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Improving utilization of poultry feedstuffs with supplemental amino acids and enzymes Chong, Chen Hiung 2000

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I M P R O V I N G U T I L I Z A T I O N O F P O U L T R Y F E E D S T U F F S W I T H S U P P L E M E N T A L A M I N O A C I D S A N D E N Z Y M E S by C H E N H I U N G C H O N G B.Sc. (Agric), The University of Manitoba, Winnipeg, Manitoba, Canada, 1995 A THESIS SUBMITTED IN PARTIAL F U L F I L L M E N T OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE F A C U L T Y OF G R A D U A T E STUDIES Department o f A n i m a l S c i e n c e We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH C O L U M B I A November, 1999 © Chen Hiung Chong, 1999 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia Vancouver, Canada DE-6 (2/88) 11 Abstract Four studies were conducted to evaluate threonine and tryptophan requirements by poultry. A purified diet based on the ideal amino acid ( A A ) profile concept for 0-3 week old broilers was established in the first study. Different ratios of threonine and tryptophan to lysine were evaluated in the second study and the results indicated that dietary digestible threonine and tryptophan for 0-3 week old broilers should be targeted at 65% and 16% of digestible lysine, respectively. Studies using reduced protein (RP) diets based on practical ingredients indicated that dietary total threonine and tryptophan for 0-3 week or 3-6 week old broilers should be targeted at 0.74% (or 4.04% of crude protein (CP)) and 0.23% (or 1.22% of CP) or 0.67% (or 3.20% of CP) and 0.17% (or 0.89% of CP) , respectively. The corresponding values for 42-50 week-old layers are 448 and 152 mg/hen/d, respectively. Crystalline A A supplementation o f the R P diets improved the A A balance in the diet, thus improving protein utilization efficiency and resulting in reduced nitrogen in the excreta. The nutritive value of palm kernel cake ( P K C ) was investigated in several studies and it was found to contain low levels of metabolizable energy (ME) and moderate quantities of most nutrients. The low A A digestibility and the low M E of P K C were attributed to nutrient entrapment within the cell, lack o f appropriate enzymes to break down mannan, as wel l as processing method. A n enzyme mixture (mannanase, cc-galactosidase and protease) significantly increased the M E of P K C and improved the performance o f both broilers and layers fed PKC-based diets. The results indicated that a 20% P K C diet could be used during the broiler starter or grower phase, but not during both phases unless supplemental enzymes were used. Laying hen performance was not different from controls when a 12.5% or 25% PKC-based diet was used. However, layers consumed more o f the P K C diets and had poorer feed efficiency than the controls. Egg yolks were paler in birds fed the 25% P K C diets. A computerized feed formulation study showed that P K C is probably best suited for layer diets under the prevailing price situation in Malaysia. K e y words: ideal amino acid concept, threonine, tryptophan, palm kernel cake, enzymes. Ill Abstract ii Tables of Content iii List of Figures vi List of Tables viii Acknowledgements xiii Contributions to Knowledge xiv I. Introduction 1 References 3 II. Literature Review 5 Threonine and Tryptophan Requirements of Poultry 5 Threonine requirements o f poultry 5 Tryptophan requirements of poultry 10 Ideal Protein Concept 15 The Use of Amino Acid-Supplemented Reduced-Protein Poultry Diets 18 Reduced protein diets - Broiler studies 18 Reduced protein diets - Layer studies 21 Palm Kernel Cake 24 A n introduction to o i l palm 24 O i l palm in Malaysia 25 Nutrient composition of palm kernel cake 25 Non-starch polysaccharides in palm kernel cake 27 Palm kernel cake - Enzyme studies 29 Palm kernel cake - Animal studies 29 References 32 III. Use of Ideal Protein Concept to Investigate the Response of 0-3 Week Old Broiler Chicks to Different Levels of Threonine and Tryptophan in Chemically Defined Diets 42 Summary 42 Introduction 43 Materials & Methods 44 B i r d Management, Diet and Data Collection 44 Experiment 1 44 Experiment 2 46 Statistical Analyses 49 iv Experiment 1 49 Experiment 2 49 Results 49 Experiment 1 49 Experiment 2 51 Discussion 60 Conclusions '. 65 References 65 IV. Responses of Broilers and Layers to Threonine and Tryptophan Supplementation in Reduced Protein Diets 70 Summary 70 Introduction 71 Materials & Methods 72 Broiler Growth Study 73 Broiler Dietary Treatment 73 Broiler Balance Study 74 Layer Production Study 79 Layer Dietary Treatment 79 Layer Balance Study 80 Calculation 80 Analyses o f Samples 83 Statistical Analyses 83 Results 84 Broiler Growth Study 84 Broiler Balance Study 89 Layer Production Study 98 Layer Balance Study 98 Discussion 103 Conclusions 108 References 109 V. Evaluation and Enhancement of Palm Kernel Cake as a Poultry Feedstuff. ...114 Summary 114 Introduction 115 Materials & Methods 116 Collection and Analyses of Palm Kernel Cake Samples 116 Scanning Electron Microscopy of Palm Kernel and Palm Kernel Cake 117 Metabolizable Energy and Amino A c i d Digestibility o f Palm Kernel Cake for Poultry 118 Selection of Suitable Enzymes for Palm Kernel Cake Saccharification 119 Effect o f PKCase on the Digestibility o f Nutrients in Palm Kernel Cake 120 Calculation 121 V Statistical Analyses : 121 Results 122 Collection and Analyses o f Palm Kernel Cake Samples 122 Scanning Electron Microscopy o f Palm Kernel and Palm Kernel Cake 124 Metabolizable Energy and Amino A c i d Digestibility o f Palm Kernel Cake for Poultry 124 Selection of Suitable Enzymes for Palm Kernel Cake Saccharification 129 Effect o f PKCase on the Digestibility of Nutrients in Palm Kernel Cake 129 Discussion 136 Conclusions 143 References 144 VI. Effects of Dietary Inclusion of Palm Kernel Cake and Enzyme Supplementation on Broiler and Layer Performance 149 Summary 149 Introduction 150 Materials & Methods 151 Broiler Growth Study 151 Broiler Dietary Treatments ..152 Digestibility o f Broiler Diets 155 Layer Production Study 155 Layer Dietary Treatments 156 Digestibility o f Layer Diets 156 Economical Analyses o f Diets 159 Analyses of Samples 159 Statistical Analyses 159 Results 160 Broiler Study 160 Layer Study 166 Economical Analyses of Diets 179 Discussion 181 Conclusions 188 References 189 VII. General Conclusions 192 References 196 vi List of Figures Figure 3.1: Body weight o f 3 week old broilers (Experiment 2 ) : The effect o f different levels o f tryptophan (Trp) when threonine (Thr) was set at 90, 100 and 110% of the N R C (1994) recommendations 54 Figure 3.2: Body weight o f 3 week old broilers (Experiment 2 ) : The effect o f different levels o f threonine (Thr) when tryptophan (Trp) was set at 90, 100 and 110% of the N R C (1994) recommendations 55 Figure 3.3: Weight gain for week 2-3 (Experiment 2) : The effect o f different levels o f tryptophan (Trp) when threonine (Thr) was set at 90,100 and 110% of the N R C (1994) recommendations 56 Figure 3.4: Weight gain for week 2-3 (Experiment 2 ) : The effect o f different levels o f threonine (Thr) when tryptophan (Trp) was set at 90, 100 and 110% of the N R C (1994) recommendations 57 Figure 3.5: Weight gain for week 1-3 (Experiment 2 ) : The effect o f different levels of tryptophan (Trp) when threonine (Thr) was set at 90, 100 and 110% of the N R C (1994) recommendations 58 Figure 3.6: Weight gain for week 1-3 (Experiment 2 ) : The effect of different levels o f threonine (Thr) when tryptophan (Trp) was set at 90, 100 and 110% of the N R C (1994) recommendations 59 Figure 4.1: Broiler weight gain for 0-3 weeks : The effect o f different levels o f tryptophan (Trp) when threonine (Thr) was set at 83, 92 and 101% o f the N R C (1994) recommendations 91 Figure 4.2: Broiler weight gain for 0-3 weeks : The effect o f different levels o f threonine (Thr) when tryptophan (Trp) was set at 102, 113 and 125% of the N R C (1994) recommendations 92 Figure 5.1: Ce l l wal l structures (700 x, 15 k V ) of palm kernel (extracted with petroleum ether for 10 seconds) under a scanning electron microscope showing honeycomb-like palm kernel cell walls covered with palm kernel o i l 125 Figure 5.2: Ce l l wal l structures (100 x, 15 k V ) o f palm kernel (extracted with petroleum ether for 10 minutes) under a scanning electron microscope showing oil-free cell walls 126 Figure 5.3: Palm kernel cake under a scanning electron microscope (3,000 x, 15 k V ) 127 Figure 5.4: Relative mannanase activity o f Alltech PKCase at different temperatures 130 vii Figure 5.5: Relative mannanase activity o f Alltech PKCase at different p H values 131 Figure 6.1: The effects o f enzyme levels at different inclusion rates o f palm kernel cake ( P K C ) on egg weight 173 Figure 6.2: The effects of dietary inclusion o f palm kernel cake ( P K C ) at different enzyme levels on egg weight 174 Figure 6.3: The effects o f enzyme levels at different inclusion rates o f palm kernel cake (PKC) on layer's feed conversion ratio 175 Figure 6.4: The effects o f dietary inclusion o f palm kernel cake ( P K C ) at different enzyme levels on layer's feed conversion ratio 176 viii List of Tables Table 2.1: Ideal amino acid ratios and digestible amino acid requirements of broiler chickens at three growth periods as proposed by the University of Illinois 17 Table 2.2: Comparison among different amino acid profiles for the 0 to 21 days starter phase of broilers 17 Table 2.3: Crude protein and amino acid composition of palm kernel cake (PKC, % dry matter) 26 Table 2.4: Digestibility of palm kernel cake (PKC) amino acids ( %) 27 Table 3.1: Experiment 1 : Amino acid ratios proposed by different researchers 45 Table 3.2: Experiment 1: Composition of diets (% of diet) 45 Table 3.3: Experiment 2: Arrangement of diets 47 Table 3.4: Experiment 2 : Composition and calculated analysis (%) of diets 48 Table 3.5: Experiment 1 : The effect of different amino acid profiles on live body weight and body weight gain by 0-1 and 1-2 week old chicks 50 Table 3.6: Experiment 1 : The effect of different amino acid profiles on feed consumption and feed conversion efficiency by 0-1 and 1-2 weeks old chicks 50 Table 3.7: Experiment 1 : Performance of chicks fed different amino acid profile diets during the first two weeks of age 50 Table 3.8: Experiment 2 : Effect of different levels of threonine and tryptophan on body weight and weight gain by broilers during weeks 1-3 post-hatch 52 Table 3.9: Experiment 2 : Effect of different levels of threonine and tryptophan on feed intake and feed/gain ratio by broilers during weeks 1-3 post-hatch 53 Table 3.10: Suggested digestible and total amino acid requirements for 0-3 week old broiler chicks after adjustments were made to Blair et al. (1977) 64 Table 4.1: Factorial arrangement of broiler diets 74 Table 4.2: Arrangement of different dietary groups in the broiler study 74 Table 4.3: Composition of broiler starter diets (0-3 week) 75 ix Table 4.4: Composition of broiler grower diets (3-6 week) 77 Table 4.5: Factorial arrangement of layer diets 80 Table 4.6: Composition of layer diets 81 Table 4.7: The effect of different dietary treatments on the performance (body weight gain, feed intake and feed/gain ratio (F/G)) of 0-3 week old broilers 86 Table 4.8: The effect of different dietary treatments on the performance (body weight gain, feed intake and feed/gain ratio (F/G)) of 3-6 week old broilers 87 Table 4.9: The effect of different dietary treatments on the overall performance (body weight gain, feed intake and feed/gain ratio (F/G)) of broilers 88 Table 4.10: Factorial comparison: The effect of different levels of threonine and tryptophan and sex on 0-3 week and 3-6 week old broilers performance (body weight gain, feed intake, feed/gain ratio (F/G)) 90 Table 4.11: The effect of different dietary treatments on nitrogen (N) excretion and retention by 2-week-old broiler chicks 93 Table 4.12: Factorial comparison: The effect of different levels of threonine and tryptophan on nitrogen (N) excretion and retention by 2-week-old broiler chicks 95 Table 4.13: The effect of different dietary treatments on nitrogen (N) excretion and retention by 5-week-old broilers 96 Table 4.14: Factorial comparison: The effect of different levels of threonine and tryptophan on nitrogen (N) excretion and retention by 5-week-old broilers 97 Table 4.15: The effect of different dietary treatments on the performance of 42-50 week old laying hens 99 Table 4.16: Factorial comparison : The effect of different levels of threonine and tryptophan on 42-50 week old laying hen performance 100 Table 4.17: The effect of different dietary treatments on nitrogen (N) excretion and retention by 47-week-old laying hens 101 Table 4.18: Factorial comparison: The effect of different levels of threonine and tryptophan on nitrogen (N) excretion and retention by 47-week-old laying hens 102 Table 5.1: Nutrient composition of Malaysian palm kernel cake (DM basis) 122 X Table 5.2: Nitrogen, crude protein and amino acid contents o f palm kernel cake ( D M basis) 123 Table 5.3: The effect o f different amounts of o i l residue (ether extract) in palm kernel cake ( P K C ) on its metabolizable energy values ( D M basis ± S.D.) 127 Table 5.4: True amino acid digestibility (%) for samples o f palm kernel cake (Mean ± S.D.) 128 Table 5.5: Release of reducing sugars (expressed as mannose equivalent) from solvent-extracted palm kernel cake ( P K C ) after incubation with different Al l tech enzymes 132 Table 5.6: Effect o f incubating solvent-extracted palm kernel cake ( P K C ) with All tech PKCase under gastro-intestinal (GI) tract conditions 132 Table 5.7: The effect of incubating several palm kernel cake ( P K C ) samples with All tech PKCase 133 Table 5.8: The effect of incubating corn, soybean meal ( S B M ) and solvent-extracted palm kernel cake ( P K C ) with All tech PKCase 134 Table 5.9: Proximate analyses of untreated palm kernel cake ( P K C ) and PKCase-treated palm kernel cake 134 Table 5.10: The effect o f enzyme (PKCase) supplementation on nitrogen corrected true metabolizable energy (TMEn), true dry matter ( D M ) retention, neutral detergent fiber (NDF) and acid detergent fiber ( A D F ) retention 135 Table 5.11: Amino acid composition (% o f CP) o f palm kernel cake ( P K C ) , corn and soybean meal ( S B M , 44%CP) in relation to the N R C (1994) requirements for the growth of 0-3 week old broilers (90% D M ) 138 Table 5.12: Amino acid composition (% of CP) o f palm kernel cake ( P K C ) , corn and soybean meal ( S B M , 44%CP) in relation to the N R C (1994) requirements for the growth o f 3-6 week old broilers (90% D M ) 138 Table 5.13: Amino acid composition (% of CP) of palm kernel cake ( P K C ) , corn and soybean meal ( S B M , 44%CP) in relation to the N R C (1994) requirements for laying hens (90% D M ) 139 Table 6.1: Nutrient composition o f broiler starter (0-3 week) diets 153 Table 6.2: Nutrient composition o f broiler grower (3-6 week) diets 154 Table 6.3: Arrangement of dietary treatments for the broiler study 155 xi Table 6.4: Arrangement of dietary treatments for the layer study 156 Table 6.5: Nutrient composition of layer diets 157 Table 6.6: The effects of dietary inclusion of solvent-extracted palm kernel cake (PKC) and enzyme supplementation on live body weight (g), weight gain (g), feed intake (g) and feed conversion ratio (FCR) of female broilers at 3 weeks of age 161 Table 6.7: The effects of dietary inclusion of solvent-extracted palm kernel cake (PKC) and enzyme supplementation on live body weight (g), weight gain (g), feed intake (g) and feed conversion ratio (FCR) of female broilers at 6 weeks of age 161 Table 6.8: Effect of dietary inclusion of solvent-extracted palm kernel cake (PKC) and enzyme supplementation on female broilers overall performance (weight gain, feed intake and feed conversion ratio (FCR)) during the 6 week experimental period 163 Table 6.9: Factorial comparison: Effect of dietary inclusion of solvent-extracted palm kernel cake (PKC) at different phases and enzyme supplementation on supplementation on female broilers overall performance (weight gain, feed intake and feed conversion ratio (FCR)) during the 6 week experimental period 164 Table 6.10: The effect of dietary enzyme supplementation on true D M retention and apparent metabolizable energy (AME) and nitrogen corrected true metabolizable energy (TMEn) of broiler starter diets 165 Table 6.11: The effect of dietary enzyme supplementation on true DM retention and apparent metabolizable energy (AME) and nitrogen corrected true metabolizable energy (TMEn) of broiler grower diets 165 Table 6.12: Effect of dietary inclusion of solvent-extracted palm kernel cake (PKC) and enzyme supplementation on broiler abdominal fat deposition at week 6 167 Table 6.13: Factorial comparison: Effect of dietary inclusion of solvent-extracted palm kernel cake (PKC) at different phases and enzyme supplementation on broiler abdominal fat deposition at week 6 168 Table 6.14: The effect of broiler grower diets on six-week-old broiler's rectal temperature 168 Table 6.15: Chi-square analysis: Comparing the effect of different broiler grower diets on broiler mortality due to heat stress during 3-6 weeks of age 169 x i i Table 6.16: The effects of dietary inclusion of solvent-extracted palm kernel cake (PKC) and enzyme supplementation on the mean performance of laying hens over 8 weeks 170 Table 6.17: Factorial comparison: The effects of dietary inclusion of solvent-extracted palm kernel cake (PKC) and enzyme supplementation on the mean performance of laying hen over 8 weeks 172 Table 6.18: The effects of dietary inclusion of solvent-extracted palm kernel cake (PKC) and enzyme supplementation on the mean egg quality over 8 weeks from 28 weeks of age 177 Table 6.19: Factorial comparison: The effects of dietary inclusion of solvent-extracted palm kernel cake (PKC) and enzyme supplementation on the mean egg quality over 8 weeks from 28 weeks of age 177 Table 6.20: Apparent and nitrogen corrected true metabolizable energy ( A M E and TMEn) and true dry matter retention of layer diets 178 Table 6.21: Factorial comparison: Apparent and nitrogen corrected true metabolizable energy ( A M E and TME„) and true dry matter retention of layer diets 179 Table 6.22: The relative costs of apparent metabolizable energy (AME) and protein (CP) in corn, soybean meal (SBM), solvent-extracted palm kernel cake (PKC), P K C + lkg/t PKCase (PKC + IE) and palm oil 180 Table 6.23: Evaluation of the effect of the price of palm kernel cake (PKC) and P K C + lkg/t PKCase (PKC + IE) on its inclusion rate in low and high apparent metabolizable energy (AME) broiler (starter and grower) and layer diets by parametric linear programming 181 Xl l l Acknowledgements The author would like to express his sincerest thanks and gratitude to his supervisor Dr. R. Bla ir for his advice, encouragement, patience, and support throughout the course o f this study. The author is also deeply indebted to his co-supervisor at the Universiti Putra Malaysia ( U P M ) Dr. Z . Idrus for his advice, criticism, enthusiasm and tireless efforts during the two years the author spent in Malaysia as an international graduate exchange student. Thanks are also extended to Dr. K . M . Cheng, Dr. J . A . Shelford, Dr . J . R. Thompson and Dr. S. Hossain for their advice, criticism and invaluable help throughout the course o f this study and writing o f this thesis. The author would like to express his gratitude to Tan Sri Dr. S. Jalaludin, the vice-chancellor o f U P M , to Dr. G . Kennedy, the director o f International Programs at The University o f British Columbia ( U B C ) , to Dr. Soekartawi, the deputy director o f S E A M E O - S E A R C A and to Dr. Z . M a n , the Head of the Department o f A n i m a l Science at U P M for making the study at U P M possible. The help from staff working at South Campus of U B C , T. Cathcart, J . Kurtu, C. Nichols, C. Shingera, and help from M r . Mazlan and M r . Ponnusamy at the poultry research farm o f U P M during the experiments are gratefully acknowledged. The author also wishes to thank Dr. S. Ibrahim, P. Wang, G . Galzy, S. Leung, S. Kumarasingham, L . Aquino, S. Motani and D . Bailey at the Department of A n i m a l Science for their help. A special thanks goes to Dr. Che Norma for her friendship and invaluable assistance at U P M . The enzymes supplied by Alltech Inc., U S A and the amino acid analysis provided by Rhone Poulenc A n i m a l Nutrition is very much appreciated. The author wishes to thank Dr. Wi lson Henderson, The University o f British Columbia, the Universiti Putra Malaysia, Canada-British Columbia Green Plan and Southeast Asian University Consortium for Graduate Education i n Agriculture and Natural Resources for funding the present study and financial assistance during the study. Finally, the author is deeply indebted to his parents, brother, sister and his fiancee Leonnie C h i n for their encouragement, confidence, patience and support during his study in Canada. xiv Contributions to Knowledge Two different approaches were used to improve the utilization of poultry feedstuffs, first, with the use of supplemental crystalline amino acids. The responses of broilers and layers to different levels of dietary threonine and tryptophan were studied in an attempt to better understand the requirement of birds for threonine and tryptophan. Secondly, supplemental enzymes were used. The nutritive quality of palm kernel cake (an agricultural by-product) and the potential of enzyme supplementation to improve its nutritive value for poultry were investigated in several studies. New information was obtained as follow: 1. The requirements of 0-3 week old broilers for threonine and tryptophan were estimated. The broiler study indicated that dietary threonine and tryptophan for 0-3 week old broilers should be targeted at 0.74% of the diet (equal to 65% of digestible lysine or 4.04% of CP) and 0.23% of the diet (equal to 16% of digestible lysine or 1.22% of CP), respectively. 2. The requirements of 3-6 week old broilers for threonine and tryptophan were estimated. The broiler study indicated that dietary threonine and tryptophan for 3-6 week old broilers should be targeted at 0.67% of the diet (or 3.40% of CP) and 0.17% of the diet (or 0.89% of CP), respectively. 3. As indicated by the layer study, the daily requirements of 42-50 week old laying hens for threonine and tryptophan were estimated to be 448 mg threonine/hen and 152 mg tryptophan/hen, respectively. 4. This new information on the requirements of poultry for threonine and tryptophan is valuable to poultry nutritionists who want to supplement crystalline threonine and tryptophan in poultry diets. 5. The efficiency of nitrogen utilization was improved in the reduced protein diets with improved amino acid balance and resulted in a 25% to 46% reduction in nitrogen content in the excreta. The use of reduced protein diets could be an option for the poultry producer who wants to reduce the nitrogen content in the excreta. 6. A purified diet based on an amino acid profile proposed by Blair et al. (1977) could support better growth than diets based on the amino acid profiles proposed by the National Research Council for poultry (1994) and The University of Illinois. This amino acid profile could be XV used by other researchers or poultry nutritionists for the development o f an ideal amino acid profile for broilers. 7. Nutrient profiles (proximate constituents, metabolizable energy, true amino acid digestibility) o f several palm kernel cakes were determined. These profiles are valuable to the Malaysian feed industry since data reported on palm kernel cake are limited. 8. A n enzyme mixture with mannanase, protease and ct-galactosidase activity was found to be able to break down the non-starch polysaccharides of palm kernel cake resulting in an increase in apparent metabolizable energy and nitrogen-corrected true metabolizable energy. 9. Dietary inclusion o f 20% palm kernel cake either during the starter or grower phase did not reduce broiler performance at the 6 t h week. However, dietary inclusion of 20% palm kernel cake during both the starter and grower phases is not recommended for broilers unless supplemental enzymes are used. 10. When 20% palm kernel cake grower diets without enzyme supplementation were fed, broilers were found to be more susceptible to heat stress under hot tropical climates. Poultry producers should not include high levels of palm kernel cake in the broiler diet during hot summers. 11. Laying performance of birds was not adversely affected when laying hens were fed either 12.5% or 25% palm kernel cake diets without enzyme supplementation. However, layers consumed significantly more palm kernel cake-based diets and had a poorer feed conversion ratio than layers fed the corn-soybean meal control diets. 12. Egg yolk color became significantly paler for layers fed the 25% palm kernel cake diets. Unless supplemented with synthetic pigmenting agents, the dietary level o f palm kernel cake should be limited to 12.5% in countries where customers demand a darker color yolk. 13. Enzyme supplementation improved the performance o f broilers and layers fed palm kernel cake-based diets. The use o f enzymes in palm kernel cake-based diets remains promising. 