as well as Ocimum americanum (Nascimento et al., 2011). Consequently, addition of aniseed (Al- Kassie, 2008), coriander seeds (EL-Shoukary et al., 2014), and hot red pepper (Al-Kassie et al., 2011b) to broiler diets increased feed consumption. However, in this experiment, OLM did not affect feed intake. It could be that due to the low dietary inclusion rates, the concentration of borneol in the feeds were not sufficient to improve feed intake. 5.2 Effects of Penicillin, FFM and OLM Supplementation on Carcass Parameters Dietary supplementation with penicillin did not affect any of the carcass variables measured in this study, in agreement with the findings of Baurhoo et al. (2009) who observed no effect of bacitracin or virginiamycin in broiler diet on the relative weights of drumsticks, thighs, whole breast, and wings. Still parallel with these observations, Bozkurt et al. (2008) supplemented broiler diet with avilamycin, and Attia et al. (2011) with flavomycin and found no impacts (p>0.05) on carcass variables. Contrarily, Onifade (1997) reported significant increases in some carcass parameters of 84 University of Ghana http://ugspace.ug.edu.gh broilers fed diets containing antibiotics. Also, antibiotics in poultry diets reduce intestine weight by shortening the gut and thinning the wall of the intestine (Gaskins et al., 2002). This report does not concur with the result of the present research since no difference (p>0.05) in intestine weight was observed between birds fed the penicillin-supplemented diet and the negative control group. The non-effect (p>0.05) of FFM and OLM on the weights of carcass cuts, internal organs, and abdominal fat conforms to the finding of Riyazi et al. (2015) who observed no significant effects of dietary supplementation with sweet basil essential oil on carcass, breast, thigh, liver, gizzard, heart, and abdominal fat weights of broilers. Hernandez et al. (2004) also found no effects of plant extracts in broiler rations on the relative weights of the liver, pancreas, proventriculus, ventriculus, and small and large intestines. Also, the inclusion of fenugreek, parsley, and sweet basil seeds in feeds for broilers did not affect any of the carcass variables measured by Abbas (2010). Still consistent with these findings, other researchers supplemented broiler diets with ginger root powder, ginger essential oil (Habibi et al., 2014), and onion extract (Aditya et al., 2017) and noticed no effects on slaughter characteristics. In contrast, Wan et al. (2017) supplemented diets of male broiler chickens with enzymatically-treated Artemisia annua whole plant and observed significant increases in carcass parameters. Similarly, Eltazi (2014) reported significant increases in the percentages of dressed carcass cuts and a decrease in abdominal fat of broilers fed diets containing a mixture of garlic and ginger powder. Likewise, dietary supplementation with 200ppm essential oil extract of either thyme or cinnamon decreased abdominal fat and increased dressing percentage as well as liver and gizzard weights of broilers (Al-Kassie, 2009). The similarity in carcass variables among the birds used in this study may be due to the similarity in their growth performance. As explained by Ferket and Gernat (2006), factors such as feed intake, feed conversion efficiency, and body weight gain of meat birds affect their carcass characteristics. 85 University of Ghana http://ugspace.ug.edu.gh 5.3 Effects of Penicillin, FFM and OLM Supplementation on Nutrient Digestibility In this experiment, dry matter, crude protein, ash, and crude fibre digestibility were not altered (p>0.05) by either the antibiotic or herbal products. This finding agrees with that of Jamroz et al. (2003) who observed no effects of avilamycin or a blend of carvacrol, capsaicin, and cinnamaldehyde in broiler diets on apparent ileal dry matter, crude ash, and nitrogen digestibility. Equally, dietary supplementation with flavomycin had no impacts on apparent total tract ash retention, and dry matter, crude protein, and crude fibre digestibility in broilers (Attia et al., 2011). Contrarily, Hernandez et al. (2004) observed improvements (p<0.05) in apparent whole tract dry matter and crude protein digestibility in finisher broiler chickens fed diets supplemented with avilamycin or plant extracts. Similarly, other researchers who supplemented broiler diets with bacitracin methylene disalicylate (Murugesan et al., 2015) and garlic powder (Issa and Omar, 2012) observed significant increases in total tract dry matter and crude protein digestibility. Likely, penicillin, FFM, and OLM did not affect the secretion of gastric and pancreatic juices or activity of the protein-digesting enzymes; chymotrypsin, elastase, pepsin, and trypsin. This could explain the similarity in protein digestibility between birds fed the supplemented and non- supplemented diet. The similarity in protein digestibility may partially explain the similarity in body weight. Also, the non-effect of the dietary treatments on fibre digestibility imply a similarity in intestinal transit time. An increase in fibre digestibility leads to a corresponding increase in intestinal transit time which makes animals consume more feed (Burrows et al., 1982). The similarity in fibre digestibility among the experimental birds may therefore explain the similarity in feed intake. The decline in fat digestibility observed among the birds fed the antibiotic and herb supplemented diets contradicts the findings of Guban et al. (2006) and Issa and Omar (2012) who observed better (p<0.05) fat digestibility among broilers fed diets supplemented with monensin and garlic powder 86 University of Ghana http://ugspace.ug.edu.gh respectively. Still contrary to the results of this study, other researchers who supplemented broiler diets with flavomycin (Attia et al., 2011) and plant extracts (Jamroz et al., 2003; Hernandez et al., 2004) found no effects on fat digestibility. The depression in fat digestibility caused by penicillin, FFM, and OLM may be due to inhibitory effects on the activity of pancreatic lipase and/or the synthesis, deconjugation, and release of bile salts to emulsify fats for lipid digestion and absorption. The tannins and saponins in FFM and OLM may be responsible for these effects, as inferred from the report of Serrano et al. (2009) who indicated that antinutritional factors can inhibit the activity of digestive enzymes and reduce nutrient availability for the host animal. This assertion contradicts the report of other scientists who indicated that some herbal feed additives improve bile flow, likewise the synthesis, secretion, and activity of pancreatic enzymes (Jang et al., 2007; Hashemipour et al., 2014). 5.4 Effects of Penicillin, FFM and OLM Supplementation on Nitrogen Excretion Some researchers indicated that antibiotics included in feed at growth-promoting levels increase nitrogen retention and utilization to form amino acids for growth enhancement and thus, reduce nitrogen excretion from animals (Wierup, 2001; Gaskins et al., 2002). Also, Jahejo et al. (2019) found that supplementing broiler diets with sweet basil seed powder significantly reduced (p<0.05) nitrogen excretion. Contrary to these reports, the relative amount of nitrogen excreted from the broilers used for this trial was not affected (p>0.05) by treatment diets. This could be due to the similarity in crude protein digestibility. Similar to the result of the current study, Jamroz et al. (2003) supplemented broiler diet with avilamycin or a blend of carvacrol, capsaicin, and cinnamaldehyde and observed no effects on nitrogen excretion. 87 University of Ghana http://ugspace.ug.edu.gh 5.5 Effects of Penicillin, FFM and OLM Supplementation on Serum Lipid Profile In the present investigation, neither did the antibiotic nor herbal products affect serum cholesterol, low-density lipoprotein (LDL), high-density lipoprotein (HDL), and triglycerides. Riyazi et al. (2015) reported a similar observation when broiler diet was supplemented with avilamycin or sweet basil essential oil. Likewise, Eslami et al. (2010) found no effects of antibiotic supplementation on the serum lipid profile of broilers. In agreement with these results, dietary inclusion of onion extract at the rates of 5, 7.5, and 10g/kg did not affect total cholesterol, high-density lipoprotein, and triglycerides in broiler chickens (Aditya et al., 2017). Contrarily, some researchers reported decreases in serum cholesterol, triglyceride, and/or LDL or an increase in HDL in birds fed diets containing phytogenic products (Kim et al., 2009; Prasad et al., 2009; Torki et al., 2018; Daramola, 2019). For example, Prasad et al. (2009) found that garlic supplementation significantly increased HDL and decreased cholesterol, triglyceride, LDL, and very-low-density lipoprotein (VLDL) in broilers. Similarly, supplementing diets of broilers with garlic bulb or garlic husk decreased serum cholesterol and LDL (Kim et al., 2009). Also, dietary supplementation with African basil leaf meal (Olumide et al., 2018), bitter leaf meal and/or moringa leaf meal (Daramola, 2019), and fenugreek, parsley, and sweet basil seeds (Abbas, 2010) significantly reduced blood cholesterol levels in broilers. The inconsistency in results may be due to differences in breed and age of birds, as well as dietary compositions. According to Toghyani et al. (2010), these factors influence the cholesterolemic effects of dietary components on farm animals. Fagara zanthoxyloides