1 CHAPTER I I n t r o d u c t i o n It is well known that methionine and lysine are the first limiting amino acids (AA) in corn-soybean meal poultry diets. Supplementation with these A A contributes to increased utilization of dietary protein and improves broiler growth and feed conversion efficiency (Peisker, 1996). Currently, feedstuffs are combined to meet the bird's needs for the most limiting A A . This usually results in a higher than required dietary protein content due to the presence of A A in excess. For example, in a typical layer diet based on corn and soybean meal, the sulfur A A are at about 100% of requirement; however, the level of other A A varies from 125% to over 300% of requirement (Davis and Austic, 1994). Excess dietary A A are utilized inefficiently by animals because the A A are deaminated and the nitrogen is excreted primarily as uric acid in avian species (Murray et al. 1993). Furthermore, the excess A A are an expensive source of metabolizable energy, yielding about 4 kcal/kg (Baker, 1997). Hence, minimizing excess A A in a diet makes economic sense. Experiments with poultry have supported the hypothesis that diets formulated to minimize an excess of A A over the chicks' known requirements improve the efficiency of protein and energy utilization (Waldroup et al, 1976). In addition, research conducted at the University of British Columbia indicated that reduced protein diets significantly improve protein utilization in feedstuffs, leading to a reduction in the daily nitrogen excretion of broilers by more than 20% and of layers by more than 30% (Blair et al, 1999). The reduction in excretion of nitrogen by poultry is one of the most important benefits of improving protein utilization in their feeds, especially in areas with intensive animal production, where pollution of groundwater by nitrogen originating from animal manure is becoming an increasing environmental burden. With the use of commercial feed-grade lysine, methionine, threonine and tryptophan, diets with a reduced protein content which meets the bird's requirement for essential A A can now be formulated. However, before nutritionists can reduce the protein content of a feed by crystalline A A supplementation, it is necessary to have an adequate knowledge of the A A requirements of the animal and an understanding of the multitude of factors that affect the A A requirements for poultry. Considerable research has been conducted to determine the requirements of poultry for lysine and methionine. Unfortunately, the requirements of poultry for 2 threonine and tryptophan are not well established, especially for layers and broilers beyond 3 weeks o f age. The National Research Council ( N R C , 1994) recommended poultry requirement values for these two A A are derived from research conducted more than 10 years ago. It should be noted that the A A requirement of the 1998 broilers is not the same as the A A requirement o f the 1978 broilers because the 1998 broilers weigh nearly twice as much as the 1978 broilers o f the same age (Dudley-Cash, 1998). Therefore, accurate information on the quantitative requirements for threonine and tryptophan needs to be revised for various ages o f poultry i n order for nutritionists to make the best economical decisions in feed formulation. The cost o f feeding poultry for production o f eggs and meat is between 70% and 80% of the total cost of production. Corn, fish meal and soybean meal, which constitute the major portion o f poultry diets in most Asian countries, are imported and are relatively expensive. Several countries in the Asian region, including Malaysia, have expressed concern over their limited feed supplies, the increasing cost o f imported feeds, and the need to increase the use of alternative feedstuff's. Inadequate availability of energy and protein-rich ingredients is the major constraint for the growth o f the poultry industry in developing countries. Nutritionists are continually searching for alternative, unconventional agro-industrial by-products to include i n poultry diets. In Malaysia alone, large quantities o f different usable by-products and residues o f varying nutritive value are produced annually. However, overall utilization remains low when compared to the total availability due to limited appreciation o f the potential value of by-products, inadequate knowledge or technology in the usage of the by-products, inadequate transfer o f technology, problems of quality and consistency o f products, as wel l as their unsuitability for non-ruminant animals due to high fiber and/or low protein content. Since many by-products or wastes have substantial potential value as ingredients in livestock diets, their utilization may be economically worthwhile, especially in developing countries, where traditional and often more expensive imported feedstuffs could be replaced. Recycling, reprocessing, and use of enzymes to increase nutrient digestibility and availability offer the possibility o f putting industrial by-products to beneficial use, as opposed to traditional methods o f disposal and relocation o f the residues. O f the huge quantities o f agricultural by-products produced in Malaysia, palm kernel cake (also referred to as palm kernel meal), the by-product of palm o i l extraction, has the greatest potential as an animal feed. 3 The world population has been growing rapidly. It is estimated that 2000 years ago the population o f the world was about 300 mill ion. In 1998, the world's population stood at 5.9 bi l l ion and it is growing at 1.3 per cent per year - an annual net addition o f 78 mil l ion people (United Nations, 1998). Based on the conservative estimations by the United Nations (1998), the world population w i l l reach 8.9 bil l ion in 2050. With this increase in population, the same agricultural lands that have been used for over 2000 years w i l l have to produce much more food. However, unlike the ever growing population, the amount of cultivable land on earth w i l l not increase, and further increases in land area devoted to cereal grains, o i l seeds and legumes w i l l be difficult to achieve, especially under the justifiable pressures from environmental groups for sustainable agriculture and nature conservation. Recently, in an anniversary lecture presented at the 6 t h Asian Pacific Poultry Congress, Sheldon (1998) emphasized the importance o f developing alternative and cheaper poultry feed ingredients in order to avoid competition with those essential for human food. Coupled with this, we have seen signs o f global warming and unexpected climatic events such as El nino and La niria that might have a negative impact on crop production. Undoubtedly, improving the energy and protein utilization of alternative feedstuffs that do not compete as food for human use and improving utilization o f current feedstuffs w i l l conserve feed resources and ease the tension resulting from human-animal food competition. During the century that is about to end, the livestock industry has faced many challenges that need to be tackled before it moves into the new millennium. Before stepping into the next millennium, livestock producers need to be made aware that animal production in the future has to be friendly to the animals, the consumers and the environment. References Baker, D . H . , 1997. Pages 1-24 in: Ideal amino acid profiles for swine and poultry and their applications in feed formulation. Biokyowa Publishing Co. , St. Louis, M O . Blair , R. , J. P. Jacob, S. Ibrahim, and P. Wang, 1999. A quantitative assessment o f the use o f reduced protein diets supplemented with amino acids to improve nitrogen utilization and reduce nitrogen pollution from broilers and layers. J. App l . Poultry Res. 8:25-47. Davis, A . J. , and R. E . Austic, 1994. Dietary amino acid balance and metabolism o f the limiting amino acid. Pages 70-81 in: Proceedings of the Cornell Nutrition Conference for Feed Manufacturers. Rochester, N Y . 4 Dudley-Cash, W . A . , 1998. Digestible amino acid requirements revised and extended for broilers. Feedstuffs 70:11,15. Murray, R. K . , D . K . Granner, P. A . Mayes, and V . W . Rodwell , 1993. Harper's Biochemistry. 23 r d ed. Appleton and Lange, Norwalk, C T . National Research Council , 1994. Nutrient Requirements o f Poultry. 9 t h rev. ed. National Academy Press, Washington, D C . Peisker, M . , 1996. Lysine requirements and amino acid profile for broilers. Zootecnica Int. 78-82. Sheldon, B . L . , 1998. Poultry and poultry products as resources for human health and food in the 21 s t century. Pages 1-8 in: Proceedings of 6 t h Asian Pacific Poultry Congress, Nagoya, Japan. Waldroup, P. W. , R . J. Mitchel l , J. R. Payne, and K . R. Hazen, 1976. Performance o f chicks fed diets formulated to minimize excess levels o f essential amino acids. Poultry Sci . 55:243-253. United Nations, 1998. Revision of the world population: Estimates and Projections. www.popin.org/popl998/Lhtm. 5 CHAPTER II Literature Review Threonine and Tryptophan Requirements of Poultry In order to maximize the utilization of dietary amino acids ( A A ) , knowledge regarding poultry requirements for these A A is crucial. Considerable amounts o f research have been conducted to determine the requirements of poultry for lysine and methionine. After methionine and lysine, threonine, tryptophan, isoleucine and arginine are the next limiting A A in most poultry diets (Elliot, 1995). However, only limited amounts o f requirement data have been published for these essential A A . Wi th the increased availability o f commercial feed-grade threonine and tryptophan, a better understanding of the requirement o f these A A for poultry is essential. Furthermore, the National Research Council ( N R C , 1994) threonine requirement value for 0-3 week old broiler chicks was derived from research done in the 1980s whereas published reports dated from 1947 to 1988 were used to derive the tryptophan requirement value. In addition, not many published data were available for use by the N R C (1994) to derive its recommendations for threonine and tryptophan for 3-6 week old broilers and for laying hens. Wi th today's highly productive birds, it is not clear whether poultry nutritionists should still follow the N R C (1994) recommended requirements for threonine and tryptophan. A s a result, more studies are required to derive the threonine and tryptophan requirements for poultry. Threonine requirements of poultry A limited number of experiments related to threonine requirements for poultry were conducted in the early 1970s. D ' M e l l o and Lewis (1970) looked at the interactions between certain essential A A . The results o f their experiment on threonine-tryptophan interaction indicated that excess threonine markedly retarded the growth of broiler chicks, the extent of the inhibition being proportional to the amount of the surplus. These adverse effects, however, were reversed by appropriate supplementation of the diet with tryptophan. A t dietary concentrations o f 0.80, 1.30, 1.80 or 2.30% threonine, the tryptophan requirement was determined to be 0.17, 0.18, 0.19 and 0.20% of the diet, respectively. It is clear that as the concentration o f threonine is increased, there is a concomitant increase in the requirement for tryptophan. The reason for this 6 kind o f interdependent relationship is not clear. Perhaps these two A A share the same A A transport system within the intestinal gut, and excesses o f one reduce the absorption o f the other. This kind o f interaction has also been reported to occur in rats (Morrison and Harper, 1960; Florentino and Pearson, 1962). Using 18% crude protein (CP) corn, soybean meal and A A diets, Hewitt and Lewis (1972) showed that the threonine requirement for 7-21 day old broiler chicks was 0.53% of the diet, while Woodham and Deans (1975) found that 2-4 week old broiler chicks required 0.50 - 0.52% o f threonine in their diet. Even though the age o f the chicks in these two separate experiments was slightly different, the derived threonine requirements were quite similar. Thomas et al. (1986) conducted two experiments with male broilers ranging from 7 to 21 days o f age that were fed graded levels o f threonine which were added to a corn, peanut meal, and soybean meal based diet supplemented with crystalline A A . The optimum level o f threonine for weight gain and feed efficiency was found to range from 0.73% to 0.77% of the diet. The threonine requirement for the feed efficiency as determined by a regression equation after a second study was 0.73%. A further study by the same researchers (Thomas et al, 1987) confirmed that the threonine requirement for male broilers was 0.72%, while female broilers tended to need less (0.67%). Data presented by Robbins (1987) clearly showed that when expressed as a percent o f the diet, the estimated threonine requirements increased as the dietary C P increased. However, when expressed as a percent o f protein, the estimated threonine requirements remained constant relative to dietary protein content. Robbins (1987) concluded that the threonine requirement was 3.7% o f dietary protein for female chicks during the starter phase. I f this value is correct, then a 23% C P starter diet would need to contain 0.851% threonine, which is 6.4% higher than the 0.80% reported by the N R C (1994). The threonine requirement of starter male broilers was determined by feeding threonine deficient (0.59%) grain sorghum-soybean meal diets either supplemented with or without crystalline threonine (Smith and Waldroup, 1988a). The threonine requirement for maximum weight gain and feed efficiency was 0.68% and 0.79%, respectively. The requirement value for feed efficiency is in agreement with the N R C (1994) recommended value of 0.80% for 0-3 week old broilers. The University of Cornell studies showed that both the source and the level of protein might influence threonine requirements (Austic and Rangel-Lugo, 1989). The threonine requirement o f a commercial strain o f broilers fed a diet containing 20% C P was 0.67 - 0.74% o f the diet. However, when the C P of the diet was increased to 25%, the requirement for threonine 7 increased by 9 to 19% (i.e. to 0.75 - 0.81% and 0.81 - 0.88%, respectively) in the two experiments based on wheat, peanut meal and crystalline A A diets. The threonine requirement was 0.68 - 0.75% when chicks were fed 25% C P diets based on corn, soybean meal and crystalline A A . Austic and Rangel-Lugo (1989) suggested that the difference in threonine digestibility in the two experimental diets might explain the observed wide variation in threonine requirement. Recently, similar experiments were carried out by the same group o f researchers (Rangel-Lugo et al. 1994). Broiler chicks were used to evaluate the threonine requirement for weight gain and feed efficiency up to 2 weeks o f age using 20 and 25% C P wheat-peanut meal based diets. Threonine requirements for maximal weight gain in the 20 and 25% C P diets were 0.67 and 0.77%, respectively. These findings are in agreement with the conclusion made by Robbins (1987) that as dietary C P is increased the threonine requirement is also increased. In addition to these studies, the threonine requirement for weight gain and feed efficiency in 16 to 28 day old broilers receiving a 20% C P diet was 0.63 and 0.69%, respectively. Studies from Mexico (Morales-Barrera et al, 1992) indicated that in 21-22% C P starter diets the threonine requirement is between 0.81 and 0.84% of the diet. This is at variance with another report from Mexico (Moreno et al. 1993) which stated that 0.70% dietary threonine was required by 0 to 3 week old broilers fed 20-22% C P sorghum-soybean meal diets and feed efficiency was worst when the level reached 0.85%. From two experiments conducted by Holsheimer et al. (1994), it was concluded that in 16% C P maize-soybean meal diets supplemented with essential A A and non-essential A A , containing 13.31 M J M E / k g , an improvement in gain and feed efficiency was observed for both sexes o f broiler chicks, when the dietary threonine content was increased to 0.725% of the diet until 3 weeks of age, and to 0.632% of the diet for females to 4 weeks o f age. Two experiments were conducted by Koide and Ishibashi (1995) to confirm the effect o f age and A A levels on female broiler requirements for threonine. Data from these studies indicated that dietary threonine requirements expressed as a percentage o f diet and C P level for maximum body weight gain and feed efficiency decreased with age and had a tendency to increase with increased dietary A A levels. In one experiment containing 19.8% C P , threonine requirements for broilers 5-15 and 25-35 days o f age were 0.818% (4.13% CP) and 0.75% (3.79% CP) , respectively. In the other experiment, threonine requirements for birds fed a low A A level diet (15.35% CP) were 0.608% (3.96% CP) and 0.586% (3.81%), respectively, for birds 8-18 and 28-35 days o f age. O n the other hand, threonine requirements for birds fed the high A A level diet (16.19% CP) were 0.667% (4.12% CP) and 8 0.621% (3.84%), respectively, for birds 8-18 and 28-35 days o f age. In another study from Japan Yamazaki et al. (1997a) reported that the available threonine requirements for 1-3 week and 4-6 week old broiler chicks were 0.65% (3.46% CP) and 0.54% o f diet (3.18% C P ) , respectively. This was equal to 0.74% (3.94% CP) and 0.60% (3.53% CP) total threonine, respectively. Both values were much lower than those reported by the N R C (1994) for broilers o f a similar age. Even though broilers consume much more feed in the second phase o f the production cycle, knowledge o f the threonine requirement for birds aged beyond 3 weeks is limited. Thomas et al. (1992) conducted two studies to determine the threonine requirement for 3-6 week old broilers. A 20% C P , 3,212 kcal M E / k g corn-peanut meal basal diet with seven graded levels of L-threonine (0.53 - 0.77%) was used. For males, the performance o f the birds receiving 0.61% threonine was similar to those fed higher levels o f threonine. The data also showed that females required less threonine, that is, 0.57% o f the diet from 4-6 weeks of age. K i d d (1996) indicated that dietary C P in 21 to 42 day old broilers may be reduced from 20% to 16.8% provided that methionine, lysine and threonine are added to the low protein diet. Performance o f the birds was not significantly different between the two diets (0.66% and 0.78% of total threonine respectively, in the 16.8% and 20% C P diet). The use o f a low protein diet would greatly reduce nitrogen (N) output in the manure. However, as reported earlier, the low protein diet (16.8%) also caused the birds to deposit significantly more abdominal fat. In another study, commercial broilers were fed threonine-deficient experimental diets composed o f sorghum, peanut meal, corn and poultry meal from 30 to 42 days of age (Kidd and Kerr, 1997). The total threonine requirement was set at 0.70% o f the diet (3.66% CP) , as maximum weight gain and feed utilization efficiency was obtained at this level. However, it is interesting to note that birds fed a diet containing lower threonine concentrations (0.65% of the diet or 3.40% of CP) had similar growth performance to the control birds (0.76% threonine). Growth and feed/gain ratios were similar for birds fed either the 0.65 or 0.70% threonine diet. K i d d and Kerr (1997) also noted that a higher level o f threonine is required to maximize breast meat yield (0.76% of diet). Four experiments were conducted to determine the digestible threonine requirement o f broiler chickens during the periods of 3 to 6 and 6 to 8 weeks posthatching (Webel et al. 1996). Basal diets deficient in threonine (0.40% digestible threonine) were used. Threonine supplementation significantly improved the performance of birds in both age groups. Max imum feed efficiency was achieved at 0.61% and 0.52% digestible threonine respectively for 3-6 and 6-8 week old birds. Extrapolating these digestible threonine requirements to total requirements for 9 chicks consuming corn-soybean meal diets (threonine digestibility = 87%) resulted in estimates o f 0.70 and 0.60% of the diets for broiler chicks during these two periods, respectively. The 3-6 week requirement was identical to that calculated by the ideal ratio procedure (70% o f 1.0% lysine = 0.70% threonine) which was lower than 0.74% reported by the N R C (1994). More recently, Penz et al. (1997) reported that a concentration o f 0.70% threonine for males and 0.60% for females appeared adequate for birds fed a diet containing 3,200 kcal M E / k g and 20% C P . This is i n good agreement with Webel et al. (1996). B y multiplying the threonine digestibility coefficient in the N R C (1994) by the analyzed threonine content o f each o f the basal diet ingredients, the digestible threonine requirement obtained for male and female was 0.59% and 0.51%, respectively. The data on the threonine requirements for laying hens are very limited. Huyghebaert and Butler (1991) conducted an experiment with medium weight laying hens to determine their threonine requirement between 28-38 weeks o f age. The daily threonine requirement of an individual laying hen was estimated by direct methods to be 8.7 mg/g egg output plus 43.49 mg/kg body weight in this experiment. That is, the threonine requirement for a 2.0 kg laying hen producing 50 g egg mass daily was 521.98 mg/d. However, a study from Japan showed that the available threonine requirement for laying hens 32 to 42 weeks o f age was 329 mg/hen/day or 385 mg total threonine/hen/day (Yamazaki et al, 1997b). This study also showed that the performance o f the laying hens decreased as dietary threonine content increased beyond requirement. Another two recent studies were carried out in Japan using 29 to 30 week old layers (Ishibashi et al, 1998). Experimental diets contained five graded levels of threonine and were fed for 21 and 58 days in Experiments 1 and 2, respectively. Threonine requirements obtained in Experiment 1 were 453, 456, and 458 mg per hen per day for egg mass, feed efficiency, and plasma threonine concentration, respectively. This agreed with the results obtained in Experiment 2 (457, 467, and 462 mg). However, the threonine requirements expressed as percentages o f diet in Experiment 1 (0.425, 0.428, and 0.430%) were higher than those i n Experiment 2 (0.395, 0.404, and 0.400%) for egg mass, feed efficiency, and plasma threonine concentration, respectively. The authors suggested that differences in feed intake were responsible for this variation. The authors also observed that as the dietary threonine exceeded the requirement level, egg mass and feed efficiency decreased with increasing dietary threonine levels, the reasons for which remain to be clarified. Meanwhile, Coon (1998) also conducted two studies to determine the digestible A A requirements for 33 and 35 week old laying hens. Corn-10 soy-meat and bone meal diets were used in the studies. The estimated digestible threonine requirements for laying hens were found to be quite variable ranging from 430 to 560 mg/hen/day. In conclusion, the estimates o f threonine requirement for poultry are very variable. For 0-3 week old broiler chicks, they range from 0.50% to 0.851% of the diet or 2.78% to 3.70% of C P . They range from 0.57% to 0.78% o f the diet or 2.85% to 3.90% o f C P for older broiler chicks (4-6 weeks old). On the other hand, laying hens were reported to need from 385 mg to 560 mg/hen/day. It is also important to note that most of the research done on threonine requirements was performed on 0-3 week old broiler chicks. Data reported for broiler chicks beyond 3 weeks o f age and for pullets and laying hens were not wel l established. A s a result, one o f the main objectives o f studies reported in this thesis is to determine the responses of broilers and layers to different levels of threonine. Tryptophan requirements of poultry There is only a small number o f published research papers on the requirement for tryptophan o f growing chickens. The reasons for this are uncertain. Perhaps the lack o f research is due to the different analytical procedures employed to measure tryptophan. Unl ike other A A , the analysis o f tryptophan requires alkaline hydrolysis and a different type o f separating column. Most laboratories do not analyze for tryptophan. The availability o f crystalline tryptophan on the market has generated a lot of interest regarding the requirements o f poultry for tryptophan and the beneficial effect of supplementing diets with crystalline tryptophan. In 1971, Boomgaardt and Baker conducted an experiment using female broiler chicks to determine the tryptophan requirements o f growing chicks (8 to 14 day post-hatching) and whether dietary C P levels affected the requirement. A crystalline A A diet devoid of tryptophan was fed at 8.7, 11.6, 14.5, 17.4 and 20.3% protein. A t each o f these protein levels L-tryptophan was supplemented to provide total tryptophan levels o f 0.414, 0.621, 0.828, 1.034, 1.241 and 1.448% of C P . When the tryptophan requirement values were expressed as a percentage o f the diet, the requirement increased with dietary protein level. However, when expressed as a percentage o f C P , the requirements remained constant at 0.87% at all five protein levels. These data agreed with those reported earlier by Griminger et al. (1956). While Boomgaardt and Baker (1971) used purified diets to derive tryptophan requirement for broiler chicks, the A A 11 requirements o f growing chicks have been determined using a diet based on soybean meal and maize meal (Hewitt and Lewis, 1972). The diet contained 18% protein in which 14% of the protein was contributed by conventional ingredients and 4% was in the form o f free A A . The results indicated that male broiler chicks aged 7 to 21 days required no more than 0.17% of tryptophan in the diet. The sex of the birds used might explain the difference in requirement for tryptophan reported for this study and that o f Boomgaardt and Baker (1971). A similar study was conducted with broiler chicks between 14 and 28 days of age (Woodham and Deans, 1975) and it was found that chicks required less than 0.14% tryptophan in the diet, or 0.78% o f C P . In experiments described by Freeman (1979), the requirements o f male and female broiler chicks for available tryptophan from 0 to 56 days o f age were determined by using the diet-dilution technique. Requirements were estimated from the dose-response relationships between dietary tryptophan and growth performance at weekly intervals throughout the growing period. The available tryptophan requirements were 2.4 (males) and 2.2 (females) g/kg o f diet from 0-7 days of age, and 1.7 g/kg (both sex) from 7-35 and 35-56 days of age. Freeman (1979) also concluded that the absolute requirement of a chick for tryptophan increased with age and was significantly different for male and female birds. These requirement (available) values were much higher than in earlier reports when converted to total tryptophan basis. The reason for this is not clear. In another experiment conducted by Steinhart and Kirchgessner (1984), birds fed a diet containing 0.22% tryptophan gave the best results with regard to weight gain, feed intake and feed efficiency. However, the differences from the groups fed 0.19% and 0.25% tryptophan were not significant. Perhaps, as the tryptophan level exceeded the requirement, it caused an imbalance o f A A in these diets. Three experiments were conducted to determine the response of male broiler chicks to tryptophan supplementation (Smith and Waldroup, 1988b). Chicks from 7 to 18 or 7 to 20 days of age were fed either a L-tryptophan supplemented sorghum-soybean meal test diet or a control 23% C P corn soybean meal diet. N o significant changes in body weight gain were observed when dietary tryptophan level was increased beyond 0.16% of the diet. Even though there were no significant differences in body weight gain between birds fed the control and the test diets, birds fed the test diets were less efficient in feed utilization. The lower protein in the test diets (20% CP) might have been deficient in one or more A A , thus causing the birds to eat more. It was concluded that a diet with 0.16% tryptophan could fulfil a growing chick's requirement for tryptophan. 12 Researchers from the University o f Reading were interested in the effects o f protein concentration on responses to dietary tryptophan by 4 to 18 day old broiler chicks (Abebe and Morris , 1990). Eight protein concentrations (16% to 30%) were combined with five tryptophan ratios (7.5 to 13.5 g tryptophan/kg CP) to provide 40 mash diets for the study. The amounts of tryptophan required for maximum growth and feed efficiency were each linear functions of dietary protein concentration. It was concluded that a fixed ratio o f tryptophan to protein (12 g/kg C P or 1.2% CP) should be used in practical diet formulation, rather than a minimum dietary concentration of tryptophan. Similar experiments conducted by other researchers confirmed that the tryptophan requirement o f broiler chicks is proportional to the dietary C P content (Rogers and Pesti, 1990). Various levels of protein and tryptophan were fed to 8-21 day old chicks. Protein in the diets ranged from 16% to 28% while tryptophan ranged from 0.34% to 2.74% of protein. Max imum gain and feed efficiency were attained when the dietary levels o f tryptophan were 0.83, 0.77, 0.77, and 0.78 of the protein for 16, 20, 24, and 28% C P , respectively. This confirmed the results reported by Boomgaardt and Baker (1971) that as the C P o f the diet increases, the requirement for tryptophan expressed as a percentage o f C P remains constant. However, the requirement value reported (0.80% of CP) reported by Rogers and Pesti (1990) was much lower than that of Boomgaardt and Baker (1971). Han et al. (1991) carried out studies to determine requirements for available histidine and tryptophan in 8 to 22 day old chicks fed a histidine and tryptophan deficient intact protein diet containing 25% C P and 3,200 kcal ME„/kg. It was found that the requirement for digestible tryptophan was 0.20% of the diet (or 0.80% of CP) for maximal weight gain and feed efficiency. This indicates that a total tryptophan level of 0.22% in the diet is necessary for chicks fed a 23% C P corn soybean meal diet. Finally, K i m et al. (1997) showed that 1-3 week old broilers required only 71.56 mg/day or 0.173% of the diet (0.99% of CP) , 69.84 mg/day or 0.168% of the diet (0.97% of CP) based on the weight gain response and N gain response, respectively. This is much lower than that o f the N R C (1994) and Han etal. (1991). Hunchar and Thomas (1976) were one o f the few research groups that embarked on a study o f the requirement o f tryptophan for chicks beyond 3 weeks o f age. Basal diets containing approximately 25% C P and 3,300 kcal M E / k g were used in the studies. Based on the regression equations for males (females), the calculated tryptophan requirement for maximum growth, optimum feed efficiency, and molted body feather count for the 4-7 week period was 0.179 (0.173), 0.170 (0.163), and 0.172% (0.172%), respectively. Using a computer model, Hurwitz et 13 al. (1978) predicted that tryptophan requirements for broilers o f different ages were 0.141% of the diet for 21-28 day old, 0.134% of the diet for 28-35 day old, and 0.118% of the diet for 35-42 day old chicks. Therefore, the average for 21-42 days o f age was 0.131% of the diet. These estimated requirement values were much lower than those suggested by the N R C (1994). Furthermore these values (Hurwitz et al, 1978) have not been rigorously examined. Early research on the requirement o f laying hens for tryptophan was conducted by Ingram et al. (1951) using a corn-corn gluten meal diet. The study showed that laying hens did not require more that 0.15% tryptophan in their diets. Further studies revealed that 0.142% tryptophan was required to maintain egg size, body weight and egg production (Ingram and Little, 1958). Using a low protein diet, Bray (1969) found that the requirement o f laying hens for tryptophan was 0.110% of the diet or 117 mg/day per hen. Two experiments were conducted with laying pullets between 32 and 47 weeks of age (Morris and Wethli , 1978). A high protein summit diet was mixed with a non-protein mix to achieve different concentrations o f tryptophan. For a flock o f laying hens with a mean body weight o f 1.5 kg producing 55 g egg mass/hen/day and consuming 110 g o f feed per day, the optimum dietary tryptophan concentration was found to be 0.17%. According to Tasaki (1983), poultry diets containing maize as the main ingredient are commonly deficient in tryptophan. White Leghorn pullets producing about 85% o f eggs were selected and fed 16% C P maize-soybean meal diets with graded levels of tryptophan (0.066% -0.721% of diet). Laying performance was lower when diets contained less than 0.1% tryptophan. Birds fed high levels of tryptophan did not show any i l l effects and were able to maintain their body weight. The author suggested that 0.11% tryptophan as recommended by the Japan Feeding Standard was too low and that the 0.17% level as recommended by Morris and Wethli (1978) should be utilized. In some cases, the author argued that it might be advantageous to raise the tryptophan content in the diet to more than 0.20%. In order to determine the tryptophan requirement o f laying hens, Ishibashi (1985) used 15 month old White Leghorn hens to carry out four experiments. The experimental diets were designed to provide the same amount o f essential A A except for tryptophan, which ranged from 0.086 to 0.32% of the diets. The hens needed to consume 210, 212 and 212 mg o f tryptophan per day in order to reach maximum egg production rate, egg production and feed intake, respectively. This was equivalent to 0.189% of the diet. In contrast to the finding by Tasaki (1983), the performance of the hens was significantly poorer at a higher dietary tryptophan concentration (0.32%). The author did not give any explanation for 14 this observation. Another report from Japan showed that supplementing L-tryptophan at 250 or 500 mg/kg to a nutritionally complete diet improved egg production and feed utilization in crossbred laying hens (Ohtani et al. 1989). Dai ly tryptophan intakes were 173, 207 and 239 mg by layers fed the basal diet, 250- and 500-mg tryptophan diets, respectively. Although a daily intake o f 173 mg tryptophan was considered adequate by Morris and Wethli (1978) and Tasaki (1983), it was not supported by data reported by Ishibashi (1985) and Ohtani et al. (1989). Several experiments were conducted to estimate the tryptophan requirement o f laying hens (Jensen et al. 1990). In the first two experiments, hens were fed a 14% C P diet supplemented with four levels o f L-tryptophan. Five levels of L-tryptophan supplementation and three levels o f protein (14, 16 and 18%) were used in two additional experiments. Even though egg production was significantly improved by tryptophan supplementation in the first two experiments, production was inferior to that expected for commercial laying hens. Egg production was only significantly increased by tryptophan supplementation to the 14% C P diet in a third experiment. The derived requirement values were 0.137% (123 mg/day), 0.118% (95 mg/day) and 0.164% (168 mg/day) for Experiments 1, 2 and 3 respectively. In the fourth experiment, the requirement changed with the protein levels, calculation from the data showing that 0.923% of C P was required. Jensen et al. (1990) concluded that the requirement values in the first two experiments might have been underestimated and the diets in the last two experiments were formulated differently. A s with the case o f threonine, data describing the tryptophan requirement for poultry are very variable. Reported requirement values for 0-3 week old broilers range from 0.14% to 0.276% of the diet or 0.78% to 1.20% of C P . On the other hand, 4-6 week old broilers were found to require 0.163% to 0.179% tryptophan in the diet or 0.65% to 0.716% of C P . Data reported for laying hens ranges from 95 mg to 212 mg/hen/day. The interpretation o f past research is quite difficult because o f variations in C P levels, energy levels, digestible threonine and tryptophan levels in the basal diets, essential and non-essential A A levels, bird age, duration o f study, and environmental conditions. Certainly, more research should be carried out in the future in order to obtain a better understanding of the requirements o f poultry for threonine and tryptophan. A s a result, it is one o f the objectives of my study to measure the responses o f broilers and laying hens to different dietary levels of threonine and tryptophan. 15 Ideal Protein Concept Ideal protein is defined as a blend o f essential A A that meets an animal's requirement for protein accretion and maintenance exactly, with no excesses and no deficiencies. The ideal protein concept was originated and developed in the work of H . H . Mitchel l and H . M . Scott at the University o f Illinois during the late 1950s and early 1960s. Amino acid requirements depend on the needs for maintenance and production. A s maintenance needs only account for 3 to 6% for young birds (Emmert and Baker, 1997), the major difference between birds growing at different rates and between birds o f different breeds and sexes is in the amount of protein they require depending on their genetic potential for lean tissue accretion. However, the relative amounts of different essential A A needed for the deposition of 1 g of lean meat tissue should not be different in every case. Therefore, it should be possible to establish an optimum balance o f essential A A for growth which, when supplied with sufficient N for the synthesis o f non-essential A A , would constitute the ideal protein. The ideal protein concept uses lysine as a reference A A , with the requirements for all other essential A A expressed as a percentage o f lysine (weight:weight basis). According to the Illinois group (Baker, 1997), lysine was chosen for several reasons: 1) after sulfur A A , lysine is the second most limiting A A in practical poultry diets, 2) data for lysine requirement under a wide range o f conditions are readily available, 3) unlike the sulfur A A and tryptophan, the analysis o f lysine in feed ingredients is straight-forward and less complicated, 4) unlike the sulfur A A and tryptophan, lysine's sole function in the body is protein accretion and maintenance (only lysine o f endogenous origin (trimethyl lysine) is used for carnitine synthesis). According to Baker et al. (1996), three important factors must be considered when using the ideal protein concept in poultry feed formulation. First of al l , ideal ratios for poultry were based upon numerous studies done with purified A A diets. A l l o f the ideal A A ratios are based on digestible levels o f dietary A A , because this eliminated differences in digestion, absorption, and utilization o f protein o f various quality and sources. Obviously, digestibility o f A A in feed ingredients must be factored into formulation schemes that use the ideal protein concept. Second, the ideal A A profile for the starter phase (0 to 21 days) differs in some respects from the ideal profile for the grower phase (21 to 42 days) of broilers. Specifically, the ideal ratios of sulfur A A , threonine and tryptophan to lysine increase as birds age, because the maintenance requirement ratios for these A A exceed protein accretion requirement ratios. Finally, since lysine 16 is the reference A A , its requirement value is very important, because it is the basis for setting requirements for all other essential A A . Even though the concept o f the ideal protein was first elaborated i n the U S A , the best ideal A A ratios were not fully developed, and not much research was done in this area. A s a result, the concept was not widely used as far as practical animal feeding was concerned. It was not until 1981 that the ideal protein concept was revived and actually put into practice in swine formulation by the Agricultural Research Council (1981). For over 30 years, work has been done at the University of Illinois on proper A A ratios for broiler chicks. Most of this work, however, was done with chicks between 0 and 21 days o f age fed purified A A diets. Based upon calculations, the Illinois group has estimated the ideal A A ratios for both early and late growth o f broilers (Baker, 1997). They have also translated these into projected A A requirements for each growth phase (Table 2.1). The digestible lysine requirement for early growth is based upon the work o f Han and Baker (1991, 1993). For the grower phase (3 to 6 weeks), the lysine requirement o f Ross x Ross broiler chicks was found to be 0.89% and 0.84% of the diet (3,200 kcal A M E / k g ) for male and female chicks respectively (Han and Baker, 1994). Recently, two chick bioassays with chemically defined A A diets were conducted to compare the Illinois Ideal Chick Protein (IICP) A A profile with that o f the N R C (1984) and the N R C (1994) (Baker and Han, 1994). Birds fed the IICP A A profile diets had higher rates o f gain per unit o f essential A A N intake than those fed the N R C (1994) profiles. Since the diet based upon IICP ratios did not show any response to further increases in A A supplementation, the authors concluded that the ratios in IICP for the 0 to 21 day growth period are not underestimated. The authors also suggested that the N R C (1994) estimate of the total lysine requirement (i.e., 1.10% of the diet) is too low. If the total lysine requirement in the N R C (1994) had been set to 1.20% of the diet, then the modified N R C A A ratios would be very close to the IICP ratios (Table 2.2). Beside the ideal A A ratio proposed by the University of Illinois, there exist other suggested A A profiles. For example, i f one expressed the requirement values o f essential A A reported by Bla i r et al. (1977), the N R C (1994), and the Rhone-Poulenc's Rhodimet Nutrition Guide (1993) to lysine, one would have three A A profiles (Table 2.2). From Table 2.2, it is obvious that there are some differences between various A A profiles. The A A ratios proposed by the University o f Illinois are quite similar to the N R C (1994), and that o f Bla i r et al. (1977) is close to the Rhodimet Nutrition Guide (1993). Blair et al. (1977) and the Rhodimet Nutrition Guide (1993) also suggested that the ratios of most A A to lysine should be higher, whereas the 17 IICP and, to a certain extent the N R C (1994) suggested the opposite. Obviously, more research is necessary to clarify these discrepancies. Instead o f using solely purified diets, research should also be extended to the use o f practical ingredients so that the results can be applied in practice. Therefore, one of the objectives of studies reported in this thesis is to establish an ideal A A profiles for 0-3 week old broilers and use the ideal A A profile to measure the responses o f 0-3 week old broilers to different dietary levels of threonine and tryptophan. Table 2.1 Ideal amino acid ratios and digestible amino acid requirements of broiler chickens at three growth periods as proposed by the University of Illinois1. 0 to 21 days 21 to 42 days 42 to 56 days Amino Ideal Requirement Ideal Requirement Ideal Requirement acid ratio Male Female ratio Male Female ratio Male Female %ofLvs % of diet %ofLvs % of diet %ofLys % of diet Lysine 100 1.12 1.02 100 0.89 0.84 100 0.76 0.73 Methionine 36 0.41 0.37 37 0.33 0.31 37 0.28 0.27 Cystine 36 0.41 0.37 38 0.34 0.32 38 0.29 0.28 TSAA 2 72 0.81 0.74 75 0.67 0.63 75 0.57 0.55 Threonine 67 0.75 0.68 70 0.62 0.59 70 0.53 0.51 Valine 77 0.86 0.79 80 0.71 0.67 80 0.61 0.58 Arginine 105 1.18 1.07 108 0.96 0.91 108 0.82 0.79 Tryptophan 16 0.18 0.16 17 0.15 0.14 17 0.13 0.12 Isoleucine 67 0.75 0.68 69 0.61 0.58 69 0.52 0.50 Leucine 109 1.22 1.11 109 0.97 0.92 109 0.83 0.80 Histidine 35 0.39 0.36 35 0.31 0.29 35 0.27 0.26 Phe + Tyr2 105 1.18 1.07 105 0.93 0.88 105 0.80 0.77 Lysine requirement data for 0 to 21 days of age were taken from Han and Baker (1993); for 21 to 42 days of age from Han and Baker (1994); for 42 to 56 days post-hatching from the NRC (1994). Ideal AA ratios for 0 to 21 days of age were obtained from Baker and Han (1994); for later growth periods, ideal AA ratios were calculated based upon projected maintenance requirements and maintenance contributions to the total requirement. Requirement values are based on air-dry basis; TSAA = methionine + cystine; Phe + Tyr = phenylalanine + tyrosine. Table 2.2 Comparison among different amino acid profiles for the 0 to 21 days starter phase of broilers. N R C Modified' NRC IICPJ Blair" Rhodimef 1994 1994 1994 1977 1993 Lysine 100 100 100 100 100 Methionine + Cystine 82 75 72 69 80 Methionine 46 42 36 55 51 Cystine 36 33 36 14 29 Threonine 73 67 67 70 66 Valine 82 75 77 86 85 Arginine 114 104 105 120 118 Tryptophan 18 17 16 20 19 Isoleucine 73 67 67 80 79 Leucine 109 100 109 140 150 Histidine 32 29 35 40 _ Phenylalanine + Tyrosine 121 112 105 140 -'NRC (1994)-1.10% lysine; *NRC (1994)-1.20% lysine; JBaker and Han (1994) - Illinois ideal chick protein; "Blair et al. (1977); SRhodimet Nutrition Guide (1993). 18 The Use of Amino Acid-Supplemented Reduced Protein Poultry Diets Traditionally, cereals used for animal feeds are low in natural lysine and have been supplemented with protein sources such as soybean meal or fishmeal. However, high levels o f protein concentrates are not always practical and have become more expensive during recent years. The higher protein contents o f these diets also over-supply many other A A that are not required by the animals for protein synthesis. Recently, with the advances in biotechnology, feed grade crystalline A A such as lysine, methionine, threonine and tryptophan are readily available for use as feed supplements by the feed industry. Supplementation with A A improves the A A balance and protein utilization o f feeds, thus allowing for the level of protein-rich feedstuffs to be reduced. Experiments with poultry have supported the hypothesis that diets formulated to minimize excesses o f A A over the chicks' known requirements would improve the efficiency of protein and energy utilization (Waldroup et al, 1976; Blair et al, 1999). The resulting decrease in N excretion is a major contribution to reducing the environmental burden, particularly in regions o f intensive animal production. B y feeding low protein-AA supplemented diets (with less excess A A ) , fewer A A have to be deaminated, converted to uric acid and excreted into the environment. A s these metabolic processes are energy driven, energy can therefore be spared for other purposes. The reduction of N in the excreta also improves air quality by lowering ammonia levels in farm buildings (Archer, 1993; Jongbloed and Lenis, 1998). Reduced protein diets - Broiler studies In 1975, Lipstein et al. found that reduced protein diets (17.5% C P sorghum-corn-soybean meal) could be fed to broilers 5 to 9 weeks old without losing performance when compared to birds fed a control diet (20.5% C P sorghum-corn-soybean meal). However, birds fed the reduced protein diets also deposited more fat than the control birds. During further studies these researchers found that when this low protein diet was supplemented with methionine and lysine to the levels in the control diets, carcass fat deposition between the two diets was not different. A year later, Waldroup et al. (1976) obtained a similar positive result with chicks grown to 21 days of age. Chicks fed a 19% crude protein (CP) corn-soybean meal diet but meeting minimum essential A A requirements gained equally when compared with chicks fed a standard 23% C P corn-soybean meal diet. Birds fed the low protein diet also use protein more efficiently. In another study with broiler finishers, U z u (1982) showed that broilers 19 could be fed a 16% C P corn-soybean meal finishing diet that was supplemented with methionine and lysine to levels present in the 24% C P control corn-soybean meal diet without reducing the weight gain. In another study, Stilbom and Waldroup (1988) fed broilers A A supplemented iso-caloric diets from 21 to 42 days o f age and found comparable 42 day body weights and feed efficiency with dietary C P levels as low as 14%. Holsheimer and Janssen (1991) conducted studies to determine i f diets based on corn-soybean meal containing 19%, 18% or 17% C P were sufficient for chicks during the period of 3 to 7 weeks o f age when compared to a 20% C P corn-soybean meal diet. A l l diets were formulated to have equal and sufficient amounts o f lysine and total sulfur A A . It was also the interest o f the authors to find out whether the addition of combinations o f threonine, tryptophan and arginine or threonine, tryptophan, arginine, isoleucine, leucine and valine to the diets low in C P would have any effect on the bird's performance. It was concluded from these studies that when the C P in diets fed during the period o f 3 to 7 weeks was decreased from 20% to 17%, weight gain and feed/gain ratios increased. The diets containing 18 and 19% C P supplemented with arginine, threonine and tryptophan to the concentrations found in the 20% C P diets showed no negative effect on growth performance. Data from this study also indicated that 0.77% threonine and 0.22% tryptophan are sufficient in finishing diets fed to broilers between 3 and 7 weeks o f age. A series o f experiments was conducted at the University o f Guelph to evaluate the performance o f 7 to 21 day old broilers fed diets in which excesses o f essential A A were minimized (Parr and Summers, 1991). A 23% C P corn-soybean meal diet supplemented with DL-methionine was used as a positive control. Protein in the corn-soybean meal diets was reduced stepwise (9 diets, with C P range from 23% to 16.5%) to the point where all essential A A were at the minimum requirement level based on the 1984 National Research Council (NRC) recommendations. A s dietary protein was reduced, crystalline essential A A were used to supplement those essential A A that became deficient. These studies showed that weight gain and feed efficiency o f birds fed the reduced protein diets were not significantly different from that o f birds fed the control diet. The authors concluded that all essential A A appeared to be adequate at levels recommended by the 1984 N R C except tryptophan, which was required at the level of 0.25% o f the diet for optimal performance. Reduced protein corn-soybean meal diets (20% C P from 0-3 weeks o f age and 17% C P from 3-6 weeks o f age) did not affect live body weights in a 6 week broiler study conducted by Moran et al. (1992); however, feed conversion was increased 20 during the grower (3-6 weeks) period when the C P was reduced. Reducing the dietary C P improved protein utilization and decreased the N content o f the litter by 23.8% in the 6 t h week. The birds fed the reduced C P diet deposited much more abdominal fat leading to lower chilled carcass weights compared to the control birds. The yields of breast pieces were also lower in the low C P group. Beside methionine and lysine, arginine, valine and threonine were also found to be limiting in the reduced protein (19% CP) corn-soybean meal diet for broiler chicks (Han et al. 1992). Weight gain, feed efficiency and fat content were not significantly different between birds fed a positive control diet with 23% C P and birds fed a 19% C P diet supplemented with the five limiting A A and amino N in the form of glutamic acid. From 3 to 6 weeks o f age, chicks fed a A A fortified 16% C P diet had a growth performance similar to chicks fed a 20% C P diet. More recently, Deschepper and De Groote (1995) found that birds fed reduced-protein wheat-sorghum-soybean meal diets supplemented with crystalline essential and non-essential A A (20% CP) to the amounts in the control diet (21% C P wheat-sorghum-soybean meal) or based on the A A profile of body protein (18% C P wheat-sorghum-soybean meal) gave similar performance when compared to birds fed the control diet. This was not achieved with reduced protein wheat-sorghum-soybean meal diets (17% CP) supplemented with crystalline A A to the amount recommended by N R C (1994). Birds fed the reduced protein diets were also more efficient in protein utilization and their N excretion was reduced by 26% similar to that reported by Moran et al. (1992). The authors concluded that it was possible to obtain the same performance with reduced protein diets supplemented with crystalline A A using an ideal A A balance. However, these diets also led to a higher carcass fat content. More recently, a series o f experiments was conducted by Ibrahim (1997) to investigate the effect o f reduced protein (wheat-soybean meal) diets on broiler performance and N excretion. In one o f the experiments, the growth over a 3-6 week period was unaffected by the reduction o f C P levels in the diet from 21% to 18%, and the N output was reduced by 20% in the reduced protein diets. In another experiment by this author, a control diet with a commercial level of C P (24% starter and 20.5% grower) was compared with three reduced-CP diets (20% starter and 17% grower) which differed in the level of limiting A A (90, 100 or 110% of industry standards). The results showed that there was no significant difference in growth performance between birds fed the control or the other dietary treatments. However, reduction in dietary C P led to a 10-27% reduction in excreted N . Broilers fed the reduced protein diets also retained significantly more 21 dietary N than broilers fed the control diet, thus indicating a better utilization o f protein and A A in reduced protein diets. Conversely, researchers at the University o f Georgia (Fancher and Jensen, 1989a,b,c; Pinchasov et al, 1990; Colnago et al, 1991; Jensen and Colnago, 1991) concluded that optimal performance o f broilers could not be achieved with reduced protein diets supplemented with crystalline A A . Despite the formulation of the reduced C P corn-soybean meal diet (15 to 16% CP) to be adequate in a l l N R C (1984) recommended essential A A requirements, body weight gain and feed efficiency were inferior in two out of three experiments to values obtained from female broilers (3-6 week of age) fed a 18% to 19% C P corn-soybean meal diet (Fancher and Jensen, 1989b). Further studies using male chicks indicated that the dietary protein requirement for maximum feed efficiency during the starter period is greater than 18%, and that feed efficiency during the grower phase appeared to decline with dietary C P levels less than 22% (Fancher and Jensen, 1989c). Pinchasov et al. (1990) showed that performance o f broiler chicks fed reduced protein corn-soybean meal diets supplemented with several essential A A was generally inferior to that of birds fed a higher protein diet in which the protein was mainly intact. These experiments also showed that reducing essential A A in proportion to C P or equalizing amino N by an inclusion o f glutamic acid failed to prevent the reduction in performance o f broiler chicks fed reduced protein diets supplemented with several crystalline A A . Similar findings were observed by Edmonds et al. (1985) when the protein level o f corn and soybean meal diets was reduced from 24% to 16%. Cabel and Waldroup (1991) found that feeding lower levels o f C P to broiler chicks had a more pronounced effect on males than on females, with the primary effects being reduced body weight, poorer feed efficiency, and increased carcass fat content. Increased abdominal fat deposition was a major problem with lower protein diets fortified with A A . With each decrease in the dietary C P content, a corresponding decrease in the dietary heat increment should have occurred. One o f the primary mechanisms involved in reducing carcass fatness by feeding higher C P diets is the associated increased energy expenditure and increased heat increment involved in degrading excess amino N to uric acid (Bartov, 1979). Reduced protein diets - Layer studies Because of the inefficiency in protein utilization, N pollution is also a major issue in the laying hen industry. In 1973, a group o f Washington State University researchers reported that a 22 corn-wheat-soybean meal diet containing 13% protein and supplemented with lysine and methionine was as effective as levels o f 15, 17 and 18% C P for supporting egg production and egg size (Fernandez et al, 1973). Layers fed a barley-wheat-corn-soybean meal diet containing 14.1% C P and supplemented with methionine and lysine had a 75% egg production when compared to 73% for layers fed a conventional diet (Blair et al, 1976). Several experiments were undertaken by Summers (1993) to investigate N excretion by laying hens fed corn-soybean meal diets varying in level of dietary protein. In one study, the performance o f 24-week-old laying birds was not jeopardized even when the dietary C P in the layer diet was lowered from 17% to 13%. Birds on the 13% protein