and Ocimum americanum extracts contain alkaloids, flavonoids, phenols, saponins, and tannins (Adesina, 2005; Anuradha, 2014). These compounds have hypocholesterolemic properties (Santoso and Fenita, 2017). Moringa leaves also contain alkaloids, 88 University of Ghana http://ugspace.ug.edu.gh flavonoids, and phenolic compounds (Moyo et al., 2012). Consequently, the addition of moringa leaf powder to broiler diets at 1.5 and 2% decreased (p<0.05) serum cholesterol and triglyceride levels (Mandal et al., 2014). However, FFM and OLM did not affect serum cholesterol and triglycerides. The low supplementation rates of both additives might have limited their ability to cause the lipid-lowering effect. Possibly, higher supplementation rates beyond 0.4% may have imparted the serum lipid profile positively. In line with this assertion, Nobakht and Moghaddam (2013) supplemented diets of laying chickens with 0.5, 1, 1.5, and 2% dried aerial parts powder of Tanacetum balsamita (costmary). In that study, only the highest inclusion rate of the herb lowered blood cholesterol. Likewise, moringa leaf powder at lower inclusion rates of 0.5 and 1% did not affect cholesterol and triglycerides but higher inclusion rates of 1.5 and 2%, significantly reduced the concentrations of both lipids (Mandal et al., 2014). Still in line with these results, hot red pepper at 0.25% in feed, did not affect blood cholesterol concentration in broilers but higher doses of 0.5, 0.75, and 1% reduced cholesterol significantly (p<0.05) (Al-Kassie et al., 2011b). Likewise, dietary supplementation with 0.5% Ocimum sanctum leaf powder did not affect serum cholesterol level but 1% lowered it (Lanjewar et al., 2009). The non-effect of FFM and OLM on serum cholesterol suggests no effect on meat cholesterol content, as inferred from the finding of Lanjewar et al. (2009) who observed a positive relationship between meat (breast and thigh muscles) and serum cholesterol level in broilers fed a diet supplemented with 1% Ocimum sanctum leaf powder. 89 University of Ghana http://ugspace.ug.edu.gh 5.6 Effects of FFM, OLM and Penicillin Supplementation on Faecal Microbial Composition 5.6.1 Effects of penicillin supplementation on faecal counts of the tested pathogenic microbes Antibiotic growth promoters decrease the populations of pathogenic bacteria in the gastrointestinal tract (Thomke and Elwinger, 1998; Gauer, 2004; Daramola, 2019). Concurrently, Jamroz et al. (2003) found that supplementing ration with avilamycin significantly reduced Escherichia coli and Clostridium perfringens counts in the recta of broilers. Similarly, Wati et al. (2015) observed that dietary supplementation with bacitracin methylene disalicylate effectively controlled Escherichia coli, Salmonella, and Clostridium in the caeca of broiler chickens. Likewise, virginiamycin in feed decreased ileal count of Escherichia coli in broilers (Darabighane et al., 2012). However, in this work, penicillin in diet did not affect the counts of the tested pathogenic bacteria strains (coliforms, Escherichia coli, and Salmonella and Shigella). This result concurs with the finding of Diarra et al. (2007) who supplemented broiler diet with bacitracin, bambermycin, penicillin, or salinomycin and observed no significant effects on Escherichia coli, Enterococcus, and Clostridium perfringens counts in litter, caecal and cloacal samples. Baurhoo et al. (2009) also found no effect of dietary supplementation with bacitracin or virginiamycin on caecal counts of Campylobacter and Escherichia coli in broilers on days 14 and 24 of the experiment. Possibly, the pathogens tested in the current study gained resistance and hence, were not eliminated. This assertion is not strange since, in some research trials, antibiotic-resistant bacteria have been isolated from poultry (Kolář et al., 2002; Diarra et al., 2007). 90 University of Ghana http://ugspace.ug.edu.gh 5.6.2 Effects of FFM and OLM supplementation on faecal counts of the tested pathogenic microbes The results of the faecal microbial analysis also reveal no effects of FFM and OLM on the counts of coliforms, Escherichia coli, as well as Salmonella and Shigella. This result agrees with the findings of Ahsan et al. (2018) who supplemented broiler diets with a commercial product containing extracts of anise, cinnamon, cumin, fennel (Foeniculum vulgare), garlic, and peppermint, and observed no effects on caecal counts of coliforms and Escherichia coli. Similarly, Murugesan et al. (2015) reported no significant effects of a blend of plant extracts in broiler diet on caecal counts of Pseudomonas aeruginosa and Staphylococcus aureus. Also, Mandal et al. (2014) found that dietary supplementation with moringa leaf powder at levels ranging from 0.5 to 1.5% did not affect coliform count in caeca content of broilers. Notwithstanding, some researchers reported significant decreases in counts of pathogenic gut microbes in birds fed diets containing phytogenic products (Khalaji et al., 2011; Goodarzi et al., 2014; Wati et al., 2015). For example, Wati et al. (2015) supplemented broiler ration with a phytogenic product containing extracts of anise, clove, melissa balm, oak, peppermint, and thyme, and found this product to be as effective as bacitracin methylene disalicylate in reducing caecal counts of Escherichia coli, Salmonella enteritidis, and Clostridium perfringens. Likewise, dietary supplementation with Artemisia sieberi leaves significantly reduced the number of coliforms and Escherichia coli in digesta samples collected from the caeca of broilers (Khalaji et al., 2011). In agreement with these findings, Goodarzi et al. (2014) observed a significant decrease in Escherichia coli population in the ilea of broilers fed diets supplemented with fresh onion bulb. The antibacterial properties of Fagara zanthoxyloides and Ocimum americanum extracts have been reported. For example, in some in-vitro studies, extracts of the fruits of Fagara zanthoxyloides 91 University of Ghana http://ugspace.ug.edu.gh inhibited the growth of Staphylococcus aureus, Salmonella enteritidis, Listeria monocytogenes (Gardini et al., 2009), Bacillus subtilis, Salmonella typhimurium, Streptococcus mutans, Micrococcus luteus, Pseudomonas aeruginosa, and Klebsiella pneumoniae (Misra et al., 2013). Ngassoum et al. (2003) also found the essential oil of dried Fagara zanthoxyloides fruits to be effective against Bacillus subtilis, Bacillus cereus, Corynebacterium glutamicum, Enterococcus faecalis, Escherichia coli, Streptococcus faecalis, and Staphylococcus aureus. These antibacterial effects are attributable to the geraniol and 3,4,5,7-tetrahydroxy-1-methoxy-10-methyl- 9-acridone contents of the fruits (Gardini et al., 2009; Wouatsa et al., 2013; Sado Kamdem et al., 2015). On the other hand, Anuradha (2014) observed excellent bactericidal effects of silver nanoparticles synthesized from aqueous Ocimum americanum leaf extract on Escherichia coli and Staphylococci aureus, which according to Gberikon et al. (2018) is caused by the cardiac glycosides and tannins contents of the leaves. The failure of FFM and OLM to lower the counts of the tested pathogens in this research work may be due to the low inclusion rates used in the feeds such that the concentrations of the antibacterial compounds were not sufficient to eliminate the bacteria from the gut. Perhaps, higher inclusion rates of FFM and OLM beyond 0.4% could have decreased the counts of the pathogens. To support this statement, Jamroz et al. (2003) observed that, at the inclusion rate of 150ppm of a mixture of capsaicin, carvacrol, and cinnamaldehyde in the ration of broilers, Clostridium perfringens population in rectal digesta samples was not affected in comparison with the control group. However, at a higher inclusion rate of 300ppm, the count of the pathogen was significantly reduced. Similarly, moringa leaf powder at inclusion rates of 0.5, 1.0, and 1.5% in broiler feeds did not affect caecal count of coliforms but at 2%, coliform count was significantly reduced (Mandal et al., 2014). 92 University of Ghana http://ugspace.ug.edu.gh Unlike the case of penicillin, it is not likely that the pathogens gained resistance to FFM and OLM. As explained in the literature, herbs are composed of a complex blend of phytochemicals with each having a different antimicrobial mode of action which makes it very difficult for bacteria to gain resistance to them (Suresh et al., 2018). Other authors equally reported that using herbs as feed additives results in little to no cases of bacteria resistance (Hashemi and Davoodi, 2011; Sethiya, 2016). 5.6.3 Effects of penicillin, FFM and OLM supplementation on faecal microbial load The decrease in faecal microbial load observed among the birds fed the penicillin, FFM and OLM supplemented diets in this study agrees with the finding of Samarasinghe et al. (2003) who reported a significant reduction in total viable microbes in the ilea of broilers fed diet supplemented with virginiamycin or turmeric root powder. Similar to this observation, Murugesan et al. (2015) recorded lower total caecal count of anaerobic bacteria in broilers fed diet containing a blend of plant extracts. These observations contradict the results of Mandal et al. (2014) who found no impact of dietary supplementation with moringa leaf powder (0.5, 1.0, and 1.5%) or bacitracin methylene disalicylate (0.02%) on caecal microbial population of broilers. Microbial load consists of pathogenic and non-pathogenic microorganisms (Diaz