diet also excreted approximately 34% less N per day than those fed the 17% protein diet. Further study with 45-week-old hens indicated that N balance was reached by feeding between 9-11% dietary protein. However, egg mass started to decline as dietary protein level was reduced below 17%. The author also concluded that there is merit to optimizing rather than maximizing egg mass output because exceptionally high intakes o f dietary protein are required to maximize egg size. A n experiment was carried out by Ibrahim (1997) to determine the effects o f reduced protein wheat-soybean meal diets on egg production and N output in excreta o f laying hens. Reducing the dietary C P level from 17% to 13.5% while maintaining the limiting A A levels at 90, 100, or 110% of industry standards had no significant effect on egg production or egg quality. Layers fed the low protein diets were also more efficient in N utilization and excreted 30% less N . Coon's (1998) research indicated that reduced protein diets with added crystalline A A can replace feeding higher protein diets to layers. The egg composition and performance of layers fed corn-soy-meat meal 14% C P diets with added methionine, lysine, isoleucine, and valine was equal to layers fed 18% C P control diets. Contrary to this, pullets receiving a 14.5% C P corn-barley-soybean meal diet in the laying period produced significantly fewer and smaller eggs than those fed a 16.5% C P corn-barley-soybean meal diet (Keshavarz, 1984). According to the author, lower intakes o f lysine and other A A might have been the reasons for the poor performance o f the birds fed the reduced protein diet. Egg weight and sometimes the rate o f egg production were significantly reduced when laying hens were fed 13% corn-soybean meal diets supplemented with adequate levels o f methionine, lysine, and tryptophan in comparison to hens fed a 16% C P corn-soybean meal diet (Jensen and Colnago, 1991). Keshavarz and Jackson (1992) conducted an experiment to determine the effect o f feeding AA-supplemented reduced protein corn-barley-soybean meal 23 diets during growing and laying periods on the layers' performance. A t 18 weeks o f age, birds fed the negative control diets (16, 13.5, and 11.5 % C P diets during the growing period and 14, 13, and 12% C P diets during the laying period) supplemented with methionine and lysine or supplemented with methionine, lysine and other deficient essential A A had comparable body weights to those fed the positive control diets (20, 16, and 14% C P diets in the growing period and 18, 16.5, and 15% C P diets in the laying period). There was no significant difference in egg weight and overall egg production between the high protein (positive control) group and the low protein AA-supplemented (negative control) group. However, egg mass output and body weight o f laying hens fed the negative control diets were inferior to laying hens fed the positive control diets. A s is in the case for broiler studies, there were conflicting results reported in the literature regarding the use o f low protein diets in the egg industry. Keshavarz (1984) suggested that difference in strains, management programs, season housed, and the extent and the age o f the initiation o f protein restriction may be factors that have contributed to the discrepancies. In conclusion, the above data show that it is possible to reduce the C P o f poultry diets without negatively influencing birds performance. However, a number o f authors showed that reduced dietary protein would lead to a poorer performance by birds. Edmonds et al. (1985) and Fancher and Jensen (1989b) stated that no clear-cut explanation could be provided as to why fortifying the reduced protein diet with al l l imiting essential A A and nonessential amino N failed to support maximal performance. Pinchasov et al. (1990) and Colnago et al. (1991) suggested that a minimal level of intact protein is necessary for optimum broiler performance. According to Pinchasov et al. (1990), this need may relate to differences in absorption o f single A A versus peptides. Matthews (1975) has demonstrated that a majority o f the intact protein consumed by monogastric animals enters the absorptive cell as small peptides and that the absorption is independent o f the uptake of free A A and is more rapid. It is also possible that different rates o f absorption o f peptides and free A A may result at times in a less than optimal availability of all essential A A at the site o f protein synthesis in the tissues. However, Han et al. (1992) stated that in the University of Georgia studies (Fancher and Jensen, 1989a,b,c; Pinchasov et al, 1990; Colnago et al, 1991; Jensen and Colnago, 1991) the reduced protein diets were formulated such that the A A nitrogen was accounted for, and this effectively lowered the soybean meal (at the expense o f corn) more than was the case in their current studies. That is, the diets might be deficient in amino N for the synthesis o f non-essential A A . Thus, chick performance on reduced protein diets was inferior in these studies. It is very important to note that in addition to the 24 requirement for essential AA, poultry diets should also supply adequate N for the synthesis o f non-essential A A . Otherwise, essential A A nitrogen would be used for the synthesis o f non-essential A A and growth and production would be jeopardized. It is important to devote more research to the area o f reduced-protein diets, so that the utilization o f protein can be further improved resulting in decreased N excretion by animals. Palm Kernel Cake The recent escalation in ingredient costs in South East As i a due to the economic crisis has stimulated a lot o f interest in re-evaluating the feeding quality o f some locally available feed ingredients for use in the poultry industry. Local ly grown food such as corn and soybean meal are not sufficient for human consumption in the South East As i a countries, not to mention the need for these ingredients for the animal feed industry. Most o f the conventional feed ingredients such as corn and soybean meal are therefore imported from the developed countries. However, most o f the South East As i a countries also produce millions o f tonnes o f agricultural by-products, which are not suitable for human consumption. Agricultural by-products such as coconut meal, palm kernel cake, rice bran, cottonseed meal and others are abundant in many countries. However, their role in the feed industry has been minimized, mainly because insufficient nutritive information is available regarding these potential ingredients. O f these, palm kernel cake ( P K C ) , which is also known as palm kernel meal or palm kernel expeller, has the greatest potential as an animal feed in countries that produce it in large quantities such as Malaysia, Indonesia, South America and Africa. An introduction to oil palm The o i l palm (Elaeis guineensis Jacq.) is a monocotyledonous plant widely believed to be native to West Africa. It belongs to the family Palmae, order Palmales and genus Elaeis. The name o f the genus Elaeis is derived from Greek word "elaion," meaning oi l . The specific name guineensis indicates its origin in the Guinea Coast. Elaeis guineensis is one o f the largest palm species. It has a stem that can reach a height of 25-35 m, topped by 35 to 60 pinnate leaves. A n o i l palm has an economically productive life o f 20-30 years, and replanting is usually carried out after about 24 years (Hartley, 1988). Almost 6 months after pollination, a fruit bunch weighing 25 15 to 20 kg and consisting o f approximately 1,000 to 1,500 fruit is produced. The shape and the size o f the fruit vary considerably. The fruit is about 2.5-5 cm in length and 2.5 cm in diameter and weigh about 3-30g (Godin and Spensley, 1971; Gascon et al, 1989). The mature fruit is a deep orange-red drupe containing pulp (mesocarp), shell (endocarp) and kernel (endosperm). The fibrous mesocarp is rich in o i l and is yellowish-orange in color, due to its high carotene content. The palm fruit produces two types o f o i l : crude palm o i l from the pulp (about 50% o i l on a fresh weight basis) and palm kernel o i l from the kernel. Oil palm in Malaysia The o i l palm was first introduced to Malaysia in 1878, while its use as a plantation crop was only developed in the early 20 t h century. During the last 20 years, o i l palm has overtaken rubber as the major plantation crop. Palm oi l has become a major vegetable o i l during the past few decades. Production has rapidly increased, more than quadrupling from 1970 to 1990. In 1996 the world production of palm oi l was reported at 16.11 M T ( P O R L A , 1997), taking second place after soybean o i l . The bulk o f palm oi l (about 82%) is produced in South East Asia , notably i n Malaysia and Indonesia ( P O R L A , 1997). Concomitant with the production o f palm oi l , palm kernel o i l also became important because of the similarity of its o i l composition to that o f coconut o i l (Gascon et al, 1989). The o i l palm considerably out-yields other o i l crops in o i l per hectare. Malaysia, the world's leading producer with more than 50% of the market share, produced 8.39 M T o f it in 1996 ( P O R L A , 1997). Nutrient composition of palm kernel cake Palm kernel meal is the major byproduct of palm kernel o i l extraction. It is a useful source o f protein and energy for livestock. However, it is highly variable in composition especially in its o i l and fiber contents. Such differences in composition may be due to o i l palm types that exist in different geographical regions, to the extent o f o i l extraction from palm kernel and also to the methods of processing used in producing the P K C (Onwudike, 1986a). Palm kernel cake generally contains 17-21% protein, 10-17% crude fiber, 4-5% ash and ether extract values of 0.7-9.0% depending on the efficiency o f o i l extraction from the kernel (Nwokolo et al., 1977; Devendra, 1978; Hutagalung et al, 1982; Onwudike, 1986a). The apparent metabolizable energy ( A M E ) for P K C has been reported to range from 2,008 to 2,999 kcal/kg (Nwokolo et al, 26 1977; Onwudike, 1986a; Ngoupayou, 1984; Longe and Tona, 1988). However, values as low as 1,482 kcal A M E / k g were reported by Yeong (1985). The protein is o f relatively good quality, but its high crude fiber content may affect the A A availability and the digestibility o f P K C . Some o f the reported A A values for P K C are listed on Table 2.3. Table 2.3 Crude protein and amino acid composition of palm kernel cake (PKC, % dry matter) Nwokolo et al. (1976b) Onwudike (1986a) Yeong (1983) Origin o f P K C Nigeria Nigeria Malaysia Crude protein 21.3 19.2 16.06 Amino acids Arginine 2.68 (12.58)1 2.65 (13.80) 2.18(13.57) Histidine 0.41 (1.92) 0.42 (2.19) 0.29(1.81) Isoleucine 0.60 (2.82) 0.62 (3.23) 0.62 (3.86) Leucine 1.23 (5.77) 1.20 (6.25) 1.11 (6.91) Lysine 0.69 (3.24) 0.68 (3.54) 0.59 (3.67) Methionine 0.47 (2.21) 0.32(1.67) 0.30(1.87) Cystine - - 0.20(1.25) Phenylalanine 0.82 (3.85) 0.74 (3.85) 0.73 (4.55) Tyrosine 0.58 (2.72) 0.53 (2.76) 0.38 (2.37) Threonine 0.66 (3.10) 0.68 (3.54) 0.55 (3.42) Valine 0.43 (2.02) 0.88 (4.58) 0.93 (5.79) Aspartic acid 1.69 (7.93) 1.72 (8.96) 1.55 (9.65) Glutamic acid 3.62 (17.00) 4.01 (20.89) 3.15(19.61) Proline 0.50 (2.35) 0.62 (3.23) 0.63 (3.92) Serine 0.90 (4.23) 0.92 (4.79) 0.69 (4.30) Glycine 0.91 (4.27) 0.92 (4.79) 0.83 (5.17) Tryptophan - - 0.17(1.06) Alanine 0.81 (3.80) 0.76 (3.96) 0.92 (5.73) 'Value in brackets is % of C P . There seem to be some differences in the A A content among samples from different countries. There is good agreement between samples from the same country (Nigeria). However, the concentration o f A A in Malaysian P K C seem to be lower than the concentration o f A A in Nigerian P K C . In general, P K C is deficient in lysine and sulfur A A . The data on the digestibility o f A A in P K C for poultry are very limited. Only a limited number o f researchers (Nwokolo et al., 1976b; Yeong, 1983; Onwudike, 1986a) have reported the digestibility o f the A A from P K C (Table 2.4). The digestibility o f A A in P K C averaged 83.3% and 84.5% according to Onwudike (1986a) and Nwokolo et al. (1976b). However, research conducted in Malaysia (Yeong, 1983) 27 reported a low value o f 64.4%. The reason for the low digestibility o f A A in P K C from Malaysia is not clear. Table 2.4 Digestibility of palm kernel cake (PKC) amino acids (%). Nwokolo et al. (1976b) Onwudike (1986a) Yeong (1983) Origin o f P K C Nigeria Nigeria Malaysia Amino acids Arginine 93.2 92.7 87.0 Histidine 90.1 88.7 66.8 Isoleucine 86.1 87.5 64.9 Leucine 88.5 90.6 66.7 Lysine 90.0 88.9 58.6 Methionine 91.0 92.1 72.1 Phenylalanine 90.5 91.6 70.4 Tyrosine 85.0 89.9 65.7 Threonine 86.5 85.3 60.7 Valine 68.4 66.7 62.8 Aspartic acid 87.6 85.3 64.4 Glutamic acid 90.1 88.6 74.4 Proline 68.0 64.2 55.0 Serine 88.7 85.4 65.0 Glycine 63.3 52.1 25.8 Alanine 85.5 83.0 67.7 Overall mean 84.5 83.3 64.4 Palm kernel cake contains a relatively high amount o f minerals, particularly calcium, phosphorus and iron (Nwokolo et al, 1976a). However, the availability o f most minerals is poor. The availability o f calcium, phosphorus, magnesium, manganese, zinc and copper was reported as 68.6, 70.8, 56.4, 45.7, 13.9, and 44.7, respectively (Nwokolo et al, 1976a). In another study, Nwokolo and Bragg (1977) found that P K C contained 1.42% phytic acid and that half o f the total phosphorus was in the form o f phytate phosphorus. The study also concluded that phytic acid reduced the availability of calcium, phosphorus, magnesium and zinc, whereas the crude fiber content depressed the availability of all minerals tested. The ratio of calcium to phosphorus in P K C is reported to be more favorable than in other oilseed meals (McDonald et al, 1982). Non-starch polysaccharides in palm kernel cake Although starch is the most-researched and economically the most important seed storage polysaccharide, it is by no means the only polymeric carbohydrate stored in seeds. There are 28 seeds o f many plant species that contain little or no starch, but are nevertheless rich in other forms o f polysaccharide reserves. The special groups o f non-starch polysaccharides are stored outside the plasmalemma, and were categorized collectively as cell wal l storage polysaccharides by Meier and Reid (1982). Mannan-type cel l wal l storage polysaccharides are al l based on a linear p-l ,4-l inked chain or "backbone." They may be subdivided into the "pure" mannans, the glucomannans in which some o f the D-mannose residues in the backbone are replaced by D -glucose, and the galactomannans in which the backbone carries a-l,6-D-galactosyl substituents. According to Meier and Reid (1982), the mannan-group polysaccharides of seeds are major reserve substances only in endosperms, as opposed to storage cotyledons. "Pure" mannans may be defined to include those polysaccharides that contain less than 10% non-mannose sugar residues. Pure mannans form the major part o f the endosperm (kernel) o f many palm seeds. They take the form of massive wall thickenings in the endosperm and are clearly the molecular basis o f the palm kernel's characteristic hardness (Meier and Reid, 1982). A group o f researchers at the Agricultural University o f Netherlands (Dusterhoft and Voragen, 1991; Dusterhoft et al., 1992) has characterized the polysaccharide components o f P K C . Palm kernel cake was found to contain mannans, cellulose and xylans, with the major part of the mannans originating from the endosperm (kernel) and xylans being almost exclusively located in the endocarp (shell). Palm kernel cake was found to contain negligible amounts o f starch (1 g/kg) and some of the protein was not digested even after the Pronase treatment, implying that the residual protein was either structurally bound in the cell wal l or present as inaccessible cytoplasmic material. There were about 726 g o f cell wal l materials per kg o f P K C containing approximately 7.3% protein, 17.5% lignin, 5% ash and 74.6% non-starch polysaccharides (Dusterhoft and Voragen, 1991). Further analysis showed that mannose (from mannan) made up about 57.1% of the cell wal l material, which means that P K C contained about 41.45% mannose in this particular study. Further study confirmed that major polysaccharides in P K C are linear mannans with very low galactose substitution (78% of total non-starch polysaccharides), followed by cellulose (12%) and small amounts o f (4-O-methyl)-glucuronoxylans and arabinoxylans (3% each) (Dusterhoft et al, 1992). Daud and Jarvis (1992) also showed linear p(l,4)-D-mannan to be the major component o f P K C cell wal l non-starch polysaccharides, and that the mannans of P K C appeared to be highly crystalline, in keeping with their insolubility. 2 9 Palm kernel cake - Enzyme studies Mannanase is found only in the endosperm and its activity increases from a negligible value shortly after imbibition to a high value and then decreases (Matheson, 1990). Even though endogenous enzyme activities (especially mannanase) were found i n the palm kernel (endosperm), the unfavourable environments (e.g. high temperature) during the o i l extraction process would denature and inactivate it. A s a result, mannanase and other enzyme activities, i f any, are not expected to be found in P K C . Several in vitro enzyme studies involving P K C have been conducted by researchers from the Netherlands (Dusterhoft et al., 1993a, b, c). Enzymes containing mannanase and cellulase activities were used in the studies. It was found that reducing particle-size, thereby increasing the surface area available for enzymes to attack, enhanced the solubilization o f P K C (Dusterhoft et al., 1993a). Since P K C ' s cell walls are highly lignified, their accessibility might be hindered by lignin and low molecular-weight phenolic compounds forming covalent linkages with sugar residues. Mannans in palm kernel cel l wal l materials were hydrolyzed by 20-50%, depending on enzyme composition, with mannose monomers and dimers as major end products (Dusterhoft et al., 1993b). Results also indicated the preferential solubilization o f the endosperm in P K C , and the resistance o f the palm kernel endocarp to enzymatic attack. Comparing the release o f glucose (cellulose) and mannose (mannan) from P K C , the authors concluded that neither cellulose hydrolysis enhances mannan-degradation, nor mannan hydrolysis enhances cellulose-degradation (Dusterhoft et al, 1993b). However, with further study they found that in the degradation o f palm kernel cell wal l materials, a synergistic action o f mannanases and glucanases was observed (Dusterhoft et al, 1993c). Daud et al. (1997) reported a 8.5% (or 230 kcal/kg) increase in the A M E content o f a PKC-based broiler diet treated with 0.1% mannanase (Alltech Inc., U S A ) . Cellulase supplementation increased the A M E value o f the PKC-based diet numerically but not significantly. A s synergistic action between cellulase and mannanase was not observed, Daud et al. (1997) concluded that mannanase is the most effective enzyme to improve the nutritive value of P K C . Palm kernel cake - Animal studies A number o f studies concerning the use of P K C in poultry diets have been conducted. In one study, three levels o f P K C (10, 20, and 30%) were used to evaluate the feeding value o f P K C 30 for broiler chicks in Malaysia (Ahmad, 1982). A corn-soybean meal diet was used as the control. Feed intake was not significantly different between diets. However, weight gains were significantly depressed when the diet contained more than 10% P K C . Feed efficiency was also poorer for those birds fed diets with more than 10% P K C . A n economic evaluation found that there was no economic advantage to use P K C in broiler diets. Nevertheless, other studies in Malaysia found that P K C has been utilized as a supplement at up to 20% in diets for broilers and layers and up to 25% for swine, without any adverse effect on their performance (Yeong, 1980; Hutagalung, 1980). Yeong and Mukherjee (1983) showed that supplementation o f a broiler diet containing 20% P K C with 9% palm oi l resulted in growth and feed efficiency similar to that obtained with the control diet. Osei and A m o (1987) reported that the inclusion o f P K C at 12.5% and 15% of the diet reduced growth and feed efficiency. A more recent work by Onifade and Babatunde (1998) showed that broiler performance was depressed even when 10% P K C was included in a diet when compared to a corn-soybean meal control diet. However, the control diet had a higher A M E content and the A M E levels in the test diets were not adjusted to that of the control. Researchers from Central America indicated that a low level o f P K C (10%) could be used in broiler diets (Garcia and Gernat, 1998) and other reports suggested that high levels o f inclusion were possible. Nwokolo et al. (1977) incorporated 30% P K C or 30% alkali-treated (3, 5, 7% NaOH) P K C into a starter diet for 2-week-old broiler chicks. There was no significant negative effect on the chicks' performance when 30% P K C or 30% alkali-treated (3% N a O H ) PKC-based diets were fed except when birds were fed 30% alkali-treated (5% or 7% NaOH) PKC-based diets. Ngoupayou (1984) demonstrated that P K C allowed good growth o f chicks when fed at a level up to 20% in diets. According to the author, this inclusion rate could replace up to 40% cottonseed cake and 25.4% corn in chick diets. In Nigeria, starter broilers were shown to be able to utilize a diet containing 28% P K C without any significant effect on their performance (Onwudike, 1986c). In a recent study, Panigrahi and Powell (1991) found that up to 50% P K C (from Malaysia) can be incorporated into broiler diets without any negative effects on their performance provided that a high level o f o i l (11.3-11.4% of the diet) is included. However, they also concluded that such diets may be uneconomical in most developing countries and, perhaps, too oi ly to be considered practical. On the other hand, finishing broilers were found to be able to utilize up to 35% P K C without any significant effect on their performance (Onwudike, 1986c). Based on these results, 31 Onwudike (1986c) concluded that the use o f up to 35% P K C in the diet o f finishing broilers would reduce the cost o f production, improve feed efficiency and also reduce the fat content o f finished broilers. For layers, Onwudike (1986b) demonstrated that diets containing up to 34% P K C could be fed to starter pullets without any adverse effects on performance. He also showed that diets containing up to 38% P K C could be fed to grower pullets without affecting the rate of egg production, egg weight, weight o f first egg dropped and feed intake. In addition, cost o f feed for one k g o f gain decreased as the level of P K C increased. Wi th laying birds, Longe (1984) using P K C from Nigeria found that layers fed a 20% P K C diet ate significantly more feed and produced less eggs than control birds. Layers were also less efficient in utilizing feed containing P K C . However, there were no negative effects on egg weight, shell thickness or cholesterol levels. Interestingly, another report from the same country found that 40% P K C could be fed without any adverse effect on laying performance and egg quality (Onwudike, 1988). A s the proportion of P K C increased beyond the 40% level, there was a significant drop in egg production, egg weight, feed intake and feed efficiency. Furthermore, birds produced very watery droppings, for a reason that has not been fully explained. This is in agreement with another report that used up to 40% P K C in layer diets (Panigrahi and Waite, 1998). West African P K C containing 17.6% C P , 14.2% crude fiber and 10.2% fat was used in a 6 week layer study. There were no significant differences in laying performance between control and P K C based diets. However, the body weight of the hens fed 40% P K C was depressed. This effect was overcome by increasing the energy value o f the diet. It was also observed that the colour o f the yolks became paler as the amount o f P K C in the diets increased. Beside P K C , palm kernel was found to be tolerated by broilers at a 5% inclusion rate, and the kernels were reported to contain 10% C P , 8% fiber, 0.21% calcium, 0.16% phosphorus, 0.44% lysine and 0.29% methionine (Oruwari et al, 1995). Fifty-six percent o f the kernel is made up o f o i l and this results in the high T M E value o f 6,400 kcal/kg. In pigs, dietary P K C tends to produce firm pork o f good quality (Gohl, 1975). Because o f low lysine availability, a higher concentration o f P K C in the diet of pigs requires balancing with supplemental lysine (Hutagalung et al, 1983). Ruminants are capable of utilizing P K C more efficiently when compared to non-ruminant. Apparent digestibility of organic dry matter ( D M ) , C P and neutral detergent fiber o f P K C for sheep was reported to be 75%, 75%, and 73%, respectively (Moss and Givens, 1994). A M E of 3,131 kcal/kg D M was also reported in this study. Palm kernel cake 32 tends to produce a firm butter when fed to dairy cattle (Gohl, 1975). Palm kernel cake also has a tendency to increase the fat content o f bovine milk (McDonald et al, 1982). From the above discussion, it seems that the maximum amount o f P K C that can be included in diets ranges from 10% to 50% for broilers and from 20% to 40% for layers. Most of the research reported in the literature concerns P K C originating from Africa. From several studies (Yeong, 1980; Hutagalung, 1980; Yeong and Mukherjee, 1983) that used P K C originating from Malaysia, 20% P K C seems to be the maximum level that can be used in poultry diets without jeopardizing the performance o f the birds. The only exception is the study by Panigrahi and Powell (1991) which is not practical due to the high level o f o i l inclusion. The major limitations o f using P K C could be attributed to its high fiber content (especially high non-starch polysaccharides), low energy content and to low digestibility o f nutrients like protein, A A , minerals, and vitamins. Palm kernel cake could be a valuable raw ingredient for the animal feed industry in countries that produce it in large quantities. Due to the differences in the methods o f processing palm kernel o i l , the duration of the studies, the sources o f P K C and the age o f the birds, the data obtained from the literature on the utilization of P K C in poultry diets are quite contradictory. Even though P K C is less digestible for poultry than for other animals, the application o f new biotechnology such as enzyme supplementation might offer hope for its use in the poultry industry. Therefore, the current studies were conducted with the objectives o f evaluating the potential o f P K C as a poultry feedstuff and the potential o f enzyme supplementation in P K C -based poultry diets. In conclusion, the above review o f the literature clearly shows that further research is necessary to determine the validity o f the N R C (1994) recommended poultry requirements for threonine and tryptophan so that protein utilization o f conventional feed ingredients could be improved and to evaluate the nutritive quality and to improve the nutrient utilization o f non-conventional feed ingredient such as P K C for poultry diets. References Abebe, S., and T. R. Morris, 1990. Effects of protein concentration on responses to dietary tryptophan by chicks. Br . Poult. Sci . 31:267-272. 