Carrasco et al., 2019). From the results, penicillin, FFM, and OLM did not affect faecal counts of the tested pathogenic microbes. The reduction in microbial load caused by penicillin, FFM, and OLM, therefore, implies that these supplements possibly inhibited beneficial bacteria in the gut. This validates the report of Hernandez et al. (2004) who indicated that plant extracts and antibiotics, in addition to inhibiting the growth and colonization of pathogenic gut bacteria, may inhibit the beneficial ones. In line with this report, Guban et al. (2006) fed broilers diets containing bacitracin methylene disalicylate and monensin alone or in combination and observed a lower ileal count of 93 University of Ghana http://ugspace.ug.edu.gh Lactobacillus salivarius. It has been explained that most antibiotics do not differentiate between beneficial and pathogenic bacteria and hence, may destroy beneficial ones in the gut (Henry Ford, 2020). Likewise, Anugom and Ofongo (2019) administered aqueous African basil leaf extract to broiler chickens and recorded significantly lower (p<0.05) counts of Lactobacillus in digesta samples collected from the crop, proventriculus, caecum, and ileum on day 35 of the experiment. These researchers concluded that the active compounds in the herb possibly have bactericidal effects also on beneficial microbes. In agreement with these results, Adefisoye et al. (2012) found that Fagara zanthoxyloides stem extracts displayed antibacterial activity against both pathogenic (Escherichia coli and Proteus vulgaris) and beneficial (Lactobacillus plantarum and Lactobacillus brevis) bacteria strains. However, other researchers observed higher counts of beneficial bacteria in chickens fed diets containing either phytogenic products or antibiotics. For instance, supplementing diet with fresh onion bulb or virginiamycin increased ileal Lactobacillus population in broilers (Goodarzi et al., 2014). Also, Darabighane et al. (2012) observed a higher ileal count of Lactobacillus in broiler chickens fed diets supplemented with Aloe vera gel. Similarly, amla fruit powder incorporated into broiler feed increased ileal Lactobacillus population (Dalal et al., 2018b). The increase in populations of beneficial gut bacteria in birds fed diets containing phytogenic products is due to the ability of some phytoconstituents to promote beneficial bacteria growth and proliferation (Windisch et al., 2008; Diaz-Sanchez et al., 2015; Suganya et al., 2016). Also, the bactericidal effects of antibiotics on pathogenic gut bacteria allow the beneficial ones to proliferate (Mehdi et al., 2018). This explains the increase in counts of beneficial gut bacteria in birds fed diets supplemented with antibiotics. 94 University of Ghana http://ugspace.ug.edu.gh 5.6.4 Effects of penicillin, FFM and OLM supplementation on faecal yeasts and moulds count The results of the faecal microbial examination reveal no effects of FFM and OLM on the count of yeasts and moulds in comparison with the negative control group (BD). However, Fagara zanthoxyloides and Ocimum americanum extracts have been shown to have antifungal properties. For example, Osho and Adelani (2012) demonstrated that aqueous and ethanolic extracts of chewing sticks made from Fagara zanthoxyloides inhibit the growth of Candida albicans, Candida krusei, and Candida tropicalis. Similarly, Gberikon et al. (2018) found Ocimum americanum leaf and flower extracts to have antifungal activity against Trichophyton mentagrophytes. In another study, silver nanoparticles synthesized from aqueous Ocimum americanum leaf extract inhibited the mycelia growth of Aspergillus niger (Anuradha, 2014). Fagara zanthoxyloides extracts are rich in flavonoids, saponins, and tannins (Adesina, 2005; Guendéhou et al., 2018). These compounds are toxic to fungi and inhibit their growth (Kim et al., 1995; Oyewole et al., 2004). In addition to flavonoids and tannins, Ocimum americanum leaves contain methyl chavicol also with antifungal properties (Anuradha, 2014). The inability of FFM and OLM to control yeasts and moulds implies that the concentrations of the anti-fungal compounds in them were not sufficient to inhibit the fungi possibly due to the low inclusion levels used in the diets. Higher inclusion rates of FFM and OLM beyond 0.4% could have decreased the yeasts and moulds count since at 0.2% FFM and 0.2% OLM, the fungi population was distinctly not affected (p>0.05) but at 0.4% FFM and 0.4% OLM, the fungi population tended to decrease (p>0.05). Only penicillin distinctly reduced (p<0.05) faecal yeasts and moulds count in this study compared to the non-supplemented group. This concurs with work done by Samarasinghe et al. (2003) who observed a significantly lower count of yeasts and moulds in the caeca of broilers fed a diet supplemented with virginiamycin. These findings disagree with the report of Mehdi et al. (2018) 95 University of Ghana http://ugspace.ug.edu.gh who indicated that antibiotics only control bacteria and not fungi or viruses. The ability of penicillin to lower the faecal yeasts and moulds population implies that it can control fungal diseases of poultry such as candidiasis, aspergillosis, cryptococcosis, dactlariosis, favus, histoplasmosis, mucormycoses, rhodotorulosis, and torulopsis which are known to cause economic losses to farmers (Dhama et al., 2013). 96 University of Ghana http://ugspace.ug.edu.gh CHAPTER SIX 6.0 CONCLUSIONS AND RECOMMENDATIONS 6.1 Conclusions The results of this study show that;  Supplementing broiler diets with FFM and OLM alone or in combination up to 0.4% does not affect growth performance, carcass characteristics, total tract dry matter, protein, ash, and fibre digestibility, nitrogen excretion, serum lipid profile, and load of pathogenic gut microbes.  Dietary supplementation with FFM, OLM, and penicillin reduces total tract fat digestibility and faecal microbial load in broilers.  FFM and OLM each at 0.4% function as an antimicrobial agent in broiler diets similar to antibiotics.  Feeding antimicrobial substances to birds raised in clean environments will have no impact on growth performance. 97 University of Ghana http://ugspace.ug.edu.gh 6.2 Recommendations  Higher dietary inclusion rates of FFM and OLM beyond 0.4% must be investigated to examine their effects on broiler growth performance, nutrient digestibility, and load of pathogenic gut microbes.  The effects of OLM and FFM on selected strains of beneficial gut microbes in poultry also need to be studied to be able to tell with certainty whether these herbs have bactericidal effects on the beneficial gut microbes.  There is a need to examine the flavouring effects of FFM and OLM on broiler meat to ascertain the acceptability of the meat by consumers.  The present study tested the efficacy of the fruits of Fagara zanthoxyloides to promote the growth of broilers. 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Poultry Science, 92(5): 1343-1347. 147 University of Ghana http://ugspace.ug.edu.gh APPENDICES Appendix A: Composition of vitamins Constituent Amount Unit Vitamin B1 7.20 mg Vitamin B2 2.74 mg Vitamin B3 14.40 mg Vitamin B5 7.20 mg Vitamin B6 3.60 mg Vitamin B7 0.57 μg Vitamin B9 0.02 mg Vitamin B12 7.20 μg Vitamin K3 10.00 mg Methionine 7.20 mg Choline chloride 11.00 mg 148 University of Ghana http://ugspace.ug.edu.gh Appendix B: Composition of broiler premix Constituent Amount Unit Vitamin A 12000000 IU Vitamin D3 2000000 IU Vitamin E 10000 mg Vitamin K3 1500 mg Vitamin B1 1500 mg Vitamin B2 4000 mg Vitamin B6 1500 mg Vitamin B12 15 mg Pantothenic acid 8000 mg Nicotinic acid 20000 mg Folic acid 500 mg Biotin 150 mg Choline chloride 120000 mg Iron 40000 mg Mn 60000 mg Cu 6000 mg Zn 50000 mg Iodate 2000 mg Selenium 150 mg Anti-oxidant (PHT) 25000 mg 149 University of Ghana http://ugspace.ug.edu.gh Appendix C: Agar media used for faecal microbial analysis Parameter Agar media used Product code Microbial load Plate Count (Tryptone Glucose Yeast Agar) Oxoid CMO325 Coliforms Violet Red Bile Agar (Not indicated) Escherichia coli Eosin Methylene Blue Agar M317-500G Salmonella and Shigella Salmonella and Shigella (SS) Agar Modified 610042 Yeast and molds Potato Dextrose Agar Oxoid CMO139 150 University of Ghana http://ugspace.ug.edu.gh Appendix D: Analysis of variance (ANOVA) tables 1. GROWTH PARAMETERS a. Variate: Body weight (Week 0) Source of variation d.f. s.s. m.s. v.r. F pr. TRT 7 317.74 45.39 1.01 0.448 REP 4 551.29 137.82 3.05 0.033 Residual 28 1263.91 45.14 Total 39 2132.94 b. Variate: Body weight (Week 1) Source of variation d.f. s.s. m.s. v.r. F pr. TRT 7 7067.8 1009.7 2.81 0.024 REP 4 4537.5 1134.4 3.16 0.029 Residual 28 10065.3 359.5 Total 39 21670.7 c. Variate: Body weight (Week 2) Source of variation d.f. s.s. m.s. v.r. F pr. TRT 7 17238. 2463. 2.14 0.072 REP 4 5886. 1471. 1.28 0.302 Residual 28 32213. 1150. Total 39 55337. d. Variate: Body weight (Week 3) Source of variation d.f. s.s. m.s. v.r. F pr. TRT 7 57177. 8168. 2.38 0.049 REP 4 13542. 3385. 0.98 0.432 Residual 28 96288. 3439. Total 39 167006. e. Variate: Body weight (Week 4) Source of variation d.f. s.s. m.s. v.r. F pr. TRT 7 51126. 7304. 1.04 0.426 REP 4 5452. 1363. 0.19 0.939 Residual 28 196497. 7018. Total 39 253075. f. Variate: Body weight (Week 5) Source of variation d.f. s.s. m.s. v.r. F pr. TRT 7 86244. 12321. 0.97 0.472 REP 4 10270. 2567. 0.20 0.935 Residual 28 355771. 12706. Total 39 452284. 151 University of Ghana http://ugspace.ug.edu.gh g. Variate: Body weight (Week 6) Source of variation d.f. s.s. m.s. v.r. F pr. TRT 7 131297. 