33 Agricultural Research Council , 1981. The nutrient requirements of pigs. Commonwealth Agricultural Bureaux, Slough, U K . Ahmad, M . Y . , 1982. The feeding value o f palm kernel cake for broilers. M A R D I Res. B u l l . 10:120-126. Archer, J. , 1993. Avoiding pollution from poultry manure. World 's Poult. Sci J. 49:167-174. Austic, R. E . , and M . Rangel-Lugo, 1989. Studies on the threonine requirement o f broiler chicks. Pages 136-143 in: Proceedings Cornell Nutrition Conference for Feed Manufacturers, East Syracuse, N . Y . Baker, D . H . , 1997. Pages 1-24 in: Ideal amino acid profiles for swine and poultry and their applications in feed formulation. Biokyowa Publishing Co. , St. Louis, M O . Baker, D . FL, C . M . Parsons, S. Fernandez, S. Aoyagi and Y . Han, 1996. Digestible amino acid requirements o f broiler chickens based upon ideal protein considerations. Zootecnica Int. 60-65. Baker, D. H . , and Y . Han, 1994. Ideal amino acid profile for broiler chicks during the first three weeks posthatching. Poultry Sci . 73:1441-1447. Bartov, I., 1979. Nutritional factors affecting quantity and quality o f carcass fat in chickens. Fed. Proc. 38:2627-2639. Blair , R. , J. P. Jacob, S. Ibrahim, and P. Wang, 1999. A quantitative assessment o f the use o f reduced protein diets supplemented with amino acids to improve nitrogen utilization and reduce nitrogen pollution from broilers and layers. J. App l . Poultry Res. 8:25-47. Blair , R. , D. J. W . Lee, C. Fisher, and C. C. McCorquodale, 1976. Responses o f laying hens to a low protein diet supplemented with essential amino acids, L-glutamic acid and/or intact protein. Br . Poult. Sci . 17:427-440. Blair , R. , S. McKenzie , and D. J. W . Lee, 1977. A purified diet for one-day-old chicks. Br . Poult. Sci . 18:129-136. Boomgaardt, J., and D. H . Baker, 1971. Tryptophan requirement o f growing chicks as affected by dietary protein level. J. A n i m . Sci . 33:595-599. Bray, D. J., 1969. Studies with corn-soya laying diets. 8. Requirements for limiting amino acids-the basal diet and the requirements for isoleucine, lysine and tryptophan. Poultry Sc i . 48:674-684. Cabel, M . C . and P. W . Waldroup, 1991. Effect o f dietary protein level and length o f feeding on performance and abdominal fat content of broiler chickens. Poultry Sci . 70:1550-1558. 34 Colnago, G . L . , A . M . Penz, Jr., and L . S. Jensen, 1991. Effect of response o f starting broiler chicks to incremental reduction in intact protein on performance during the grower phase. Poultry Sci . 70(Suppl. l):153.(Abstr.) Coon, C , 1998. Amino acid requirements o f commercial laying hens. Pages 70-75 in: Proceedings of 6 t h Asian Pacific Poultry Congress, Nagoya, Japan. Daud, M . J. , and M . C . Jarvis, 1992. Mannan of palm kernel. Phytochem. 31:463-464. Daud, M . J. , N . Samad, and S. Rasool, 1997. Specific commercial enzymes for nutritive value improvement o f palm kernel cake for poultry diets. Pages 137-138 in: 19 t h M S A P Annual Conference. Y . W . Ho , M . Z . Saad, F . Y . Chin, I. Zulki f l i , and H . K . Wong ed. Johor Bahru, Johor, Malaysia. Deschepper, K . and G . De Groote, 1995. Effect of dietary protein, essential and non-essential amino acids on the performance and carcass composition of male broiler chickens. Br . Poult. Sci . 36:229-245. Devendra, C . 1978. Utilization of feeding stuffs from oi l palm. Pages 116-131 in: Feedingstuffs for Livestock in South East Asia . C . Devendra and R. I. Hutagalung, eds. Malaysian Society o f Animal Production, Kuala Lumpur. D ' M e l l o , J. P. F. , and D . Lewis, 1970. Amino acid interactions in chick nutrition. III. Interdependence in amino acid requirements. Br . Poult. Sci . 11:367-385. Dusterhoft, E . M . and A . G . J. Voragen, 1991. Non-starch polysaccharides from Sunflower (Helianthus annuus) Mea l and Palm Kernel (Elaeis guineenis) Mea l - Preparation o f cell wal l material and extraction of polysaccharide fractions. J. Sci . Food Agric . 55:411-422. Dusterhoft, E . M . , M . A . Posthumus, and A . G . J. Voragen, 1992. Non-starch polysaccharides from Sunflower {Helianthus annuus) Mea l and Palm Kernel {Elaeis guineenis) Mea l -Investigation o f the structure o f major polysaccharide. J. Sci . Food Agric . 59:151-160. Dusterhoft, E . M . , F . M . , Engels, and A . G . F. Voragen, 1993a. Parameters affecting the enzymic hydrolysis o f oil-seed meals, lignocellulosic by-products o f the food industry. Bioresource Technol. 44:39-46. Dusterhoft, E . M . , A . W . Bonte, and A . G . J. Voragen, 1993b. Solubilisation of non-starch polysaccharides from oil-seed meals by polysaccharides - degrading enzymes. J. Sci . Agric . 63:211-220. Dusterhoft, E . M . , A . W . Bonte, J. C . Venekamp, and A . G . J. Voragen, 1993c. The role of fungal polysaccharidases in the hydrolysis o f cell wal l materials from sunflower and palm-kernel meals. World J. Microbiology and Biotechnology 9:544-554. Edmonds, M . S., C . M . Parsons, and D . H . Baker, 1985. Limit ing amino acids in low protein corn-soybean meal diets fed to growing chicks. Poultry Sci . 64:1519-1526. 35 Elliot , M . A . , 1995. Feeding and managing pullets to optimize genetic potential. Pages 149-175 in: Proceedings o f the California Nutritional Conference. Emmert, J. L . , and D . H . Baker, 1997. Use o f the ideal protein concept for precision formulation o f amino acid levels in broiler diets. J. App l . Poult. Res. 6:462-470. Fancher, B . I. and L . S. Jensen, 1989a. Influence on performance o f three to six-week-old broilers o f varying dietary protein contents with supplementation o f essential amino acid requirements. Poultry Sci . 68:113-123. Fancher, B . I. and L . S. Jensen, 1989b. Dietary protein level and essential amino acid content: Influence upon female broiler performance during the grower period. Poultry Sci . 68:897-908. Fancher, B . I. and L . S. Jensen, 1989c. Male broiler performance during the starting and growing periods as affected by dietary protein, essential amino acids, and potassium levels. Poultry Sci . 68:1385-1395. Fernandez, R. , A . J. Salman, and J. McGinnis , 1973. Effect o f feeding different protein levels and o f changing protein level on egg production. Poultry Sci . 52:64-69. Florentino, R . F. , and W . N . Pearson, 1962. Effect of threonine-induced amino acid imbalance on the excretion o f tryptophan metabolites by the rat. J. Nutr. 78:101-108. Freeman, C . P., 1979. The tryptophan requirement o f broiler chicks. Br . Poult. Sci . 20:27-37. Garcia, C. A . , and A . G . Gernat, 1998. The effect o f using different levels o f palm kernel meal in broiler diets. Poultry Sci . 77(Suppl. l):44.(Abstr.) Gascon, J. P., J. M . Noiret, and J. Meunier, 1989. O i l palm. Pages 475-495 in: O i l Crops of the World . G . Robbelen, R. K . Downey, and A . Ashri , ed. New York, M c G r a w - H i l l . Godin, V . J. , and P. C . Spensley, 1971. T. P. I. Crop Product Digest No . 1. London, Tropical Products Institute. Gohl , B . , 1975. Tropical feeds. Pages 375-376 in: Feed Information Summaries and Nutritive Values. F A O Animal Production and Health Series. F A O , Rome. Griminger, P., H . M . Scott, and R. M . Forbes, 1956. The effect o f protein level on the tryptophan requirement o f the growing chick. J. Nutr. 59:67-76. Han, Y . , and D . H . Baker, 1991. Lysine requirement o f fast and slow growing broiler chicks. Poultry Sci . 70:2108-2114. Han, Y . , and D . H . Baker, 1993. Effects of sex, heat stress, body weight and genetic strain on the lysine requirement o f broiler chicks. Poultry Sci . 72:701-708. 36 Han, Y . , and D . H . Baker, 1994. Digestible lysine requirement of male and female broiler chicks during the period three to six weeks posthatching. Poultry Sci . 73:1739-1745. Han, Y . , H . Suzuki, and D . H . Baker, 1991. Histidine and tryptophan requirement of growing chicks. Poultry Sci . 70:2148-2153. Han, Y . , H . Suzuki, C . M . Parsons, and D . H . Baker, 1992. Amino acid fortification of a low protein corn and soybean meal diet for chicks. Poultry Sci . 71:1168-1178. Hartley, C . W . S., 1988. The O i l Palm, 3 r d ed., Longman, London. Hewitt, D . , and D . Lewis, 1972. The amino acid requirements o f the growing chick. I. Determination o f amino acid requirements. Br . Poult. Sci . 13:449-463. Holsheimer, J. P., and W . M . M . A . Janssen, 1991. Limit ing amino acids in low protein maize-soyabean meal diets fed to broiler chicks from 3 to 7 weeks of age. Br . Poult. Sci . 32:151-158. Holsheimer, J. P., P. F . G . Vereijken and J. B . Schutte, 1994. Response o f broiler chicks to threonine-supplemented diets to 4 weeks of age. Br . Poult. Sci . 35:551-562. Hunchar, J. G . , and O. P. Thomas, 1976. The tryptophan requirement o f male and female broilers during the 4-7 week period. Poultry Sci . 55:379-383. Hurwitz, S., D . Sklan, and I. Bartov, 1978. New formal approaches to determination o f energy and amino acid requirements of chicks. Poultry Sci . 57:197-205. Hutagalung, R. I., 1980. Availabili ty of feedstuff's for farm animals. Proc. Abstr. First As ia -Australasia Animal Science Congress. 40:15.(Abstr.) Hutagalung, R. I., M . D . Mahyuddin, and S. C. Jalaludin, 1982. Feeds for farm animals from the o i l palm. Pages 609-622 in: The Oil Palm in Agriculture in the Eighties, V o l . 11. E . Pushparajah and C. Poh-Soon, eds., Incorporated Society of Planters, Kuala Lumpur. Hutagalung, R. I., M . D.Mahyuddin, P. Vijchulata, S. Jalaludin, and J. Zainal, 1983. Nutrient availability and utilization o f feedstuff's for farm animals, in: Feed Information and Animal Production, G . G . Robards, and R. G . Packham. eds. Slough: Commonwealth Agricultural Bureaux. Huyghebaert, G . , and E . A . Butler, 1991. Optimum threonine requirement o f laying hens. Br . Poult. Sci . 32:575-582. Ibrahim, S. B . , 1997. Modified poultry diets: A n approach to sustainable animal production. Ph.D. Thesis, Univ . o f British Columbia, Vancouver, Canada. Ingram, G . R. , W . W . Cravens, C. A . Elvehjem, and J. G . Halpin, 1951. Studies on the lysine and tryptophan requirements o f the laying and breeding hen. Poultry Sci . 30:426-430. 37 Ingram, G . R., and P. L . Little, 1958. Further studies on the methionine, tryptophan and lysine requirements of laying hens. Poultry Sci . 37:1214-1215. (Abstr.) Ishibashi, T., 1985. Tryptophan requirement of laying hens. Jpn. Poult. Sci . 22:256-263. Ishibashi, T., Y . Ogawa, T. Itoh, S. Fujimura, K . Koide, and R. Watanabe, 1998. Threonine requirements o f laying hens. Poultry Sci . 77:998-1002. Jensen, L . S., V . M . Calderon, and C. X . Mendonca, Jr., 1990. Response to tryptophan o f laying hens fed practical diets varying in protein concentration. Poultry Sci . 69:1956-1965. Jensen, L . S., and G . L . Colnago, 1991. Amino acids and protein for broilers and laying hens. Pages 29-36 in: Proceedings Maryland Nutrition Conference, College Park, M D . Jongbloed, A . W. , and N . P. Lenis, 1998. Environmental concerns about animal manure. J. A n i m . Sci . 76:2641-2648. Keshavarz, K . , 1984. The effect of different dietary protein levels in the rearing and laying periods on performance of white Leghorn chickens. Poultry Sci . 63:2229-2240. Keshavarz, K . , and Jackson, M . E . , 1992. Performance o f growing pullets and laying hens fed low protein, amino acid-supplemented diets. Poultry Sci . 71:905-918. K i d d , M . T., 1996. L-threonine for poultry. K y o w a Hakko Technical Review-8. Nutri-Quest, Inc. Chesterfield, M O , U S A . K i d d , M . T., and B . J. Kerr, 1997. Threonine responses in commercial broilers at 30 to 42 days. J. A p p l . Poultry Res. 6:362-367. K i m , J. H . , W . T. Cho, I. S. Shin, C. J. Yang, and In K . Han, 1997. Partition o f amino acids requirement for maintenance and growth o f broiler. III. Tryptophan. A J A S 10:284-288. Koide, K . , and T. Ishibashi, 1995. Threonine requirement in female broilers affected by age and dietary amino acid levels. Jpn. Poult. Sci . 32:329-336. Lipstein, B . , S. Bornstein, and I. Bartov, 1975. The replacement o f some o f the soybean meal by the first-limiting amino acids in practical broiler diets. 3. Effects o f protein concentrations and amino acids supplementations in broiler finisher diets on fat deposition in the carcass. Br . Poult. Sci . 16:627-635. Longe, O. G . , 1984. Effects of increasing the fiber content o f a layer diet. Br . Poult. Sci . 25:187-193. Longe, O. G . , and G . O. Tona, 1988. Metabolizable energy values o f some tropical feedstuffs for poultry. Trop. Agric . (Trinidad) 65:358-360. 38 Matheson, N . K . , 1990. Mannose-based polysaccharides. Pages 371-413 in: Methods In Plant Biochemistry. P. M . Dey and J. B . Harborne, ed. V o l . 2. Carbohydrares. P. M . Dey, ed. Academic Press Ltd. , London, U K . McDonald , P., R. A . Edwards, and J. F . D . Greenhalgh, 1982. Animal nutrition, 3 r d Ed . Harlow, Essex: Longman. Meier, H . , and J. S. G . Reid, 1982. Reserve polysaccharides other than starch in higher plants. Pages 418-471 in: Plant Carbohydrates. F . A . Loewus and W . Tanner, ed. Springer-Verlag, N Y . Morales-Barrera, E . , E . Avila-Gonzalez, and J. L . Laparra-Vega, 1992. Effect o f threonine supplementation on practical broiler diets with different levels of arginine. Veterinaria-Mexico 23:223-226. Moran, JR., E . T., R. D . Bushong, and S. F . B i l g i l i , 1992. Reducing dietary crude protein for broilers while satisfying amino acid requirements by least-cost formulation: L ive performance, litter composition, and yield o f fast-food carcass cuts at six weeks. Poultry Sci . 71:1687-1694. Moreno, J. R., G . M . Cuca, and H . J. G . Herrera, 1993. Study of the effects o f threonine supplementation in broiler chickens from 0 to 3 weeks old. Archivos-Latinoamericanos-de-Produccion-Animal. 1:129-138. Morris , T. R., and E . Wethli, 1978. The tryptophan requirements of young laying pullets. Br . Poult. Sc i . 19:455-466. Morrison, M . A . , and A . E . Harper, 1960. Amino acid balance and imbalance: I V . Specificity of threonine in producing an imbalance in diets deficient in niacin and tryptophan. J. Nutr. 71:296-302. Moss, A . R., and D . I. Givens, 1994. The chemical composition, digestibility, metabolizable energy content and nitrogen degradability o f some protein concentrates. A n i m . Feed Sci . Technol. 47:335-351. National Research Council , 1984. Nutrient Requirements o f Poultry. 8 t h rev. ed. National Academy Press, Washington, D C . National Research Council , 1994. Nutrient Requirements of Poultry. 9 t h rev. ed. National Academy Press, Washington, D C . Ngoupayou, Ngou J. D . , 1984. Nutritional value o f palm kernel cake in broiler diets. Poult. Sci . 63 (Suppl. 1):155-156. (Abstr.) Nwokolo, E . N . , D . B . Bragg, and W . D . Kitts, 1976a. A method for estimating the mineral availability in feedstuffs. Poult. Sci . 55:2217-2221. Nwokolo , E . N . , D . B . Bragg, and W . D . Kitts, 1976b. The availability o f amino acids from palm kernel, soybean, cotton seed and rapeseed meal for the growing chick. Poult. Sci . 55:2300-2304. 39 Nwokolo , E . N . , and D . B . Bragg, 1977. Influence of phytic acid and crude fiber on the availability o f minerals from four protein supplements in growing chicks. Can. J. A n i m . Sci . 57:475-477. Nwokolo , E . N . , D . B . Bragg, and H . S. Saben, 1977. A nutritive evaluation o f palm kernel meal for use in poultry rations. Trop. Sci . 19:147-154. Ohtani, H , S. Saitoh, H . Ohkawara, Y . Akiba , K . Takahashi, and M . Horiguchi, 1989. Research Note: Production performance o f laying hens fed L-tryptophan. Poultry Sci . 68:323-326. Onifade, A . A . , and G . M . Babatunde, 1998. Comparison of the utilization o f palm kernel meal, brewers' dried grains and maize offal by broiler chicks. Br . Poult. Sc i . 39:245-250. Onwudike, O. C , 1986a. Palm kernel as a feed for poultry. 1. Composition o f palm kernel meal and availability of its amino acids to chicks. A n i m . Feed Sci . Technol. 16:179-186. Onwudike, O. C , 1986b. Palm kernel as a feed for poultry. 2. Diets containing palm kernel meal for starter and grower pullets. A n i m . Feed Sci . Technol. 16:187-194. Onwudike, O. C , 1986c. Palm kernel as a feed for poultry. 3. Replacement o f groundnut cake by palm kernel meal in broiler diets. A n i m . Feed Sci . Technol. 16:195-202. Onwudike, O. C , 1988. Palm kernel as a feed for poultry. 4. Use of palm kernel meal by laying birds. A n i m . Feed Sci . Technol. 20:279-286. Oruwari, B . M . , B . T. Sese, and O. O. Mgbere, 1995. The effect o f whole palm kernel on broiler performance and production cost: energy protein ratio. J. A n i m . Sci . 10:115-120. Osei, S. A . , and J. A m o , 1987. Research note: palm kernel cake as a broiler feed ingredient. Poult. Sci . 66:1870-1873. Panigrahi, S., and C . J. Powell , 1991. Effects of high rates of inclusion o f palm kernel meal in broiler chick diets. A n i m . Feed Sci . Technol. 34:37-47. Panigrahi, S., and B . S. Waite, 1998. Use of rations up to forty per cent palm kernel meal for egg production. Br . Poult. Sci . 39(Suppl): S37-S38. Parr, J. F. , and J. D . Summers, 1991. The effect o f minimizing amino acid excesses in broiler diets. Poultry Sci . 70:1540-1549. Penz, A . M . Jr., G . L . Colnago, and L . S. Jensen. 1997. Threonine supplementation o f practical diets for 3- to 6-wk-old broilers. J. App l . Poultry Res. 6:355-361. Pinchasov, Y . , C . X . Mendonca, and L . S. Jensen. 1990. Broiler chick response to low protein diets supplemented with synthetic amino acids. Poultry Sci . 69:1950-1955. 40 P O R L A , 1997. Porla palm oi l statistics. 16 t h ed. Palm O i l Registration and Licensing Authority. Ministry of Primary Industries, Malaysia. Rangel-Lugo, M , C . L . Su, and R. E . Austic, 1994. Threonine requirement and threonine imbalance in broiler chickens. Poult. Sci . 73:670-681. Rhodimet Nutrition Guide, 1993. Feed ingredients formulation in digestible amino acids, 2 n d edition 1993 Rhone-Poulenc Animal Nutrition. Robbins, K . R., 1987. Threonine requirement of the broiler chick as affected by protein level and source. Poultry Sci . 66:1531-1534. Rogers, S. R. , and G . M . Pesti, 1990. The influence o f dietary tryptophan on broiler chick growth and l ip id metabolism as mediated by dietary protein levels. Poultry Sci . 69:746-756. Smith, N . K . , Jr., and P. W . Waldroup, 1988a. Investigation of threonine requirements o f broiler chicks fed diets based on grain sorghum and soybean meal. Poultry Sci . 67:108-112. Smith, N . K . , Jr., and P. W . Waldroup, 1988b. Estimation o f the tryptophan requirement o f male broiler chickens. Poultry Sci . 67:1174-1177. Steinhart, H . , and M . Kirchgessner, 1984. Investigations on the requirement of tryptophan for broilers. Arch . Geflugelkd. 48:150-155. Stilborn, H . L . , and P. W . Waldroup, 1988. Min imum levels of dietary protein for growing broilers. Poultry Sci . 67(Suppl. l):36.(Abstr.) Summers, J. D . , 1993. Reducing nitrogen excretion of the laying hen by feeding lower crude protein diets. Poultry Sci . 72:1473-1478. Tasaki, I. 1983. Influence o f tryptophan deficiency and excess on egg production i n laying pullets. Jpn. Poult. Sci . 20:103-108. Thomas, O. P., A . I. Zuckerman, M . Farran, and C . B . Tamplin, 1986. Updated amino acid requirements o f broilers. Pages 79-85 in: Proceedings Maryland Nutrition Conference, College Park, M D . Thomas, O. P., M . Farran, C. B . Tamplin, and A . I. Zuckerman, 1987. Broiler Starter Studies: I. The threonine requirements of male and female broiler chicks. II. The body composition of males fed varying levels of protein and energy. Pages 38-42 in: Proceedings Maryland Nutrition Conference, College Park, M D . Thomas, O. P., M . Farran, and C. B . Tamplin, 1992. Broiler nutrition update: Threonine requirement for 3-6 week-old broilers. Pages 45-53 in: Proceedings Maryland Nutrition Conference, College Park, M D . U z u , G . , 1982. Limi t of reduction of the protein level in broiler feeds. Poultry Sci . 61:1557-1558. 41 Waldroup, P. W. , R. J. Mitchel l , J. R. Payne, and K . R. Hazen, 1976. Performance o f chicks fed diets formulated to minimize excess levels o f essential amino acids. Poultry Sci . 55:243-253. Webel, D . M . , S. R. Fernandez, C. M . Parsons, and D . H . Baker, 1996. Digestible threonine requirement o f broiler chickens during the period three to six and six to eight weeks posthatching. Poultry Sci . 75:1253-1257. Woodham, A . A . , and P. S. Deans, 1975. Amino acid requirements o f growing chickens. Br . Poult. Sci . 16:269-287. Yamazaki, M . , Y . Oka, H . Murakami, M . Takemase, M . Ando, and M . Yamazaki, 1997a. Available threonine requirement o f broiler chickens at two growing stages. Jpn b Poult. Sc i . 34:45-51. Yamazaki, M . , H . Ohguchi, H . Murakami, M . Takemase, S. Hijikuro, M . Ando, and M . Yamazaki, 1997b. Available threonine requirement o f laying hens. Jpn. Poult. Sci . 34:52-57. Yeong, S. W. , 1980. The nutritive value of palm oi l by-products for poultry. Proc. Abstr. First Asia-Australasia Animal Science Congress. 45:17. (Abstr.) Yeong, S. W. , 1983. Amino acid availability of palm kernel cake, palm oi l sludge and sludge fermented product (Prolima) in studies with chickens. M A R D I Res. B u l l . , 11:84-88. Yeong, S. W. , 1985. Palm oi l by-products as feeds for poultry. Pages 175-186 in: Proc. Nat l . Symp. On oi l palm by-products for agro-based industries, 5-6 November, 1985. Malaysia. Yeong, S. W. , and T. K . Mukherjee, 1983. The effect o f palm oi l supplementation in palm kernel cake-based diets on the performance o f broiler chickens. M A R D I Res. B u l l . , 11:378-384. 4 2 CHAPTER III Use of the Ideal Protein Concept to Investigate the Response of 0-3 Week Old Broiler Chicks to Different Levels of Threonine and Tryptophan in Chemically Defined Diets Summary Two starter broiler experiments were conducted to compare the efficacy o f diets based on essential amino acid ( A A ) profiles proposed by Blai r et al. (1977), N R C (1994) and Illinois Ideal Chick Protein (IICP) (Exp. 1) and to evaluate the responses o f broilers to three levels o f threonine and three levels of tryptophan (Exp. 2) by using the A A profile derived in Exp. 1. During a study o f broilers 0-2 weeks old (Exp. 1), three semi-purified diets (Diet 1 = N R C (1994); Diet 2 = IICP; Diet 3 = Blai r et al. (1977)) were formulated by using isolated soy protein and crystalline A A . The body weights of the birds fed Blair ' s diet were significantly higher (P < 0.05) at the end o f weeks 1 and 2 when compared with the other two diets. It was concluded that the diet based on A A profile proposed by Bla i r et al. (1977) could support better growth and it was appropriate for a follow-up study to examine the responses o f broiler chicks to threonine and tryptophan in a purified diet based on crystalline A A . In Exp. 2, three levels (90%, 100% and 110% o f N R C (1994)) of threonine and tryptophan were arranged in a 3 x 3 factorial manner to give nine chemically defined diets containing crystalline A A as the only source o f A A . The amounts o f the other A A in the diets were based on the Blai r et al. (1977) A A profile. Broiler chicks were fed a commercial diet during the first week o f age and experimental diets were fed from 1 to 3 weeks of age. The interactions between threonine and tryptophan for body weight (week 3), weight gain (week 2-3) and overall weight gain (week 1-3) were significant (P < 0.05). The differences of both body weight and body weight gain increased as the levels o f threonine and tryptophan increased. Weight gain of the birds was significantly lower (P < 0.05) at the highest levels o f both A A (110% threonine and 110% tryptophan). There were indications that the A A in the diets became imbalanced at high levels of both threonine and tryptophan. The results of Exp. 2 indicate that when formulating diets for 0-3 week old broilers, dietary levels o f threonine and tryptophan should be targeted at 65% of lysine (equivalent to 0.63% of digestible threonine) and 16% of lysine (equivalent to 0.16% of digestible tryptophan), respectively. These 43 data and other recent data indicate that N R C (1994) has overestimated the threonine requirements and underestimated the tryptophan requirements for 0-3 week old broiler chicks. K e y words: ideal protein, broiler, threonine, tryptophan. Introduction The requirements o f broilers and layers for commonly limiting amino acids ( A A ) such as lysine and methionine are well established as a result of numerous investigations. However, the recent availability o f feed grade threonine and tryptophan has increased the need for accurate estimates o f the requirements for these two potentially limiting A A . The published literature on threonine and tryptophan provide widely varying estimates of the requirements that cannot be readily explained. The requirement values o f 0-3 week old broilers for threonine range from 0.50 to 0.85% of the diet (or 2.8 to 3.7% o f CP) (Woodham and Deans, 1975; Robbins, 1987). On the other hand, tryptophan should be formulated at 0.14 to 0.28% of the diet (or 0.78 to 1.2% of CP) (Woodham and Deans, 1975; Abebe and Morris, 1990). The National Research Council ( N R C , 1994) recommended requirement values (0-3 weeks old broiler chicks) are 0.80% of the diet (3.48% of dietary crude protein) for threonine and 0.20% o f the diet (0.87% of dietary crude protein) for tryptophan. The N R C (1994) threonine requirement values for 0-3 week old broiler chicks was derived from research done in the 1980s, whereas published reports dated from 1947 to 1988 were used to derive tryptophan requirements for animals o f markedly different productive potential than the one existing today. Therefore, it is not certain whether poultry nutritionists in the feed industry can still use the requirement values for threonine and tryptophan recommended by the N R C (1994) in feed formulation. D ' M e l l o and Lewis (1970c) concluded that it is impossible to determine the chick's actual requirement for essential A A unless the diet is in good A A balance, since the A A pattern of the diet w i l l affect the chick's response to supplementation. The usual method o f determining the A A requirements involves the technique of adding graded concentrations of a single A A until a maximum response is obtained. According to D ' M e l l o and Lewis (1970c), this method is inadequate in providing an accurate assessment o f A A requirements, because the requirements for A A are interdependent. The interdependence in A A requirements have been reported between lysine and arginine (D 'Me l lo and Lewis, 1970a), leucine, isoleucine and valine 44 ( D ' M e l l o and Lewis, 1970b) and threonine and tryptophan ( D ' M e l l o and Lewis, 1970c). Furthermore, A A requirements o f poultry are influenced by a multitude o f dietary, environmental and genetic factors. A l l o f these factors have led to the development o f the ideal protein concept. Ideal protein is defined as the perfect A A profile or balance in terms o f dietary concentrations among the essential A A to meet all A A requirements for a particular species or age without any excesses or deficiencies. In addition, formulating diets according to the ideal protein concept allows for the most efficient and economical use of dietary protein by maximizing nitrogen utilization and minimizing nitrogen excretion (Mack et al, 1999). Therefore, the objectives o f the present experiments were: 1) to compare the efficacy o f the essential A A profiles present in the Blai r et al. (1977), N R C (1994) and Illinois Ideal Chick Protein (IICP) (Baker and Han, 1994) recommended diets and; 2) using the A A profiles that support better growth, to evaluate the responses o f 0-3 week old broiler chicks to different dietary levels o f threonine and tryptophan. Materials & Methods Bird Management, Diet and Data Collection Experiment 1 A total o f 108 day old male (Peterson x Arbor Acres) broiler chicks was obtained from a local commercial hatchery. A l l birds were vaccinated against Marek's disease and housed in battery brooding units (Petersime Incubators Co. , Gettysburg, O H 45328) with 23 h o f light daily (7:00 a.m. to 6:00 a.m.). Group feed intake and individual body weights were recorded at the start o f the experiment and then weekly for the two week study. Three semi-purified diets (Diet 1 = N R C 1994; Diet 2 = IICP and Diet 3 = Blai r et al. (1977)) with different A A profiles (Table 3.1) were used and the compositions of the diets are shown in Table 3.2. There were four replications (nine chicks per replication) for each dietary treatment. Cornstarch and corn o i l served as the main energy source, whereas isolated soy protein (NURISH® 1500) and crystalline A A were used as the only source o f A A . Hardwood sawdust was used in the original study by Bla i r et al. (1977). Therefore, it was also included in this current study and the sawdust used in Diet 3 was obtained from a local lumberyard and ground to pass through a 2 mm screen size 45 (Tyler Standard Screen Scale, Ohio, U S A ) . It was later extracted with dichloromethane for 24 h to remove any resin or toxin. Table 3.1 Experiment 1: Amino acid ratio proposed by different researchers N R C 1 I I C P Z Blair et al.i Lysine 100 100 100 Arginine 114 105 120 Histidine 32 32 40 Methionine 46 36 55 Cystine 36 36 14 Phenylalanine 66 55 70 Tyrosine 56 50 70 Threonine 73 67 70 Leucine 109 109 140 Isoleucine 73 67 80 Valine 82 77 86 Tryptophan 18 16 20 Glycine 114 65 100 Proline 55 44 70 'National research council for Poultry, 1994 I^llinois ideal chick protein. Baker and Han, 1994. 