18757. 0.75 0.633 REP 4 39853. 9963. 0.40 0.808 Residual 28 700963. 25034. Total 39 872113. h. Variate: ADG (Week 1) Source of variation d.f. s.s. m.s. v.r. F pr. TRT 7 99.450 14.207 3.26 0.012 REP 4 42.714 10.679 2.45 0.069 Residual 28 122.014 4.358 Total 39 264.178 i. Variate: ADG (Week 2) Source of variation d.f. s.s. m.s. v.r. F pr. TRT 7 113.142 16.163 1.74 0.140 REP 4 57.262 14.316 1.54 0.217 Residual 28 259.814 9.279 Total 39 430.218 j. Variate: ADG (Week 3) Source of variation d.f. s.s. m.s. v.r. F pr. TRT 7 302.29 43.18 1.82 0.123 REP 4 50.13 12.53 0.53 0.716 Residual 28 664.82 23.74 Total 39 1017.24 k. Variate: ADG (Week 4) Source of variation d.f. s.s. m.s. v.r. F pr. TRT 7 62.89 8.98 0.22 0.976 REP 4 170.67 42.67 1.06 0.393 Residual 28 1122.99 40.11 Total 39 1356.55 l. Variate: ADG (Week 5) Source of variation d.f. s.s. m.s. v.r. F pr. TRT 7 675.88 96.55 1.50 0.207 REP 4 280.00 70.00 1.09 0.380 Residual 28 1797.60 64.20 Total 39 2753.49 152 University of Ghana http://ugspace.ug.edu.gh m. Variate: ADG (Week 6) Source of variation d.f. s.s. m.s. v.r. F pr. TRT 7 1427.5 203.9 1.38 0.252 REP 4 339.2 84.8 0.57 0.684 Residual 28 4134.6 147.7 Total 39 5901.3 n. Variate: ADFI (Week 1) Source of variation d.f. s.s. m.s. v.r. F pr. TRT 7 299.11 42.73 3.01 0.017 REP 4 106.78 26.70 1.88 0.142 Residual 28 397.77 14.21 Total 39 803.67 o. Variate: ADFI (Week 2) Source of variation d.f. s.s. m.s. v.r. F pr. TRT 7 487.95 69.71 1.33 0.271 REP 4 458.34 114.58 2.19 0.095 Residual 28 1462.39 52.23 Total 39 2408.68 p. Variate: ADFI (Week 3) Source of variation d.f. s.s. m.s. v.r. F pr. TRT 7 429.77 61.40 0.75 0.632 REP 4 766.31 191.58 2.34 0.079 Residual 28 2290.65 81.81 Total 39 3486.73 q. Variate: ADFI (Week 4) Source of variation d.f. s.s. m.s. v.r. F pr. TRT 7 396.3 56.6 0.42 0.882 REP 4 829.9 207.5 1.53 0.219 Residual 28 3786.0 135.2 Total 39 5012.1 r. Variate: ADFI (Week 5) Source of variation d.f. s.s. m.s. v.r. F pr. TRT 7 1006.3 143.8 1.07 0.410 REP 4 577.8 144.4 1.07 0.389 Residual 28 3774.6 134.8 Total 39 5358.7 153 University of Ghana http://ugspace.ug.edu.gh s. Variate: ADFI (Week 6) Source of variation d.f. s.s. m.s. v.r. F pr. TRT 7 879.3 125.6 0.51 0.819 REP 4 669.0 167.3 0.68 0.612 Residual 28 6894.0 246.2 Total 39 8442.4 t. Variate: FCR (Week 1) Source of variation d.f. s.s. m.s. v.r. F pr. TRT 7 0.08105 0.01158 0.98 0.468 REP 4 0.00183 0.00046 0.04 0.997 Residual 28 0.33241 0.01187 Total 39 0.41529 u. Variate: FCR (Week 2) Source of variation d.f. s.s. m.s. v.r. F pr. TRT 7 0.18376 0.02625 0.94 0.492 REP 4 0.13843 0.03461 1.24 0.317 Residual 28 0.78117 0.02790 Total 39 1.10337 v. Variate: FCR (Week 3) Source of variation d.f. s.s. m.s. v.r. F pr. TRT 7 0.45730 0.06533 1.29 0.293 REP 4 0.08063 0.02016 0.40 0.809 Residual 28 1.42281 0.05081 Total 39 1.96074 w. Variate: FCR (Week 4) Source of variation d.f. s.s. m.s. v.r. F pr. TRT 7 0.21264 0.03038 0.42 0.883 REP 4 0.58731 0.14683 2.02 0.119 Residual 28 2.03945 0.07284 Total 39 2.83941 x. Variate: FCR (Week 5) Source of variation d.f. s.s. m.s. v.r. F pr. TRT 7 1.3473 0.1925 1.24 0.314 REP 4 1.1209 0.2802 1.81 0.156 Residual 28 4.3421 0.1551 Total 39 6.8103 154 University of Ghana http://ugspace.ug.edu.gh y. Variate: FCR (Week 6) Source of variation d.f. s.s. m.s. v.r. F pr. TRT 7 15.726 2.247 1.79 0.128 REP 4 3.553 0.888 0.71 0.592 Residual 28 35.049 1.252 Total 39 54.328 z. Mortality % (d 1-42) Source of variation d.f. s.s. m.s. v.r. F pr. TRT 7 360.00 51.43 1.45 0.224 REP 4 250.00 62.50 1.77 0.163 Residual 28 990.00 35.36 Total 39 1600.00 2. CARCASS PARAMETERS a. Abdominal fat weight Source of variation d.f. s.s. m.s. v.r. F pr. TRT 7 283.6 40.5 0.38 0.903 REP 4 728.2 182.1 1.73 0.172 Residual 28 2948.1 105.3 Total 39 3960.0 b. Breast weight Source of variation d.f. s.s. m.s. v.r. F pr. TRT 7 121532. 17362. 1.28 0.296 REP 4 20892. 5223. 0.38 0.818 Residual 28 379924. 13569. Total 39 522348. c. Defeathered weight Source of variation d.f. s.s. m.s. v.r. F pr. TRT 7 568215. 81174. 1.52 0.203 REP 4 321980. 80495. 1.50 0.228 Residual 28 1499612. 53558. Total 39 2389807. 155 University of Ghana http://ugspace.ug.edu.gh d. Dressed weight Source of variation d.f. s.s. m.s. v.r. F pr. TRT 7 445832. 63690. 1.38 0.252 REP 4 178217. 44554. 0.97 0.441 Residual 28 1290739. 46098. Total 39 1914788. e. Drumsticks weight Source of variation d.f. s.s. m.s. v.r. F pr. TRT 7 5330. 761. 0.54 0.794 REP 4 7868. 1967. 1.41 0.258 Residual 28 39178. 