3B\air etal. 1977 Table 3.2 Experiment 1: Composition of diets (% of diet). Ingredient N R C 1994 IICP (1994) Blai r et al. (1977) Corn starch 54.44 52.71 38.16 Isolated soy protein 1 15.71 12.57 16.80 Amino acid mixtures 2 3.69 4.00 3.86 L-glutamic acid 10.64 14.97 9.26 Corn oi l 5.85 6.08 12.25 Cellulose 3.00 3.00 3.00 Mineral-vitamin mixtures 3 6.67 6.67 6.67 Extracted saw dust 4 0.00 0.00 10.00 'NURISH® 1500, a gift from Protein Technologies International™. Checkerboard Square, St. Louis, MO, USA. 2A11 diets were made isonitrogenous (3.68% N) by adjusting the level of L-glutamic acid. Dietary percentages of all amino acids can be calculated by multiplying the ratios in Table 3.1 by 1.2%. 3Supplied per kg of diet: 2 g choline chloride (100%), 20 mg thiamin HCL, 10 mg riboflavin, 30 mg calcium pantothenate, 50 mg niacin, 6 mg pyridoxine HCL, 4 mg folacin, 0.6 mg biotin, 0.04 mg vitamin B12, 100 mg inositol, 2 mg para-aminobenzoic acid, 5,200 IU vitamin A, 600 ICU vitamin D 3 , 20 IU vitamin E, 2 mg vitamin K, 125 mg antioxidant (ethoxyquin), 5 g NaCI, 33 g Ca3(P04)2, 0.6 g MgO, 4.5 g K 2 C 0 3 , 5 g NaHC0 3, 5 g Al(OH)3, 650 mg MnS0 4.H 20, 50 mg ZnO, 567 mg FeS04.7H20, 20 mg CuS04.5H20, 0.4 mg Na2Se03, 40 mg KI, 1 mg CoS04.7H20, 9 mg H 3 B0 3 , 9 mg Na2Mo04.2H20. 4Extracted with dichloromethane for 24 h. Isolated protein was used at such levels that the need for supplementation with crystalline A A was minimized. The isolated soy protein contained 83.2% protein and the following A A 46 content (% o f product): alanine (3.5%), arginine (6.2%), aspartic acid (9.6%), cysteine (1.0%), glutamic acid (15.7%), glycine (3.4%), histidine (2.2%), isoleucine (4.0%), leucine (6.7%), lysine (5.2%), methionine (3.0%), phenylalanine (4.3%), proline (4.2%), serine (4.3%), threonine (3.1%), tryptophan (1.1%), tyrosine (3.1%), valine (4.2%). A l l A A were supplied as L-isomers except methionine, which was supplied as the D L -isomer. Free-base forms o f A A were used with the exception o f lysine which was provided as the hydrochloride. Feed-grade sources were used for lysine HC1 (78.8%), threonine (98.5%), and methionine (98%); the remaining A A were pharmaceutical grade. The true digestibilities of A A in the isolated soy protein and free A A were assumed to be 100% (Chung and Baker, 1992). A l l diets were made isonitrogenous (3.68% N) by adjusting the level o f L-glutamic acid. Apparent metabolizable energy and digestible lysine were set at 3,500 kcal/kg and 1.2% (of diet), respectively. Feed and water were offered ad libitum during the 14-day experiment. Experiment 2 A total o f 180 male day old broiler chicks (Peterson x Arbor Acres) was used in the study. A l l birds were vaccinated against Mareks disease and housed in battery brooding units (Petersime Incubators Co. , Gettysburg, O H 45328) with 23 h of light daily (7:00 a.m. to 6:00 a.m.). A l l o f the chicks were wing-banded at day old and fed a commercial broiler starter diet for the first week of life. On the morning o f Day 8, all chicks were individually weighed. The chicks o f approximately same weight were randomly assigned to battery pens. Individual body weight and group feed intake were measured weekly. There were a total o f nine diets fed to birds from 1-3 weeks o f age (Table 3.4), with four replications o f five birds per diet. Since locally available feed ingredients were used to formulate a practical broiler diet, it was not possible to formulate a diet with less than 90% of the National Research Counci l ( N R C , 1994) broiler recommendations for threonine and tryptophan. A s a result, levels o f 90%, 100% and 110% of the N R C (1994) recommendations for threonine and tryptophan were used. In the previous ideal protein study (experiment 1), we found that the Blai r et al. (1977) A A profile gave the best growth response. Therefore, the Blai r et al. (1977) A A profile was used in this experiment but the amounts o f threonine and tryptophan in the diets were adjusted to 90%, 100% and 110% of the N R C (1994) recommendations. Three levels o f threonine and three levels o f tryptophan were arranged in a factorial manner as shown in Table 3.3. 47 Table 3.3 Exper iment 2: Arrangement of diets \ T H R : 9 0 % N R C o r 0 .72% of the diet T H R : 1 0 0 % N R C o r 0 .80% of the diet T H R : 1 1 0 % N R C o r 0 .88% of the diet T R P : 9 0 % N R C o r 0 .18% of the diet 0.72%THR 10.18% TRP Diet 1 0.80%THR/0.18%TRP Diet 2 0.88%THR/0.18%TRP Diet 3 T R P : 1 0 0 % N R C o r 0 .20% of the diet 0.72%THR /0.20% TRP Diet 4 0.80%THR /0.20% TRP Diet 5 0.88%THR /0.20% TRP Diet 6 T H R : 1 1 0 % N R C or 0 .22% of the diet 0.72%THR /0.22% TRP Diet 7 0.80%THR /0.22% TRP Diet 8 0.88%THR /0.22% TRP Diet 9 'Threonine = Thr; Tryptophan = Trp; NRC (1994). Nutrient composition o f the diets is shown in Table 3.4. Cornstarch and o i l served as the main energy sources, whereas crystalline A A were used as the only source o f A A . Contrary to the previous study, the diets were based on crystalline A A rather than partly on isolated soy protein. The intention was to provide more exact details of the A A contents o f the diets, and to aid with the interpretation of the results. A l l A A were supplied as L-isomers except methionine, which was supplied as the DL-isomer. Free-base forms of A A were used with the exception o f lysine which was provided as the hydrochloride. Feed-grade sources were used for lysine HC1 (78.8%), threonine (98.5%), and methionine (98%); the remaining A A were pharmaceutical grade. The true digestibility o f free A A was assumed to be 100% (Izquierdo et al, 1988; Chung and Baker, 1992; Zhang and Parsons, 1993). A l l diets were made isonitrogenous (2.83% N) by adjusting the level of L-glutamic acid. Apparent metabolizable energy and digestible lysine were set at 3,644 kcal/kg and 1.10% (of diet), respectively. The digestible lysine level was reduced from 1.20% to 1.10% of the diet, because several recent publications indicated that 1.10% lysine is required by starter broilers (Han and Baker, 1991, 1993; Rhodimet Nutrition Guide, 1993; Vazquez and Pesti, 1997). Feed and water were offered ad libitum during the experiment. 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C5, O a 'o i n (D >> o O CM 60 60 , > a a vo in 49 Statistical Analyses Experiment 1 This was a completely randomized design experiment. Data were subjected to analysis of variance ( A N O V A ) procedures appropriate for completely randomized design by using the General Linear Models ( G L M ) procedure o f SAS® software (SAS Institute, 1996). I f treatments were found to be significantly different, Tukey's multiple range test (Snedecor and Cochran, 1980) was used to determine the significant differences between treatment least-square means. Experiment 2 This was a completely randomized design with a factorial arrangement o f treatments. Data were subjected to analysis o f variance ( A N O V A ) procedures using the General Linear Models ( G L M ) procedure o f SAS® software (SAS Institute, 1996). I f treatments were found to be significantly different, Tukey's multiple range test (Snedecor and Cochran, 1980) was used to determine the differences between treatment least-square means. Results Experiment 1 There were no significant differences in body weight among day-old chicks in the different treatment groups (Table 3.5). L ive body weight o f the birds fed Diet 3 (Blair et al, 1977) was significantly higher (P < 0.05) at days 7 and 14 when compared with the birds fed the other two diets (Table 3.5). N o significant differences in body weight were found between Diets 1 ( N R C , 1994) and 2 (IICP). Body weight gain and feed consumption followed the same trend (Tables 3.5 and 3.6). Feed conversion efficiencies were not significant among birds fed Diets 1-3. Overall, during the 2 week study (Table 3.7) birds fed diet based on the A A profile suggested by Blai r et al. (1977) gained significantly more weight (P < 0.05), and ate significantly more (P < 0.05) than those fed diets based on the N R C (1994) and IICP A A profiles, while the differences in feed conversion efficiency were small. 50 Table 3.5 Experiment 1: The effect of different amino acid profiles on live body weight and body weight gain by 0-1 and 1-2 weeks old chicks. Live body weight (g) Body weight gain (g) Day 1 Day 7 Day 14 Week 0-1 Week 1-2 N R C (1994) 41.8 86.3" 175 b 44.3 b 88.4 b IICP (1994) 1 40.3 82.6 b 185 b 41.7" 103.3 b Blair et al. (1977) 40.7 103.5 8 233 a 61.2 a 131.5 a Overall mean 40.9 90.8 198 49.1 107.7 Overall S E M 2 0.4 2.0 9.7 2.2 7.2 Illinois ideal chick protein (Baker and Han, 1994) Standard error of the mean; data represent mean of four replications of nine chicks. 'bTreatment means with different superscripts within a column are significantly different at PO.05. Table 3.6 Experiment 1: The effect of different amino acid profiles on feed consumption and feed conversion efficiency by 0-1 and 1-2 week old chicks. Feed intake (g/bird) Feed conversion efficiency Week 0-1 Week 1-2 Week 0-1 Week 1-2 N R C (1994) 53.9 b 115.5 b 0.82 0.74 IICP (1994) 1 48.2 b 125.6 b 0.85 0.82 Bla i r et al. (1977) 64.2* 176.2 a 0.95 0.74 Overall mean 55.4 139.1 0.87 0.77 Overall S E M 2 ••i — 2.4 9.2 0.03 0.02 Illinois ideal chick protein (Baker and Han, 1994) Standard error of the mean; data represent mean of four replications of nine chicks. ''"Treatment means with different superscripts within a column are significantly different at PO.05. Table 3.7 Experiment 1: Performance of chicks fed different amino acid profile diets during the first two weeks of age1. Weight gain (g) Feed intake (g/bird) Feed conversion efficiency (gain/feed) N R C (1994) 132.7 b 169.4 b 0.77 IICP (1994) 2 145.0 b 173.9 b 0.83 Bla i r et al. (1977) 192.8 a 240.4 a 0.79 Overall mean 156.8 194.6 0.80 Overall S E M 3 9.9 10.7 0.03 Data represent mean of four replications of nine chicks. Illinois ideal chick protein (Baker and Han, 1994) 3Standard error of the mean a'bTreatment means with different superscripts within a column are significantly different at PO.05. 51 Experiment 2 The level of threonine and tryptophan did not significantly (P > 0.05) affect body weight o f birds at 2 weeks o f age, weight gain during weeks 1-2, daily feed intake or overall feed intake (week 1-3) (Tables 3.8 and 3.9). When the birds reached 3 weeks o f age, their body weight was not affected by threonine levels. However, body weight was significantly lower (P < 0.05) when tryptophan in the diet reached 110% of the N R C recommendation. The interaction between threonine and tryptophan for body weight (week 3) was also significant (P < 0.05). Weight gain during weeks 2-3 was significantly lower (P < 0.05) at the 110% threonine and 110% tryptophan levels. Overall weight gain (weeks 1-3) was not significantly affected (P > 0.05) by the threonine level as shown in Table 3.8, but it decreased significantly (P < 0.05) as the level o f tryptophan increased. The gain/feed ratio was not significantly affected (P > 0.05) by the threonine level but it was significantly decreased (P < 0.05) with the increased tryptophan levels (Table 3.9). The interactions between threonine and tryptophan for body weight (week 3), weight gain (week 2-3) and overall weight gain (week 1-3) were significant (P < 0.05). A s a result, each situation was interpreted separately by using Figures 3.1 and 3.2 for body weight (week 3), Figures 3.3 and 3.4 for weight gain (week 2-3) and Figures 3.5 and 3.6 for the overall weight gain (week 1-3). Body weight at 3 weeks o f age was significantly lower (P < 0.05) i n birds fed diets containing 110% tryptophan when threonine level reached 110% of the N R C (1994) recommendation (Figure 3.1) and it was significantly lower (P < 0.05) in birds fed diets containing 110% threonine when tryptophan level reached 110% of the N R C (1994) recommendation (Figure 3.2). Diets based on tryptophan at 100% and 110% of the N R C (1994) recommendations significantly reduced (P < 0.05) weight gain (week 2-3) o f birds when threonine was set at 100% and 110% of N R C (1994) recommendation (Figure 3.3). On the other hand, only birds fed diets containing the highest level of both threonine and tryptophan (110% N R C ) had significantly lower (P < 0.05) weight gain during weeks 2-3 (Figure 3.4). Overall weight gain (weeks 1-3) was significantly reduced (P < 0.05) when the diets contained 100% of threonine and 110% of tryptophan or 110% of threonine and 110% o f tryptophan (Figure 3.5). 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In fact, Baker and Han (1994) have shown that using digestible A A rather than total A A requirement data for the N R C (1994) did not lower any of the ratios in Table 3.1 below those calculated on a total A A basis. A n earlier study by Baker and Han (1994) showed that there were no significant differences in growth performance when birds were fed a diet based on either the N R C (1994) or IICP A A profiles. Since the IICP had also lower A A ratios relative to lysine, the Illinois group concluded that some of the A A ratios set by the N R C (1994) were too high and the ratios set by the IICP were better. However, the results of the present study disagreed with the conclusion of Baker and Han (1994) and showed that birds fed the Baker and Han (1994) diet did not have a better growth performance than those fed the N R C (1994) diet. Compared to the A A profiles proposed by the N R C (1994) and the Illinois group (IICP), the profile suggested by Blai r et al. (1977) contained a higher ratio of most essential A A relative to lysine except total sulfur A A , threonine and glycine. The diet based on Blair ' s A A profile was the diet that gave the best weight gain for the chicks. A s shown in Tables 3.6 and 3.7, birds fed the Bla i r ' s diet ate significantly more (P < 0.05) throughout the 2-week study than birds fed the other two diets. It is obvious that lower feed intake reduced the weight gain o f birds fed diets based on the N R C (1994) and IICP A A profiles. The sawdust in the Blair ' s diet may have improved the texture o f the diet, but this does not fully explain the greater feed intake. Moreover, all diets were formulated to have the same energy and nitrogen levels. It is not likely that the differences in feed consumption were due to a deficiency of energy or nitrogen in one o f the diets. However, it is wel l known that the A A balance in general also plays an important role in maximizing the feed consumption of diets. Studies regarding how A A imbalances depress feed intake are well-documented (Knight et al, 1991; D ' M e l l o , 1993; 1994). I f the diet is not well balanced in A A , birds are not likely to consume enough of it to grow optimally. A A imbalances could be a serious problem, especially for birds fed a semi-purified or purified crystalline A A diet. Most A A in a practical type diet are presented as intact proteins, and 61 excess A A (up to 150% of the requirement) under these conditions do not seem to depress the birds' performance (Baker, 1997). Bach Knudsen and J0rgensen (1986) concluded that the absorption of free crystalline A A is more rapid than that of peptide-bound A A due to the fact that the rate o f passage o f the free A A from the stomach to the small intestine is faster than that o f the peptide-bound A A . A s a result, in crystalline A A diets where 100% of all A A are assumed to be immediately available for absorption, small changes i n A A balance and A A excesses could have profound effects on the overall efficiency of the diet and particularly on the ad libitum feed intake (Dudley-Cash, 1998). The daily live-weight gain of the birds fed Blair ' s diet was 13.77 g in the present experiment which was close to the gain by birds fed the practical diet (14.4 g) (Blair et al, 1977). On the other hand, birds fed diets based on the A A profiles o f the N R C (1994) and IICP gained only 9.55 and 10.36 g per day, respectively, in the present experiment. Some o f the A A ratios proposed by Blair et al. (1977), however, might have also been too high. A t this point, it is difficult to know whether some o f the A A ratios could be reduced, however, the ratios are very similar to those o f the Rhodimet Nutrition Guide (1993). The interactions between threonine and tryptophan shown in Figures 3.1 to 3.6 of Experiment 2 clearly show that both body weight and weight gain o f the birds decreased as the level o f threonine and tryptophan in the diets increased. The differences o f both body weights and body weight gains of birds increased as the level o f threonine and tryptophan increased. Weight gain was lowest at the highest levels of both A A (110% of threonine and 110% of tryptophan). This finding agrees with that reported by D ' M e l l o and Lewis (1970c), who showed that there were interactions between threonine and tryptophan. Interactions involving other A A have been reported to exist between lysine and arginine ( D ' M e l l o and Lewis, 1970a), and leucine, isoleucine and valine (D 'Mel lo and Lewis, 1970b). D ' M e l l o and Lewis (1970c) also stated that the adverse effects o f excess threonine were reversed by appropriate supplementation o f the diet with tryptophan. This effect o f tryptophan, however, was not observed in our study. The results o f the present study indicate that as the level o f threonine and tryptophan increase, A A in the diets become unbalanced, leading to a reduction in feed intake as shown in Table 3.9. This agrees with the findings of Knight et al. (1991) and D ' M e l l o (1993; 1994) that A A imbalance affects animal performance by depressing feed intake. Excess threonine has been shown by Moreno et al. (1993) and Ishibashi et al. (1998) to reduce the performance o f broilers and layers. Similarly, Tasaki (1983) reported poorer layer performance with excess tryptophan. 62 Based on the observations of the threonine and tryptophan interaction on weight gain (Figures 3.5 and 3.6) and gain/feed ratio (Table 3.9), the results obtained from the present study (Experiment 2) indicate that the dietary level of threonine and tryptophan in 1-3 week old broiler diets should be targeted at 65% and 16% of lysine, respectively. That is, if the dietary level of digestible lysine was set at 0.95% (equivalent to the requirement value of 1.1% total lysine in the diet recommended by the NRC (1994)) (Table 3.10), then dietary level of digestible threonine and digestible tryptophan should be set at 0.63% and 0.16%, respectively. When converted to total requirement values (Table 3.10), this is equal to 0.73% for threonine and 0.19% for tryptophan in the diet or 4.13% of CP for threonine and 1.07% of CP for tryptophan. Using a milo-soybean meal-corn gluten meal basal diet, Yamazaki et al. (1997) concluded that 1-3 week broiler chicks require 0.65% available threonine in the diet, which is the same as the estimate obtained in this study. Thomas et al. (1986, 1987) found that the optimum level of total threonine for feed efficiency was 0.73% (1986), and in a further study Thomas et al. (1987) confirmed that the threonine requirement for male broilers was 0.72%. Recently, Thomas et al. (1992) reported that male and female broilers aged 0-3 week need 0.77% and 0.71% of dietary threonine (average 0.74%). On the other hand, Holsheimer et al. (1994) concluded that in a 16% protein maize-soybean meal diet supplemented with essential AA and non-essential AA, an improvement in gain and feed efficiency was observed for both sexes of broiler chicks, when the dietary threonine content was increased to 0.725% until 3 weeks of age. In another study, Kidd et al. (1996) found that threonine levels ranging from 92% (0.736% of diet) to 112% (0.896% of diet) of NRC (1994) recommendations failed to improve weight gain of 1-21 day old broiler chicks. The estimated requirement for total threonine found in the present study also falls into the range of 0.67% to 0.77% reported by Rangel-Lugo et al. (1994). The estimate derived from the present study, however, is higher than those of Hewitt and Lewis (1972) (0.53% of diet), Woodham and Deans (1975) (0.50-0.52% of diet) and Leeson and Summers (1997) (0.70% of diet). On the other hand, Morales-Barrera et al. (1992), Rhodimet Nutrition Guide (1993), Austic (1994) and the NRC (1994) reported that about 0.80% dietary threonine was required by starting chicks, which is higher than the estimated threonine requirement obtained in the present study. When expressed as % of CP, the estimated requirement value obtained in the present study is higher than most of the reported values, except that of Holsheimer et al. (1994) and Koide and Ishibashi (1995). A value of 4.53% of CP was reported by Holsheimer et al. (1994). Recently, data presented by Koide and Ishibashi (1995) 63 indicate that dietary threonine requirements expressed as a percentage of C P range from 3.96% to 4.13% C P . Based on the findings o f the present and other recent studies, there are indications that the N R C (1994) may have overestimated the requirement o f 0-3 week old broilers for threonine. The National Research Council ( N R C , 1994) recommendation for tryptophan requirement o f 0-3 week old broilers is 0.20% of the diet. However, Hewitt and Lewis (1972) indicated that male broiler chicks aged 7 to 21 days require not more than 0.17% tryptophan i n their diet. Woodham and Deans (1975) found that chicks required less than 0.14% tryptophan in their diet and Smith and Waldroup (1988) concluded that a diet containing 0.16% tryptophan could fulfil a growing chick's requirement for tryptophan. On the other hand, several researchers have reported a higher requirement value for tryptophan (Freeman, 1979; Steinhart and Kirchgessner, 1985; Abebe and Morris, 1990; Parr and Summers, 1991; Han et al, 1991). More recently, tryptophan requirement values o f 0.225%, 0.23% and 0.24% of diet were reported by Thomas et al (1992), Rhodimet Nutrition Guide (1993) and Austic (1994), respectively. Boomgaardt and Baker (1971) discovered that when tryptophan requirement was expressed as percent o f the diet, the requirement increased with dietary protein level. However, when expressed as a % o f C P , the requirement stayed constant at 0.87% at five different protein levels. Smith and Waldroup (1988) reported a value o f 0.80% of C P , whereas Hewitt and Lewis (1972) suggested 0.94% of C P . A l l o f these values are lower than the findings obtained from the present study. However, Abebe and Morris (1990) concluded that the amounts o f tryptophan required for the maximum growth and feed efficiency were each linear functions o f dietary protein concentration (12 g/kg C P or 1.2% CP) . Abebe and Morris (1990) also concluded that a fixed ratio o f tryptophan to protein should be used in practical diet formulation, rather than a minimum dietary concentration of tryptophan. More recently, Austic (1994) and Leeson and Summers (1997) suggested a value of 1.1% and 0.9% of C P , respectively. Even though the estimated requirement value o f 0-3 week old broilers for tryptophan found i n the present study agrees with that of the N R C (1994) recommendations, several recent studies indicate that the N R C (1994) may have underestimated the requirement o f 0-3 week old broilers for tryptophan. Further study is necessary to explain these disagreements. Based on the findings o f experiment 2, the ratios o f threonine and tryptophan relative to lysine were revised from 70% and 20% of lysine to 65% and 16% o f lysine, respectively. The ratio o f threonine relative to lysine (65% of lysine) obtained in the present study is slightly 64 higher than 62% reported by Austic (1994), but lower than 67% reported by Baker (1997). The ratio o f tryptophan relative to lysine (16% of lysine) obtained in the present study agrees with the finding o f Baker (1997), but is lower than the 18% (of lysine) recommended by Austic (1994). The revised A A ratios were used as the base for a deductive approach to estimate the A A requirements (Table 3.10) Table 3.10 Suggested digestible and total amino acid requirements for 0-3 week old broiler chicks after adjustments were made to Blair et al. (1977)1,1 Amino acids ( A A ) Average D i g . (%) 3 Blair et al. (1977) Blair et al. (1977) Blai r et al. (1977) N R C (1994) Ratio of A A t o lysine (%) Dig . req. (% of diet) Total req. (% o f diet) Total req. (% o f diet) Lysine 87.8 100 0.97 1.10 1.10 Methionine 91.3 55 0.53 0.58 0.50 T S A A 4 87.5 69 0.67 0.77 0.90 Threonine 86.7 65 0.63 0.73 0.80 Tryptophan 85.0 16 0.16 0.19 0.20 Arginine 93.8 120 1.16 1.24 1.25 Isoleucine 90.2 80 0.78 0.86 0.80 Leucine 90.9 140 1.36 1.50 1.20 Valine 88.2 86 0.83 0.94 0.90 The amino acid ratios of threonine and tryptophan proposed by Blair et al. (1977) were adjusted to reflect the results obtained in this study 2 Digestible lysine requirement was set at 0.97% of the diet (or 1.10% total Lys) so that comparison with the NRC (1994) is possible. Digestible amino acid requirements for the rest of the amino acids were derived from each profile by multiplying the ratios by 0.97% digestible lysine. In converting from digestible amino acid requirement to total amino acid requirements, it was assumed that a corn-soybean meal diet with 22 to 23% CP would be fed (83% of CP from soybean meal and 17% of CP from corn) (Baker et al., 1993) 3 Average true amino acid digestibility in a 22 to 23% CP corn-soybean meal diet (Rhodimet Nutrition Guide, 1993) 4 Total sulfur amino acid (methionine + cystine) There were some differences between the A A levels recommended by the N R C (1994) and the adjusted Blai r et al. (1977) estimated requirements (Table 3.10). The proposed requirements for arginine and tryptophan were very close between N R C (1994) and the adjusted Bla i r A A profiles. Based on the adjusted Blair ' s A A profile, the estimated requirements for methionine, isoleucine, leucine and valine were higher than those o f the N R C (1994) but lower than those o f the N R C (1994) for total sulfur A A and threonine. Recently, Beck et al. (1998) reported that the cystine requirement recommended by the N R C (1994) is too high. They found that cystine supplementation o f a basal diet (0.25% cystine) did not improve the performance o f birds. The great discrepancy for the estimated leucine requirement between the two A A profiles indicates that further research in this area is warranted. 