1399. Total 39 52376. f. Empty gizzard (ventriculus) weight Source of variation d.f. s.s. m.s. v.r. F pr. TRT 7 271.10 38.73 0.84 0.568 REP 4 244.15 61.04 1.32 0.288 Residual 28 1298.65 46.38 Total 39 1813.90 g. Full gizzard (ventriculus) weight Source of variation d.f. s.s. m.s. v.r. F pr. TRT 7 841.50 120.21 1.25 0.310 REP 4 551.35 137.84 1.43 0.249 Residual 28 2692.25 96.15 Total 39 4085.10 h. Heart weight Source of variation d.f. s.s. m.s. v.r. F pr. TRT 7 20.700 2.957 1.15 0.362 REP 4 4.750 1.188 0.46 0.763 Residual 28 72.050 2.573 Total 39 97.500 156 University of Ghana http://ugspace.ug.edu.gh i. Empty intestine weight Source of variation d.f. s.s. m.s. v.r. F pr. TRT 7 1279.60 182.80 2.09 0.078 REP 4 71.65 17.91 0.20 0.934 Residual 28 2447.15 87.40 Total 39 3798.40 j. Full intestine weight Source of variation d.f. s.s. m.s. v.r. F pr. TRT 7 2435.0 347.9 1.93 0.101 REP 4 297.7 74.4 0.41 0.797 Residual 28 5035.2 179.8 Total 39 7767.8 k. Legs weight Source of variation d.f. s.s. m.s. v.r. F pr. TRT 7 1359.6 194.2 0.68 0.689 REP 4 1329.8 332.5 1.16 0.349 Residual 28 8012.5 286.2 Total 39 10702.0 l. Live weight Source of variation d.f. s.s. m.s. v.r. F pr. TRT 7 781239 111606. 1.70 0.150 REP 4 379605 94901. 1.44 0.246 Residual 28 1841302 65761. Total 39 3002146 m. Liver weight Source of variation d.f. s.s. m.s. v.r. F pr. TRT 7 316.18 45.17 1.61 0.173 REP 4 31.15 7.79 0.28 0.890 Residual 28 784.45 28.02 Total 39 1131.78 157 University of Ghana http://ugspace.ug.edu.gh n. Neck weight Source of variation d.f. s.s. m.s. v.r. F pr. TRT 7 1828.2 261.2 1.94 0.100 REP 4 748.2 187.1 1.39 0.262 Residual 28 3760.9 134.3 Total 39 6337.4 o. Stomach (Proventriculus) weight Source of variation d.f. s.s. m.s. v.r. F pr. TRT 7 27.775 3.968 1.04 0.428 REP 4 10.900 2.725 0.71 0.590 Residual 28 107.100 3.825 Total 39 145.775 p. Thighs weight Source of variation d.f. s.s. m.s. v.r. F pr. TRT 7 6922 989. 0.68 0.686 REP 4 7338 1834. 1.27 0.307 Residual 28 40569 1449. Total 39 54828 q. Wings weight Source of variation d.f. s.s. m.s. v.r. F pr. TRT 7 8753.8 1250.5 1.94 0.101 REP 4 4470.0 1117.5 1.73 0.171 Residual 28 18079.6 645.7 Total 39 31303.4 3. APPARENT TOTAL TRACT NUTRIENT DIGESTIBILITY a. Ash digestibility Source of variation d.f. s.s. m.s. v.r. F pr. TRT 7 680.6 97.2 0.81 0.592 REP 2 306.2 153.1 1.28 0.309 Residual 14 1676.1 119.7 Total 23 2663.0 158 University of Ghana http://ugspace.ug.edu.gh b. Crude protein digestibility Source of variation d.f. s.s. m.s. v.r. F pr. TRT 7 285.10 40.73 1.09 0.420 REP 2 10.09 5.05 0.13 0.875 Residual 14 523.76 37.41 Total 23 818.95 c. Fat digestibility Source of variation d.f. s.s. m.s. v.r. F pr. TRT 7 2713.85 387.69 22.67 <.001 REP 2 10.16 5.08 0.30 0.748 Residual 14 239.40 17.10 Total 23 2963.41 d. Dry matter digestibility Source of variation d.f. s.s. m.s. v.r. F pr. TRT 7 56.443 8.063 1.09 0.418 REP 2 16.033 8.017 1.09 0.364 Residual 14 103.337 7.381 Total 23 175.813 e. Fibre digestibility Source of variation d.f. s.s. m.s. v.r. F pr. TRT 7 539.39 77.06 1.97 0.133 REP 2 134.20 67.10 1.72 0.215 Residual 14 547.20 39.09 Total 23 1220.80 4. EXCRETED NITROGEN Excreted nitrogen Source of variation d.f. s.s. m.s. v.r. F pr. TRT 7 285.10 40.73 1.09 0.420 REP 2 10.09 5.05 0.13 0.875 Residual 14 523.76 37.41 Total 23 818.95 159 University of Ghana http://ugspace.ug.edu.gh 5. SERUM LIPID PROFILE a. Cholesterol Source of variation d.f. s.s. m.s. v.r. F pr. TRT 7 8.8818 1.2688 2.03 0.086 REP 4 0.6522 0.1631 0.26 0.900 Residual 28 17.4967 0.6249 Total 39 27.0308 b. High-density lipoprotein Source of variation d.f. s.s. m.s. v.r. F pr. TRT 7 1.07746 0.15392 2.04 0.085 REP 4 0.02797 0.00699 0.09 0.984 Residual 28 2.11175 0.07542 Total 39 3.21718 c. Low-density lipoprotein Source of variation d.f. s.s. m.s. v.r. F pr. TRT 7 3.3915 0.4845 1.92 0.103 REP 4 0.5394 0.1349 0.54 0.711 Residual 28 7.0508 0.2518 Total 39 10.9817 d. Triglycerides Source of variation d.f. s.s. m.s. v.r. F pr. TRT 7 0.34754 0.04965 0.76 0.622 REP 4 0.09207 0.02302 0.35 0.839 Residual 28 1.82178 0.06506 Total 39 2.26138 6. FAECAL MICROBIAL COMPOSITION a. Coliform count Source of variation d.f. s.s. m.s. v.r. F pr. TRT 7 29.400 4.200 2.19 0.066 REP 4 7.300 1.825 0.95 0.449 Residual 28 53.637 1.916 Total 39 90.338 160 University of Ghana http://ugspace.ug.edu.gh b. Escherichia coli count Source of variation d.f. s.s. m.s. v.r. F pr. TRT 7 53.557 7.651 2.05 0.083 REP 4 46.938 11.734 3.15 0.029 Residual 28 104.289 3.725 Total 39 204.783 c. Microbial load Source of variation d.f. s.s. m.s. v.r. F pr. TRT 7 60.5070 8.6439 9.44 <.001 REP 4 18.0628 4.5157 4.93 0.004 Residual 28 25.6516 0.9161 Total 39 104.2213 d. Salmonella and Shigella count Source of variation d.f. s.s. m.s. v.r. F pr. TRT 7 43.034 6.148 2.14 0.071 REP 4 8.180 2.045 0.71 0.590 Residual 28 80.252 2.866 Total 39 131.466 e. Yeasts and moulds count Source of variation d.f. s.s. m.s. v.r. F pr. TRT 7 114.705 16.386 4.68 0.001 REP 4 26.427 6.607 1.89 0.141 Residual 28 98.073 3.503 Total 39 239.205 161