65 Conclusions Broiler chicks fed diets based on AA profiles proposed by the NRC (1994) and the Illinois group (Baker and Han, 1994) did not grow at the same rate as those fed diets based on the AA profile proposed by Blair et al. (1977). The poorer performance of the birds fed diets based on the NRC (1994) and the IICP (Baker and Han, 1994) was likely caused by the significantly lower feed intake that might be due to an imbalance of AA in these diets. The results of experiment 2 indicate that when formulating diets for 0-3 week old broilers, dietary levels of threonine and tryptophan should be targeted at 65% of lysine (equivalent to 0.63% of digestible threonine in the diet or 0.73% of total threonine in the diet or 4.13% of CP) and 16% of lysine (equivalent to 0.16% of digestible tryptophan in the diet or 0.19% of total tryptophan in the diet or 1.07% of CP), respectively. Recent studies conducted here and elsewhere point strongly to the fact the NRC (1994) has overestimated the requirement of threonine for 0-3 week old broilers. On the other hand, even though the results of the present study supported the NRC (1994) tryptophan recommendation for 0-3 week old broilers, several recent studies disagree and indicate that broiler chicks require higher levels of tryptophan in their diets. Since data on threonine and tryptophan requirement for broiler chicks are not well established, more research, especially for broilers beyond 3 weeks of age, should be conducted in the future. Moreover, since the present two experiments were not conducted using practical ingredients, another study should be conducted to evaluate the responses of poultry to different levels of threonine and tryptophan in practical diets. The results from these studies should have implications with regard to the supplementation of normal and reduced protein diets with commercial AA. References Abebe, S., and T. R. Morris, 1990. Effects of protein concentration on responses to dietary tryptophan by chicks. Br. Poult. Sci. 31:267-272. Austic, R. E., 1994. Update on amino acid requirements and ratios for broilers. Pages 114-120 in: Proceedings of the Maryland Nutrition Conference, College Park, MD. Bach Knudsen, K. E., and H. Jorgensen, 1986. Use of synthetic amino acids in pig and poultry diets. Pages 215-225 in: Recent Advances in Animal Nutrition. W. Haresign and D.J.C. Cole, ed. Butterworths, London. 66 Baker, D . H . , and Y . Han, 1994. Ideal amino acid profile for broiler chicks during the first three weeks posthatching. Poultry Sci . 73:1441-1447. Baker, D . H . , C . M . Parsons, S. Fernandez, S. Aoyagi , and Y . Han, 1993. Digestible amino acid requirements o f broiler chickens based upon ideal protein considerations. Pages 22-32 in: Proceedings o f the Arkansas Nutrition Conference, Fayetteville, A R . Baker, D . H . , 1997. Pages 1-24 in: Ideal amino acid profiles for swine and poultry and their applications in feed formulation. Biokyowa Publishing Co. , St. Louis, M O . Beck, C . R. , R. H . Harms, and G . B . Russell, 1998. Is the cystine content of the diet o f concern for broilers from 0 to 21 days o f age? J. App l . Poultry Res. 7:233-238. Blair , R. , S. McKenzie , and D . J. W . Lee, 1977. A purified diet for one-day-old chicks. Br . Poult. Sci . 18:129-136. Boomgaardt, J., and D . H . Baker, 1971. Tryptophan requirement of growing chicks as affected by dietary protein level. J. A n i m . Sci . 33:595-599. Chung, T. K . , and D . H . Baker, 1992. Apparent and true digestibility o f a crystalline amino acid mixture and o f casein: comparison of values obtained with ileal-cannulated pigs and cecectomized cockerels. J. A n i m . Sci . 70:3781-3790. D ' M e l l o , J. P. F. , 1993. Amino acid supplementation o f cereal-based diets for non-ruminants. A n i m . Feed Sci . Technol. 45:1-18. D ' M e l l o , J. P. F. , 1994. Amino acid imbalances, antagonisms and toxicities. Pages 63-97 in: Amino Acids In Farm Animal Nutrition. J. P. F . D ' M e l l o , ed. C A B Intl., Wellingford, Oxon, U K . D ' M e l l o , J. P. F. , and D . Lewis, 1970a. Amino acid interactions in chick nutrition. I. The interrelationship between lysine and arginine. Br . Poult. Sci . 11:299-311. D ' M e l l o , J. P. F. , and D . Lewis, 1970b. Amino acid interactions in chick nutrition. II. Interrelationship between leucine, isoleucine and valine. Br . Poult. Sci . 11:313-323. D ' M e l l o , J. P. F . , and D . Lewis, 1970c. Amino acid interactions in chick nutrition. III. Interdependence in amino acid requirements. Br . Poult. Sci . 11:367-385. Dudley-Cash, W . A . , 1998. Application o f ideal amino acid profiles for formulation examined. Feedstuffs 70(1):10-11,18. Freeman, C . P., 1979. The tryptophan requirement o f broiler chicks. Br . Poult. Sci . 20:27-37. Han, Y . , and D . H . Baker, 1991. Lysine requirement o f fast and slow growing broiler chicks. Poultry Sci . 70:2108-2114. 67 Han, Y . , and D . H . Baker, 1993. Effects of sex, heat stress, body weight and genetic strain on the lysine requirement o f broiler chicks. Poultry Sci . 72:701-708. Han, Y . , H . Suzuki, and D . H . Baker, 1991. Histidine and tryptophan requirement of growing chicks. Poultry Sci . 70:2148-2153. Hewitt, D. , and D. Lewis, 1972. The amino acid requirements o f the growing chick. I. Determination o f amino acid requirements. Br . Poult. Sci . 13:449-463. Holsheimer, J. P., P. F . G . Vereijken and J. B . Schutte, 1994. Response o f broiler chicks to threonine-supplemented diets to 4 weeks o f age. Br . Poult. Sci . 35:551-562. Ishibashi, T., Y . Ogawa, T. Itoh, S. Fujimura, K . Koide, and R. Watanabe, 1998. Threonine requirements of laying hens. Poultry Sci . 77:998-1002. Izquierdo, O. A . , C . M . Parsons, and D . H . Baker, 1988. Bioavailability o f lysine in L -lys ine .HCL. J. A n i m . Sci . 66:2590-2597. K i d d , M . T., B . J. Kerr, J. D . Firman, and S. D . Bol ing, 1996. Growth and carcass characteristics of broilers fed low-protein, threonine-supplemented diets. J. App l . Poultry Res. 5:180-190. Knight, C . D . , J. J. Dibner, and F. J. Ivey, 1991. Crystalline amino acids diets for chicks: History and future. Pages 19-29 in: Proceedings Maryland Nutrition Conference, College Park, M D . Koide, K . , and T. Ishibashi, 1995. Threonine requirement in female broilers affected by age and dietary amino acid levels. Jpn. Poult. Sci . 32:329-336. Leeson, S., and J. D . Summers, 1997. Commercial poultry nutrition. Leeson, S., and J. D . Summers ed. 2 n d edition. University Books, Guelph, Ontario, Canada. Mack, S., D . Bercovici , G . De Groote, B . Leclercq, M . Lippens, M . Pack, J. B . Schutte, and S. V a n Cauwenberghe, 1999. Ideal amino acid profile and dietary lysine specification for broiler chickens of 20 to 40 days o f age. British Poult. Sci . 40:257-265. Morales-Barrera, E . , E . Avila-Gonzalez, and J. L . Laparra-Vega, 1992. Effect o f threonine supplementation on practical broiler diets with different levels of arginine. Veterinaria-Mexico 23:223-226. Moreno, J. R. , G . M . Cuca, and H . J. G . Herrera, 1993. Study of the effects o f threonine supplementation in broiler chickens from 0 to 3 weeks old. Archivos-Latinoamericanos-de-Produccion-Animal. 1:129-138. National Research Council , 1994. Nutrient Requirements o f Poultry. 9 t h rev. ed. National Academy Press, Washington, D C . Parr, J. F. , and J. D . Summers, 1991. The effect o f minimizing amino acid excesses in broiler diets. Poultry Sci . 70:1540-1549. 68 Rangel-Lugo, M , C . L . Su, and R. E . Austic, 1994. Threonine requirement and threonine imbalance in broiler chickens. Poult. Sci . 73:670-681. Rhodimet Nutrition Guide, 1993. Feed ingredients formulation in digestible amino acids, 2 n d edition 1993 Rhone-Poulenc Animal Nutrition. Robbins, K . R , 1987. Threonine requirement o f the broiler chick as affected by protein level and source. Poultry Sci . 66:1531-1534. S A S Institute, 1996. SAS® User's Guide: Statistics. Version 6 Edition. S A S Institute Inc., Cary, N C . Smith, N . K . , Jr., and P. W . Waldroup, 1988. Estimation o f the tryptophan requirement o f male broiler chickens. Poultry Sci . 67:1174-1177. Snedecor, G . W . , and W . G . Cochran, 1980. Statistical methods. 8 t h ed. Iowa Press, Ames, Iowa. Steinhart, H . , and M . Kirchgessner, 1984. Investigations on the requirement o f tryptophan for broilers. Arch . Geflugelkd. 48:150-155. Tasaki, I. 1983. Influence o f tryptophan deficiency and excess on egg production in laying pullets. Jpn. Poult. Sci . 20:103-108. Thomas, O. P., M . Farran, and C . B . Tamplin, 1992. Broiler nutrition update: Threonine requirement for 3-6 week-old broilers. Pages 45-53 in: Proceedings Maryland Nutrition Conference, College Park, M D . Thomas, O. P., A . I. Zuckerman, M . Farran, and C. B . Tamplin, 1986. Updated amino acid requirements o f broilers. Pages 79-85 in: Proceedings Maryland Nutrition Conference, College Park, M D . Thomas, O. P., M . Farran, C . B . Tamplin, and A . I. Zuckerman, 1987. Broiler Starter Studies: I. The threonine requirements o f male and female broiler chicks. II. The body composition of males fed varying levels o f protein and energy. Pages 38-42 in: Proceedings Maryland Nutrition Conference, College Park, M D . Vazquez, M . , and G . M . Pesti, 1997. Estimation of the lysine requirement o f broiler chicks for maximum body gain and feed efficiency. J. App l . Poultry Res. 6:241-246. Woodham, A . A . , and P. S. Deans, 1975. Amino acid requirements o f growing chickens. Br . Poult. Sci . 16:269-287. Yamazaki , M . , Y . Oka, H . Murakami, M . Takemase, M . Ando, and M . Yamazaki , 1997. Available threonine requirement o f broiler chickens at two growing stages. Jpn. Poult. Sci . 34:45-51. 69 Zhang, Y . , and C. M . Parsons, 1993. Effect of crystalline lysine and methionine intake on amino acid excretion by precision-fed cockerels. Poultry Sci. 72:1180-1183. 70 C H A P T E R IV Responses of Broilers and Layers to Threonine and Tryptophan Supplementation in Reduced Protein Diets Summary A total o f 2,000 mixed sex broilers and 1,020 layers was used in a 6 week broiler study and an 8 week layer study. Each o f the two experiments was also composed o f two studies that ran concurrently: a growth or production study and a balance study. The objectives of the experiments were to evaluate the responses of broilers and layers to different levels o f threonine and tryptophan in reduced protein diets, while maintaining the levels of other essential amino acids ( A A ) at the N R C (1994) recommendations for growth, egg production and excretion of nitrogen by poultry. The broiler study consisted of a factorial arrangement o f treatments in a completely randomized design with 3 levels o f threonine (90, 100, and 110% N R C (1994)) and 3 levels o f tryptophan (90, 100, and 110% N R C (1994)) to give nine experimental diets (21% C P for starter diets and 18.5% C P for grower diets). Commercial starter (23 % CP) and grower diets (20% CP) were used as control. The layer study used the same design except that the levels of threonine and tryptophan were set at 95, 100, and 105% of the N R C (1994). A commercial layer diet (17% CP) was used as control. The protein contents o f the layer experimental diets were reduced to 12.4% C P . Except for threonine and tryptophan, all other A A and nutrients were formulated according to the N R C (1994) recommendations for broilers and layers, respectively. Amino acid analyses revealed that the starter experimental diets contained 83%, 92% and 101% of the N R C (1994) threonine recommendations and 102%, 113% and 125% of the N R C (1994) tryptophan recommendations. The grower experimental diets also contained higher levels o f tryptophan than planned (96%, 107% and 118%) of the N R C (1994) recommendation. The levels o f threonine in the grower experimental diets and the levels o f threonine and tryptophan in the layer experimental diets were as planned. The broiler study indicated that dietary threonine and tryptophan for 0-3 week old broilers should be targeted at 0.74% of the diet (or 4.04% of CP) and 0.23% of the diet (or 1.22% of CP) , respectively. For 3-6 week old broilers, dietary threonine and tryptophan should be targeted at 0.67% o f the diet (or 3.40% of CP) and 0.17% of the diet (or 0.89% of CP) , respectively. Laying hens aged 42-50 weeks, as indicated by the layer 71 study, should be targeted at a daily intake of 448 mg threonine/hen and 152 mg tryptophan/hen. The results o f the balance studies clearly showed that crystalline A A supplementation of the reduced-protein poultry diets improved the A A balance in the diet, hence improving protein utilization efficiency and resulting in a reduction of nitrogen in the excreta. K e y words: reduced protein diets, threonine, tryptophan, requirement, nitrogen excretion Introduction When feed ingredients and crystalline amino acids ( A A ) are combined to supply the essential A A required by poultry, the crude protein (CP) content o f the diet is usually high because there are excesses o f many A A . For example, in a typical layer diet based on corn and soybean meal, sulfur A A are at about 100% of the requirement. However, the level o f other A A varies from 125% to over 300% of the requirement (Davis and Austic, 1994). According to Macleod (1997), the excretion of nitrogen (as uric acid) from A A required six mol o f A T P / m o l A A for most A A containing one nitrogen atom, but as much as 18 mol of A T P for histidine, which contained three nitrogen atoms. The high energy cost of uric acid synthesis and excretion implies several nutritional advantages o f balancing the A A composition o f absorbed protein close to the requirement. Experiments with poultry have supported the hypothesis that diets formulated to minimize the excess o f A A over the chicks' known requirements would improve the efficiency of protein and energy utilization (Waldroup et al, 1976). One o f the most important factors affecting the utilization o f dietary protein is the balance o f A A in the feed. The closer the A A composition o f the diet matches the requirement for maintenance and growth, the less protein the animal needs and wastes. A better utilization of protein could be achieved with reduced protein diets that were supplemented with crystalline A A such as lysine, methionine, threonine and tryptophan (Blair et al, 1999). Supplementing with crystalline A A to create an A A balanced diet allows the level o f dietary protein to be reduced. The performance o f the birds was not jeopardized while achieving a 10-27% reduction in the total nitrogen excreted during the six-week broiler production cycle and a 30-35% reduction in daily nitrogen output was achievable for layers (Blair et al, 1999). The growing public concerns about the impact of animal production on the environment have increased the interest o f poultry nutritionists in improving protein utilization and the use o f reduced protein diets. However, 72 before the nutritionists can reduce the protein content of a feed, they must have an adequate knowledge o f A A requirements of animals. Wi th the advance in biotechnology, feed grade threonine and tryptophan are now available for use in the feed industry. Unfortunately, the requirements o f poultry for threonine and tryptophan are not wel l studied. More over, there are great variations in reported threonine and tryptophan requirements for both broilers and layers. For instance, estimates o f requirement values o f 0-3 week old broiler for threonine range from 0.50 to 0.85% of diet (or 2.8 to 3.7% o f CP) (Woodham and Deans, 1975; Robbins, 1987). On the other hand, the estimates o f requirements for tryptophan ranged from 0.14 to 0.28% of the diet (or 0.78 to 1.2% of CP) (Woodham and Deans, 1975; Abebe and Morris , 1990). The National Research Counci l ( N R C , 1994) recommended requirement value for threonine is 0.80%, 0.74% and 0.43% of the diet, respectively, for 0-3 week old, 3-6 week old broilers and laying hens, respectively. For tryptophan, the N R C (1994) recommended requirement value is 0.20%, 0.18% and 0.15% of the diet, respectively, for 0-3 week old, 3-6 week old broilers and laying hens, respectively. The N R C (1994) recommendations for threonine and tryptophan were based on research conducted at least ten years ago. Clearly, solid requirement data are not available for most essential A A , particularly during the growth period beyond 21 days of age for broilers and laying hens. It would be in the interests of the nutritionists to question the validity o f the N R C (1994) recommended requirement values for essential A A , especially for threonine and tryptophan. Using chemically defined diets containing A A as the sole source o f dietary nitrogen, the previous study (Chapter III) clearly showed that the estimated requirement o f 0-3 week old broiler chicks for digestible threonine and digestible tryptophan was 0.63% and 0.16% of the diet, respectively. This equates to 0.73% (4.13% of CP) and 0.19% (1.07% of CP) o f total threonine and tryptophan in the diet, respectively. Therefore, present studies were conducted to determine the responses o f broilers (0-3 and 3-6 week of age) and layers to different levels o f threonine and tryptophan in a reduced protein diet based on practical ingredients. Materials & Methods A 6 week broiler study and an 8 week layer study were conducted. Each o f the two experiments was composed o f two studies that ran concurrently: a growth or production study and a balance study. 73 Broiler Growth Study A total o f 2,000 mixed sex day-old broilers (Peterson x Arbor Acres) was used in the study with two replications of males and two replications o f females for each diet. The broiler chicks were sexed and vaccinated against Marek's disease at the hatchery and were randomly distributed, according to sex, among 40 1.5 x 4.0 m floor pens located in the broiler unit at The University o f British Columbia Av ian Research and Teaching Facility. Chicks were raised to 42 days o f age in floor pens in groups of 50 birds. Temperature in the pens at litter level was maintained at 33 °C at the start o f the experiment, reducing to 21°C when the birds reached 42 days o f age. The lighting program was o f increasing photoperiod after 3 days, i.e. 0-3 days, 23 h light ( L ) : l h dark (D); 4-14 days, 6L:18D; 15-21 days, 10L:14D, 22-28 days, 14L:10D; 29-35 days, 18L:6D; 36-42 days, 23I/.1D. Light intensity measured 23 cm above the litter was maintained at 20 lx during days 0-6 and 5 lx during days 7-42. A t 43 days o f age, the birds were marketed and slaughtered at a commercial processing plant. Max imum and minimum temperatures were recorded daily at two locations in the building. Dai ly mortalities were recorded and the carcasses saved for post-mortem examinations. Individual body weights of the birds were measured at 1, 21 and 42 days o f age. Fresh water and feed were supplied ad libitum during the study. Feed conversion ratios were calculated as the ratio o f feed consumption in grams to weight gain in grams. Broiler Dietary Treatment Using locally available feed ingredients, the level o f threonine and tryptophan in a practical broiler diet could not be reduced to less than 90% of the N R C (1994) broiler recommendations. Therefore, levels of 90%, 100% and 110% of the N R C (1994) recommendations were selected for the current study. A 3 x 3 x 2 (threonine x tryptophan x sex) factorial arrangement o f treatments in a completely randomized design (Table 4.1) was employed with three levels of threonine (90, 100 and 100% of the N R C ) and three levels of tryptophan (90, 100 and 100% of the N R C ) . Except for threonine and tryptophan, all other A A and nutrients were formulated according to the N R C (1994) recommendations. In addition, commercial broiler starter and grower diets were also brought in and used as the control (Diet 1). Therefore, there was a series of 10 starter diets (fed from 0-3 weeks o f age) and a series o f 10 74 grower diets (fed from 3-6 weeks of age). Broilers were assigned to 10 different groups (Table 4.2). The nutrient compositions of broiler diets are presented in Tables 4.3 (Starter) and 4.4 (Grower). Table 4.1 Factorial arrangement of broiler diets1 90% NRC Threonine (Thr) 100% NRC Threonine (Thr) 110% NRC Threonine (Thr) 90% NRC Tryptophan (Trp) 90%Thr-90%Trp Diet 2 100% Thr-90% Trp Diet 5 110% Thr-90% Trp Diet 8 100% NRC Tryptophan (Trp) 90% Thr-100% Trp Diet 3 100% Thr-100% Trp Diet 6 110% Thr-100% Trp Diet 9 110% NRC Tryptophan (Trp) 90% Thr-110% Trp Diet 4 100% Thr-110% Trp Diet 7 110% Thr-110% Trp Diet 10 Diet 1 = control starter or control grower diets Table 4.2 Arrangement of different dietary groups in the broiler study. Dietary group Starter diet (0-3 week) Grower diet (3-6 week) 1 Control (diet 1) Control (diet 1) 2 Diet 2 (90% Thr - 90% Trp) Diet 2 (90% Thr - 90% Trp) 3 Diet 3 (90% Thr - 100% Trp) Diet 3 (90% Thr - 100% Trp) 4 Diet 4 (90% Thr -110% Trp) Diet 4 (90% Thr - 110% Trp) 5 Diet 5 (100% Thr - 90% Trp) Diet 5 (100% Thr - 90% Trp) 6 Diet 6 (100% Thr -100% Trp) Diet 6 (100% Thr - 100% Trp) 7 Diet 7 (100% Thr -110% Trp) Diet 7 (100% Thr -110% Trp) 8 Diet 8 (110% Thr-90% Trp) Diet 8 (110% Thr - 90% Trp) 9 Diet 9 (110% Thr -100% Trp) Diet 9 (110% Thr -100% Trp) 10 Diet 10 (110% Thr -110% Trp) Diet 10 (110% Thr -110% Trp) Broiler Balance Study In addition to the growth study, 320 day-old broilers (160 males and 160 females) were used in a balance study. During the first 3 weeks of the balance study, the broilers were housed in battery brooders. A total of 40 pens was used with each pen housing eight broilers (eight males or eight females). 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Feed consumption and overall body weight changes for the same 48-h period were also recorded. For both excreta collection periods (during the second and fifth weeks) excreta were collected every 12 h and were frozen until analyzed. L a y e r Product ion Study A total o f 1,020 42-week-old layers (510 Hyline and 510 H & N ) was used. The trial was composed o f two parts: a production study and a balance study. Both studies ran concurrently for a period o f 8 weeks. For the production study, 960 layers (480 Hyline and 480 H & N ) were used. Twenty rows o f battery cages were used with each row representing a replicate. There were two replications (one row o f 44 Hyline layers and one row o f 44 H & N layers) per diet. The layers were housed in battery cages (0.3 m wide x 0.4 m deep) with two layers per cage (0.67 ft2 or 0.06 m 2 per bird). The layers received 15!/2 h o f light each day throughout the trial period. The individual body weight o f each layer was recorded at the start and the end o f the trial. Dai ly mortalities were removed and recorded. Maximum and minimum temperatures were recorded daily in at least two locations within the building. Feed intake per row was determined weekly by weighing back unconsumed feed. Eggs were collected once daily and egg production recorded by row. Eggs collected were classified as normal, cracked, deformed shell, soft-shell or no shell. Every two weeks, all eggs produced during a 2-day period were individually weighed. L a y e r Dietary Treatment A s in the broiler study, the study was intended to evaluate the effects of reduced dietary protein with 3 levels of threonine (90, 100 and 110% of the N R C (1994) estimated requirement) and 3 levels o f tryptophan (90, 100 and 110% of the N R C (1994) estimated requirement) while maintaining the levels of other essential A A at the N R C (1994) recommendations on growth and excretion o f nitrogen by layers. It proved to be impossible to formulate a layer diet with 90% of the N R C (1994) estimated requirements for threonine and tryptophan using the ingredients available locally. Thus, the treatment levels were adjusted to 95, 100 and 105% of the N R C estimated requirements. Similar to the broiler study, a 3 x 3 x 2 (threonine x tryptophan x strain) factorial arrangement o f treatments in a completely randomized design (Table 4.5) was 8 0 employed with three levels o f threonine (95, 100 and 105% of the N R C ) and three levels of tryptophan (95, 100 and 105% of the N R C ) . Except for threonine and tryptophan, all other A A and nutrients were formulated according to the N R C (1994) layer recommendations. The nutrient compositions of layer diets were presented in Table 4.6. In addition, a commercial layer diet was also brought in and used as the control (Diet 1). Therefore, there was a series often layer diets. Table 4.5 Factorial arrangement of layer diets 1 95% N R C Threonine (Thr) 100% N R C Threonine (Thr) 105% N R C Threonine (Thr) 95% N R C Tryptophan (Trp) 95% T h r - 9 5 % Trp Diet 2 100% T h r - 9 5 % Trp Diet 5 105% T h r - 9 5 % Trp Diet 8 100% N R C Tryptophan (Trp) 95% T h r - 1 0 0 % Trp Diet 3 100% T h r - 1 0 0 % Trp Diet 6 105% T h r - 1 0 0 % Trp Diet 9 105% N R C Tryptophan (Trp) 95% T h r - 1 0 5 % Trp Diet 4 100% T h r - 1 0 5 % Trp Diet 7 105% T h r - 1 0 5 % Trp Diet 10 Diet 1 = commercial control layer diets Layer Balance Study In addition to the production study, 80 layers (four Hyline layers and four H & N layers per diet) were used in the balance study. The layers were housed individually in layer cages. Five weeks after the introduction of the test diets, individual measurement o f feed intake and excreta output over a 48-h period were recorded. The layers were also individually weighed at the start and at the end o f the 48-h collection period. A l l excreta were frozen until analyzed. The guidelines o f the Canadian Council on Animal Care were followed and the protocol for this experiment was approved by The University of British Columbia Animal Care Committee. Calculation Nutrient Retention (%) = [(a-b) / a] x 100 where, a = diet intake ( D M ) x % nutrient in diet ( D M ) b = excreta voided ( D M ) x % nutrient in excreta ( D M ) 81 co CD CJ> rs c o "55 o a E o O CO © CO 00 a> Q CO Q CD a> a a> -| <u ^ •— O ) Q a> a CM a> O a> a c CO v. O ) c 00 o • 00 CO o CT) O m T -co cn q d o • 00 cn in T -o °> • 00 CO q d cn o in t-co CD O CD O m 1 -o . • CD co fj> o o CO co CM CD cn in CM d o o q d cn o in o ^ P • CD O m cn o " I d r co CO o d d cn o m T -0 0 0 m" iri A CM CM T -o q q d d fl CM N r o q o iri iri fl CM CM T -0 0 0 iri iri fl CM CM T -0 0 0 iri iri fl CM CM r O O O iri iri fl CM CM T -0 0 0 iri iri fl CM CM T -0 0 0 iri iri fl CM CM T -0 0 0 iri iri fl CM CM T -m CM CM in CM •n CM o iri CM o •ri CM in CM in CM o d CM m q q q o d d d co CP fl fl in eq q CM fl q fl co T - o o T -o P P ° . P m o o o > i - A o ' o o 0 1 n J CO fl fl T - ~ m CO T— O O T— m o d m o d T~ o 9 9 9 ° i n o o in © O CD CO co fl fl fl CO T -o fl o in o 0 0 0 m o o cn co CO fl fl T -in w q CM CM q in q q A CO T— O O T - O T " O P P P P W O O C D T -0 q q q q •n O O OJ CO 1 0 CO fl fl r-m eo o» fl" d T™ CM q in o d d CM q in q 0 T-1 d 0 0 0 0 m m o o cn co —* CO fl fl T " m 00 CD CM co T - o 0 0 0 0 d d d d CO fl fl T -o 0 . 9 0 . ° . i n c o o > o d g 2 2 £ f l d - « - : d U) CO 0) r fl cd T - d o o q d d d M »»* w •»* in fl q o o d d CM T -o fl 9 9 o o n 01 2 % CM d CM ^ q in o o T~ C) T~" V * o in o o T - O T~ T -o in q o T— O T— T— q in q q T— O T— T— CO CM CO CM CM £ o o o o CO CM 00 (\j CM O . fl O "* ° S 2 » o o o 0 -n 2 o A 2 £J CO f\j • • " ^ N r ° ° 6 d co o CO d o co d co o CM ^ CO CM CM £ s i 00 co o o o o cp d c*> 2 o fl f 5* o o o 00 co 2 CM ^ CO CM CM T -CM O fl P <°. fl . o O o o co CM ^. CO CM CM £ O fl to d d m 00 in co fl 1-; d d co o CM 00 CM CM T -P * S 8 CO d d o o co CM .^ t-00 CM CM o fl co o o in CM in m fl T-; d d co d co d o co d o CO d CD T £ 75 « ® S c a d) £ -O h 3 U> _ 75 E 5 5 o 2 J _ S I «= s « o. — 2 E 55 co ™ g» a> a) « i _ ^ = = C0 < O O 3 E co S o> « Tog? c £ S r o - s i 5 ] l " ^ " 5 g o * - t ji >, t c c = i c c g s » E 5 m o S i L w o o i o i - < J J Q W J < c (0 CO J= c f l II o .5 CO — CM O » o I s N 3 5 ~ < -I E E 2 2 a. a. 1 2 2 .E > S w rs o C CM •o CO o Q. E o o CM 1 -00 CO O O o o co co in o o fl f l CO c 3^ (A + 0) P» z £ * • o E o 3 o n O =- co » .a 5 i2 UJ "5 ~ £ 5> o. m i * * 0) 3 H H O 82 o o o 00 o CO co •n rd d d o o o 00 o co C O in co d d T - CO d oi 00 T -* o s co d oi CD T -00 N. CO i -co m m n- r - f - 00 T -O r CD O O O O O h - C O T t O C O N ( O I O * C D S S ( D ( O T ^ * * T ^ i n * C D i n dddddt^oimd o o o co o r- co co in" co d d o o o oo O S CD f ) in co d d o o o oo o s co co in co d d o o o oo q s to co m" co d d o o o co o s co co in co d d o o o oo o h- co CO m' cd d d o o o oo q s to c i in oo d d o o CO T -s co s * co co d d -—- co sS E » £ E o. C= e « 5? ,^ x: ° o ? w ^ XI *~ o 0) «= E f i s "5 £ s « L It O > O O h- < co co e o h - c o i n c O T - c D c o e o ( O r * * r i n i O O ) I O § £J o d d o d s d r i o T - C~ t> CO d oi C D 1 -T- O oo in d oi oo T -h» CD N- CM d oi CD T -C0 CM CO CO d oi CD 1 -oi r-h- CM d oi CD r -CO d oi CD T-"co 5 oi oi • ° m o d s e o r » c o c o m c D c o h - o t f l r * * T ; t o i q o i » dddddi^oieid C O N t O C O ' t O l C N S S dddddi^aitod eOSCOCOCOOOCOCDOl t O r - f T f T - T - T f O l O d d d d d s o i T f d O N l O O i n t O T f ^ r t O r ; ^ T t r ; q t O C N C D dddddooai^d co h> CO T -co o * co oo co oo * * T - co m o m o o o o o t » o o * o co s CO T - ; d d co o co CM in o to * * r CN Oj CO in d d d N cd co d S c o "5 o a a a a g z z z CO c (0 co" £ * d 8 g | ~ r- » 2 S? C | | Q. - C aJ .Ex: Q Q O J S c co ca x: oj CL "D _ 'u • £ C t ID B S h h O < O o> E c! a E . . o p i - 2 o C o o 6 £ c «* E c ca — ft SZ 0 ° °. o p E E TT to o d £ * g 1 £ ro a > b) = E UJ -5 c .2 E = 3 en > E 5 £ o 2 o o in n T" O 2 S 0 2 S £ » a. 1 » u E d £ o d § ~ cn O < 2 £ b> E E 2 r -> u i : u <i> ra =5 o ° y a m t E .E 2L •o , E « .3> > o a. e " Q. O 'S CO C ^ 83 Analyses of Samples Before the start o f the experiment, main feed ingredients were collected from the feedmill, ground to pass through a 1 mm mesh screen and then analyzed for C P ( N x 6.25), A A composition, and other proximate constituents prior to formulation of the diets. Proximate analysis for C P , crude fiber, ether extract, ash and calcium were carried out following the procedures outlined by the Association of Official Analytical Chemists ( A O A C , 1984). Phosphorus was measured using a spectrophotometric method (Estrin and Brammell , 1968). Amino acids were analyzed commercially (Heartland Lysine lnc, Chicago, Illinois 60631, U S A ) , using methods outlined by Spackman et al. (1958). Hydrolysates for the determination of methionine as methionine sulfone and cysteine/cystine as cysteic acid were prepared by performic oxidation o f the protein followed by 6 N HC1 hydrolysis (Moore, 1963), whereas feed ingredients were subjected to alkaline hydrolysis and high performance liquid chromatography for tryptophan determination (Jones et al, 1981). During the entire experimental period feed samples (approximately 250 g) from each experimental broiler and layer diets were collected every week and stored in a freezer until analyzed. These were then pooled, mixed and sub-sampled for proximate analysis as described above. The excreta samples obtained from the broiler and layer balance studies were dried at 60 °C, re-weighed (dry weight) and ground to pass through a 1 mm mesh screen. Total N content in dry excreta was analyzed according to A O A C (1984) procedures. Statistical Analyses Both the broiler and layer studies were factorial experiments in a completely randomized design. A l l data generated from the experimental diets were subjected to analysis o f variance ( A N O V A ) procedures. I f treatments were found to be significantly different, Tukey's multiple range test (Snedecor and Cochran, 1980) was used to determine the statistical significance among treatment least-square means. The results obtained for birds fed the control commercial diet were compared to those from birds fed the experimental diets using a one-way A N O V A by using the General Linear Models ( G L M ) procedure of SAS® software (SAS Institute, 1996). I f treatments were found to be significantly different, the Bonferroni (Dunn) T test (SAS Institute, 1996) was used to 84 determine the statistical significance among treatment least-square means. The Bonferonni (Dunn) T test was used because it w i l l avoid detecting random differences when comparing a large group o f treatments (ten dietary treatments in both the broiler and layer study). Results Bro i l e r G r o w t h Study Experimental diets for the broiler study were prepared at the Agriculture and Agri -Food Canada feed-mill at Agassiz, British Columbia. The broiler starter (0-3 week) diets did not prove to be of the planned standard since a mould infestation was observed in them shortly after the broiler experiment was initiated. In addition, the determined N contents o f the broiler starter (0-3 week) and broiler grower (3-6 week) diets did not compare favorably with the planned protein contents (Tables 4.3 and 4.4), even though care was taken to analyze the ingredients for protein and A A prior to the diets being formulated. Experimental diets for the layer study were prepared by a commercial feedmill (Pro-form, lnc, Chill iwack, Canada) and the N contents of the layer diets were o f planned standard. In order to confirm the amount o f A A in the diets, feed samples from the broiler and layer studies were sent to the University o f Manitoba for another A A analysis. Feed samples were analyzed for A A content with a L K B 4151 Alpha Plus A A Analyzer. Feed samples were prepared by acid hydrolysis using the method o f Andrews and Baldar (1985). A c i d hydrolysis involves digestion in 6 N hydrochloric acid for 24 h at 110 °C. Samples for methionine and cystine determination were subjected to performic acid pretreatment and later analyzed by the method of Andrews and Baldar (1985). Tryptophan in the feeds was analyzed following alkaline hydrolysis (Hugh and Moore, 1972). The C P content of the control commercial broiler diets and all o f the layer diets were as planned. However, C P was lower than planned in the starter experimental diets whereas it was higher in the grower diets. The results of the A A analysis from the University of Manitoba indicated that the amount o f threonine in the broiler starter diets was lower than planned, whereas tryptophan was higher than planned in both the broiler starter and grower diets. The levels o f threonine in the broiler grower diets and the levels of threonine and tryptophan in the layer diets were as planned. Because o f the variations i n calculated and analyzed values, the levels o f threonine and tryptophan in the starter diets were changed from 90%, 100% and 110% 85 of the N R C (1994) to 83%, 92% and 101% of the N R C (1994), respectively, for threonine, and to 102%, 113% and 125% of the N R C (1994) respectively for tryptophan. Likewise, the levels of tryptophan in the grower diets were changed from 90%, 100% and 110% o f the N R C (1994) to 96%, 107% and 118% of the N R C (1994), respectively. Growth performance, feed intake and feed/gain ratios o f broilers during the starter and grower periods are presented in Tables 4.7 and 4.8, respectively. Even though feed intake was not significantly different (P > 0.05) among starter diets, broilers fed the control starter diet gained significantly more (P < 0.05) weight than broilers fed the experimental starter diets. Significant differences (P < 0.05) in weight gain were found among broilers fed different experimental starter diets. Broilers fed the control starter diet were also more efficient in utilizing the starter feed than broilers fed the experimental starter diets (Table 4.7). The male broilers consumed significantly more (P < 0.05) feed and gained significantly more (P < 0.05) weight than the female broilers from 0-3 weeks of age. Growth performance by broilers during the grower period (3-6 week o f age) followed the same trends as that o f the broilers during the starter period (Table 4.8). Broilers fed the control grower diet, however, gained significantly more (P < 0.05) weight and consumed significantly more (P < 0.05) feed than the broilers fed the experimental grower diets. Contrary to the starter period, no significant differences in weight gain was detected among broilers fed the different experimental grower diets. Feed/gain ratios among broilers fed different grower diets were not significantly different (P > 0.05) during the grower period. Male broilers were superior to female broilers in performance from 3-6 weeks o f age. For the overall performance (0-6 week) of the birds, birds in the dietary control group gained significantly more (P < 0.05) weight than the birds in the dietary groups 2-10. However, no significant differences in feed intake were found among birds assigned to dietary groups 1 (control), 4 (83% threonine and 125% tryptophan in starter diet and 90% threonine and 118% tryptophan in grower diet), 6 (92% threonine and 113% tryptophan in starter diet and 100% threonine and 107% tryptophan in grower diet) or 8 (101% threonine and 102% tryptophan in starter diet and 110% threonine and 96% tryptophan in grower diet). Birds in the dietary control group were significantly more (P < 0.05) efficient in converting feed into body tissues (Table 4.9) than birds in dietary groups 2 (83% threonine and 102% tryptophan in starter diet and 90% threonine and 96% tryptophan in grower diet), 4 and 6. co IO o fe £ „ D /—s CD 5 -lb ? • l a 50 co •a o T C ex o c ' 3 o ID M H loo O N O vo V© T-H <n vo vo IT) fN vo 1 0 m CN ro in A-i > co o vo o t— t- 0 0 C N CN O N 0 0 VO r -A © CN t-- w-i t-~ r - t- r-~ r - o 0 0 1 0 •a •O •O •a a u xi . 0 £> .O XI VO 0 0 T-H VO _ l ON 0 0 ro ON ON t— 0 O ON 0 0 ON m 1 0 IT) r-~ 1 0 O 0 0 IO fl o i i o U 1 o U CN ro O T-H </-> CN CN ro O T-H CN CN CO o ^ CN ai (U s ro 0 0 ro 0 0 ro 0 0 CN ON 0 s - 0 s -CN CN O N O N T-H CN CO i O VO 0 0 O N 2 T f r T-H ir> O T ^ © 0 0 r~-vo VO ON VO CN B w c3 TH > o T3 u o fe o I d cd a % T—I e D . U |H cd cl s •a IS o I  PH a '1 o u u ON ON o IS? 1 s 1 S <<H S o a in fi £ cd £ & cvs IH n DJ) cu T3 a cs CU « .2° >> T3 O V w a S c2 cu <u a o a cu S cs <u u C3 cu •3 "S "» S >-CU CU s -s 2 •-3 * «~ ° 5 C M • cu cn is © H ^ oo O * £5 cu w S .2 VO fa £ CD •a -29 vo T3 CD CD fa •s 1 CS H a, o CD a '3 o <D "•3 <D CN 0 0 0 0 m 0 0 vo 0 0 ro oo 0 0 0 0 c s 0 0 m 0 0 0 0 o 0 0 0 0 0 0 .o JS JS JS JS JS JS a O O N O N 0 0 0 0 O N i n oo VO O N O S O N </N O N m vo o oo oo oo oo 0 0 oo r-- O vo m CN CN CN CN CN CN CN CN CN cn CN JS .Q JS J3 .O oo O N VO m O N m CN i n i n cn >n r- m 0 0 vo cn CN r- m i n m m i n m m i n i n l> t—4 r-4 i • — i 1 o U VO O N C > o-v S = O 2 g _1 _ , O N 0 s -o o v 0s - o s oo r-~ oo ~ £ 2 =: CD 13 B ( D fa O J 3 C o U o O N o O N o s O O N 0 s -O O O o 0s- 6 s o^ - 6 s o o o o O CN cn i n vo oo O N 2 0 0 o O N © oo CN rn <D s 1 CD > o w TJ "3 o PM o a, J o CJ G JT i l § CD ll-H 11 a -o a 8 cn | T 3 \& a CQ a o o <u ||ON U " ' 2 » s w a • S a l ca g £ .2 H « u °i a * -a CU OX) IS "B CD CD CD CD CD ••€> VH CD 00 T—i £50 f -oo o oo vo r-- O N vo m r-- V O 1 o U c o V O O N I J3 H 0s-O O N v= v o o so 0s-o O N 0s-00 0s-O ON & & & H H H ^ =^ CN m i n O >—1 cs £ J3 & H H H H o^ o 0s-O O & H 0s- o x CN co O T-H c5 J3 m c o oo oo c o oo so \0 o x 0s-CN CS O N O N H 0s-oo J3 H 0s-O O & H 0s-m CS J3 H 0s-CS O N & H o x V O O N I J3 H o x O N ? N ° 0s- 0s-r~- oo O T-H H H 0s- 0s-O O H H o x 0s-CS CO & H 6s-i n CS £ j a J a H H H c £ c £ T-H T-H T—I o o o CS CO m vo 00 O N 2 Xl CS A o r-~ oo Xl Xi Xi cs cs ja cs XI a Xl XI cs Xl c s ON C - c o V O CS 00 CO T-H m CO o ON V O c o 00 V O c o ON vo T-H oo V O i n V O i n V O i n V O m i n 00 CO CO c o CO CO CO c o CO CO c o CO CO J3 XI Xl Xl Xi XI X, Xl es Xl r- O N O N o co co o CS T-H wo co cs A- T-H 00 co 00 m CO cs T-H cs o o o o o o o o o cs as cs cs cs cs cs cs cs cs cs cs cs ca JD B CD fe r-~ T-H f- o T ^ © c o 00 S oo c o r o V O o cs ON CD B CD > o w 00 o fe 89 N o significant differences in weight gain, feed intake and feed/gain ratio were observed among birds assigned to dietary groups 2-10 for the overall 0-6 week period. Overall performance o f the male broilers was superior to the performance by the female broilers, as expected. When the results obtained from the experimental starter diets (diets 2-10) were compared i n a factorial manner, no significant differences (P > 0.05) in weight gain and feed intake were observed with birds (0-3 weeks o f age) fed diets containing the three levels of threonine (Table 4.10). However, feed/gain ratios were significantly lower (P < 0.05) for birds fed starter diets containing 92% or 101% of the N R C (1994) recommendations for threonine. Young broiler chicks (0-3 week of age) gained significantly more (P < 0.05) weight when they were fed diets containing 113% or 125% of the N R C (1994) recommendations for tryptophan (Table 4.10). There was also a significant threonine and tryptophan interaction for 0-3 week weight gain (Table 4.10). When the dietary level o f threonine was set at 92% o f the N R C (1994) recommendation, birds fed diets containing 102% of the N R C (1994) recommended tryptophan level gained significantly less (P < 0.05) weight than birds fed diets containing 113% of the N R C (1994) tryptophan level (Figure 4.1). However, there were no significant differences in weight gain among birds fed diets containing different levels o f threonine when dietary tryptophan was set at 102%, 113% or 125% of the N R C (1994) recommendations (Figure 4.2). Feed intake was similar among experimental diets during the 0-3 week period. During the starter period, male broilers performed significantly better (P < 0.05) in all o f the parameters measured except for the feed/gain ratio, in which there was no difference between the males and the females. When the results obtained from the experimental grower diets (diets 2-10) during the grower period were compared in a factorial manner, no differences were observed in any of the parameters measured (weight gain, feed intake and feed/gain ratio), but the male broilers did perform significantly better (P < 0.05) than the female broilers (Table 4.10). B r o i l e r Balance Study Broiler chicks (2-week-old) fed the control starter diet excreted significantly more (P < 0.05) excreta and N than chicks fed the experimental starter diets (Table 4.11). The percentage of N in the excreta excreted by birds fed the control starter diet was significantly higher (P < 0.05) than that excreted by birds fed the experimental starter diets 2-10. 90 ro T3 a « it <u CD co • o fl o X cu co T J fl cs fl es JS Cu . TJ O o CQ CS CU ~ fl fl '3 si O SD O CU CA "3 .3 > fl +- T J cu .»* S « fl CM . M TJ OX CU ' M fc £ « TV 5 ° I fl ^ tw fl •c g 6 <2 O U cu cu •2 2 CU o cs u bu X ! 0 TJ 1 "o cu cu es £ H vo VO | 3 fl "9 <D cr cu fa . •IB CD CD CD VO i ro ro O o fa £ CD ^ •a £9 CD CD S fa ^ 3 6 0 co -a1 Si CD CD c o ^ t t 00 00 CO ro ro oo r t C O C N 00 00 00 C N C N C N in in vo in in ca •5 s I g - vP sP g o o ° 2 £ R ON r t r t in uo <n .—4 ,—< O I s VI 00 vo vo t~- i> i> -vi- r t 00 ON ON i t -it r t •5 g u u N? N? o^- r_, c o C N S 00 ON 2 co m in 00 00 00 CO H ON VI f l H 00 00 00 C N C N C N r t 00 >—i VO V N r t m m in t-» m vo m in >n o C N —i oo vo r - t- r--ft, C N ro in £ ^ ^ ^ O 00 00 00 r~ C N C N r t O vo ro C N a JD ON >—i VO -vf CD fa vo vo m in O C N ON in a a a JS ro m ro O 00 ON ON O 00 t r t r t m r t cd CD CCJ s CD fa r t 00 o i—i i—l ' H O ro J ; oo C N c o in f~-CD a 1 (D > o w J H o VO « M m o «-<" d vo C N r t c3 ^ CD <t S W ^ CO a> o o £ ca s • s o Si 5 .C3 8 c» R S O Si i> * &3 &3 co s S3 &3 Co * S3 &3 & g &3 &2 &5 * * * * I 6 9 * Co g cS CO g "cl o C3 O II o rt p C4H O . 3 o o o § v S •s 'S C3 rt • £ u o ? (L> *tn OT n cn O 1 a •2 43 * * •a A fl rt a S ° B g e m >-• Q \ 5 rt a a rt u fl g ^ rt q> cn ? 8 w o • WD • P * CU CU CM cu 2N CU w a> cu pfl fl H S •• o & h OJ CU fl pfl • P H o p* © o A p2 u fl WD E • P N »22 CA fl #o CM -O fl cu o cu cu p« as as cu o 0 s fl CM f N O N m oo CM cu CA H I O <n C S I H 6 0 s -c n & H I U CN o I o V a. a u fc '•3 >> a on a o Q. S o C3 U V C a I '•3 O o O 6 fc; » f l H i U fc C N as H I u fc; cn oo o o in o o o o in cn o o cn (§) ure§ ;H§I9AV Apog 9 2 CM p f l O H fl Os 0\ fc; p f l A b CM a> WD fl 2 1 ^ fa " « O ^ N© ID f N O 2 -s CQ cp i z fl fl <g WD <tt p f l to 7 3 fl CM fl E & o CM CM o o HH r~ O IT) u fc v a .SP "5 cu ca "5" o CA 'E ca Ch E o cu u cu a o o o o o o m CO o o H 6 CN H i U fc H i U fc CN O 0 P C a o +^  & P H O 'a> p > a> P - P . (§) ure§ ; q § i 9 A \ Apog 93 o 1 -t-< > H at ,—• g Q .S ^ CM a, fl o 5 £ Q oo t f CN Q 60 Si - M s s o S J3 JS a es ja « J3 a a « ja a JS a ja a VO Os OS OS vq T-H vq 0 0 I> cn fl m OS m vd VO CN VO oi VO vo «ri VO CN CN VO CN VO a ja JS ja ja ja ja ja ja cn cn 0 0 0 0 0 0 fl cn oo cn O OS r-© m IT) O in OS in 0 0 iri fl vi in fl iri iri iri cn i — i vo cn o o U 1 o U CO © OS T-H * — i CN »—i i—i os o ja ja ja © fl O CN CN CN q U U U U q q q q \ ° N? o x o N \ ° CN cn m CN cn m CN cn m o T-H CN O CN O CN U q u U U q q q q c N v o 0 S N O o x \ ° o N \ ° \ ° cn 0 0 cn 0 0 cn 0 0 CN OS CN OS CN OS O T-H r—1 © * - H T-H o CN cn fl in VO 0 0 OS o CN CN i > cn CN CN vo VO VO a ja ja ja ja ja ja ja ja ja ja a CN T—1 O fl T-H cn VO fl m CN fl fl VO T-H VO VO fl O fl 0 0 cn O r-~ iri fl fl fl fl fl fl fl fl fl fl fl VO in o m m VO T-H CN CN "el c3 a CD 0 0 d fl in fl d in o cn o VO <ri d fl o 2 cn" a 1 > O W CO O o PH a u PH tH ej a, CO :§ 6 0 OJ CO o C3 U CM O co a CO PH '« 3 a s a ^ §1 k A 5 C A 8 h os w 53 w 6 « a U c«J •3 S o ja U "P C3 ea u •o S S 't-is n to a C M J - 1 rn tS 94 Broilers fed the control starter diet also retained significantly less (P < 0.05) N when compared to the birds fed experimental starter diets 3 (83% threonine and 113% tryptophan), 6 (92% threonine and 113% tryptophan) and 7 (92% threonine and 125% tryptophan) (Table 4.11). N o significant differences were observed in any of the parameters measured between the birds fed experimental starter diets 2-10. The N content in the excreta excreted by the female chicks was significantly higher (P < 0.05) than that by the male chicks. When data (starter diets 2-10) were analyzed in a factorial manner, no significant differences were observed in any of the parameters measured and calculated, except in the percentage of N retained by 2-week-old broiler chicks (Table 4.12). The broiler chicks fed starter diets containing 113% of the N R C (1994) recommended dietary tryptophan level retained significantly more (P < 0.05) N than the chicks fed diets containing 102% of the N R C (1994) recommendation for tryptophan. N o significant difference in excreta output, N output and N retained was detected between the sexes except that the percentage of N in the excreta from the female birds was significantly higher (P < 0.05) than that from the male birds. In the second balance study, broilers (5 weeks old) fed the control grower diet excreted significantly more (P < 0.05) excreta (except birds fed diet containing 100% threonine and 96% tryptophan) and N than broilers fed the experimental grower diets (Table 4.13). However, there was no significant difference (P > 0.05) in the percentage of N in the excreta excreted by birds fed different grower diets. When compared with the birds fed diets 2 (90% threonine and 96% tryptophan), 5 (100% threonine and 96% tryptophan), 7 (100% threonine and 118% tryptophan), 9 (110%o threonine and 107% tryptophan) and 10 (110% threonine and 118% tryptophan), the proportion of N retained was also significantly (P < 0.05) lower by the birds fed the control grower diet. There were significant differences (P < 0.05) between sexes in all o f the parameters measured and calculated (Table 4.13). When data (grower diets 2-10) were analyzed in a factorial manner, no significant differences in all o f the parameters measured and calculated were observed in birds fed different grower diets (Table 4.14). Male birds produced significantly more (P < 0.05) excreta than female birds but the percentage of N was significantly higher (P < 0.05) in the females. 95 fl o ••a cu M cu X fl CU DX) O M fl o fl « O H O fl M cu fl ' f l o CU M CU > CU fl cu .cu CU CA v .a CU -H X5 « ^ cu 2 - ® I * s s •a *E ™ o >, fa o <N '3 i - H fl • eu r f M cu JS £ * fl H 5 fl H-> § m ecJ 0) ,—-d Q .3 ^ P H r t o 5 Q so •3 § 3 fa C N —< vq cn in C N V O V O V O O N r t O N cn «n r t r t r t r t m CN 10 n H in in C N cn r t C N —1 C N ca •5 ! Q o a N ? 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CS • M C U '•3 fl cu <M '•3 C M O - M u S M C U cu 43 H co T - l C U 3 es H S3 o '•fl CD H—» <u >H CD s f l D O fl w J3 eS J3 es J3 es « J3 a « JS es eg es ja CO fl N O <"H m i n CN 00 CN i n i n N O l > V O O N N O r » O N N O r - H O N N O d d O N N O d O N N O c o i n fl © NO NO c o CN c o fl fl NO CN fl NO NO NO i n i n i n i n i n i n in" i n i n i n es ja XI Xi ja JB ja ja Xl es ja NO r - c o oo >n O c o 00 ON r - < CN NO r—1 O CN O i n O CN o m CN i n fl fl fl fl fl fl* fl fl fl fl fl Q D O O H o CD fl •a o u CD Q es ja ja JS es ja ja ja ja J3 00 c o r- CO c o O fl ON ON H ON ON fl r -NO NO c o oo i n ON r--i n l > NO O PH M S o U fl o U u u NO ON t~- 00 O H t~- oo O r H ° r- oo O r ^ NO ON o u q o x o \ ° O O O O N O N O N CN c o fl u U U U u U \ ° 0s-o N v ? 0s- --° o x 0s- 0s-o O O O O o o O O r-H r-H i n NO 00 ON O r H CN CN es 00 CN 00 " c 3 NO J3 00 CO CD " c 3 § U-i © CN d i n fl NO © NO ON CO O NO 00 m a 1 > O w C O r 2 "o o PH fl C U M cu X fl cu OJD O U fl o fl C3 xa a o D-•»-< •a a CJ CU fl fl o cu M CU fl cu M CM C M '•3 C M O - M C U -4> J M C M C U C U © M .2 £ M - M a, o i b u fl o ' M CD cu C U •e o >, tS X! fa o i M fl H « "2 -E? 0 0 fl r t o S 00 M "S o cd •*-> CD » H o X w 00 ca l-i o fa o d d r - r -NO co cs r—I r t -tf © m cs co i o KI vi oo co c*^  vi ON co r-~ t-~ •s CS g y -p 0 s -O O N y y N ° N O O s 0 s -O O O ^ O N - - H O I O N H O N O C — co <—i r~-co <—i cs rt' ^ r t T-H NO ON co co i n i n vi vi •n ON r t oo i n r t t> r~ 5 £ -R e ** O N y y r - oo o —i t-~ O N o ON NO NO C S CO i—> r t r t i-* CO C S NO i n i n a J Q O N C S <D •3 B fa CO d C N d r t r» cs o r t O C S N O r t q i n d ™ co 8 1 CD > o w C/j U o P H g g g g g § § § g g Cn * g ; g •I * ? ^ 2! s> g Si a s a •R t «8 CO to S O " « cj •5 "3 CO CO u "c3 a m o © V PH a p -o o « H '3 •SP *co u 43 a, o o u .S § fl •a 'S in <*H O o © § v « PH a * | I-l r « H § 3 11 •43 "3 * CO * •n a * fl « s CU CO O 8 E g g u <=> A IH O fl, /—S I -r t O * ; ON JS g O « a CO CO •3 fl o o ^ (2 z , &0 u & 3 3^ % CO 3 rt 98 L a y e r Product ion Study The C P contents of layer diets were closer to the calculated values (Table 4.6). Results from A A analysis indicated that the levels of threonine and tryptophan in the experimental layer diets (diet 2-10) were same as planned (Table 4.6). There were no strain differences (Hyline versus H & N ) in any o f the parameters measured. Therefore, the data were pooled together and treated and analyzed as one strain. N o significant differences (P > 0.05) were found in egg production, mean egg weight, daily egg mass and feed conversion ratio during the 8 week layer study (Table 4.15). The control birds ate significantly more (P < 0.05) feed than the birds fed diet 9 (105% threonine and 100% tryptophan). When the data (layer diets 2-10) were analyzed in a factorial manner, no significant differences were found in all o f the parameters measured among layers fed diets contained different levels o f threonine and tryptophan (Table 4.16). L a y e r Balance Study The percentage o f N in the excreta and the amount of N excreted by layers fed the control layer diet were significantly higher (P < 0.05) than that of layers fed the experimental layer diets 2-10 (Table 4.17). Laying hens fed the high protein control diet also excreted significantly more (P < 0.05) excreta than laying hens fed diets 4, 5, 6, 7, 9 and 10. The proportion o f N retained by layers fed the control diet was significantly lower (P < 0.05) than that o f layers fed the experimental diets (Table 4.17). There were no significant differences (P > 0.05) in any o f the parameters measured when the data (layer diets 2-10) were analyzed in a factorial manner (Table 4.18). 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H g. = * c o "55 -4-» CD >—> g Q .5 f l fl o Ctf. -*-> CD I-i CD X w oo on co ( H O •*-» % PH ON fl" VO oo cu 0s-OS ro CS co VO cs oo vo wo co vo C— I-H t-H A- vq no CO CO CO V O wo d U U 0s-wo O CN co VO fl CO vo co vo co VO vo vo OS oo 0s-O o cs iri vo fl co co cs r-vq vo os os A fl wo o 00 CO vo v-> d CO "Tl co fl o OS fl o r H O OS d wo CD s > O W oo •d "o o PH § g g 1 S a CD S R s c o cs fl c-> s 8 o -a X It. fl OS OS I "5 <4H o as 103 Discussion The growth performance of the broilers on the experimental broiler diets used in this study was not as good as that obtained with the commercial control broiler diets, contrary to our previous experience. Commercial broiler diets purchased from a feed manufacturer gave superior results during both stages of the experiment. A l l starter feeds (infestation o f mold was less serious in the commercial feed) became infested and this factor undoubtedly had an influence on the feed intake o f the birds on the growth trial. This problem was attributed to the feeds being bagged before being cooled sufficiently after pelleting. Furthermore, the C P contents in the experimental starter diets were much lower than planned (21% versus 18.4%). Fritz et al. (1973) and Sharby et al. (1973) reported that broiler chicks fed diets containing corn infested by various fungi showed a depressed weight gain and feed efficiency. In another study, Beasley et al. (1980) found that chicks fed corn inoculated with Penicillium lanosum developed diarrhea and grew at a slower rate than the controls. The metabolizable energy o f the experimental starter diets in the present study might have been lower than planned as Bartov et al. (1982) showed that a decreased energy level in diets containing ground moldy grains (not containing mycotoxins) is an important factor for their reduced nutritional value. In addition, the C P content in experimental starter diets was 3% C P lower than planned. The mean body weight gain and mean feed intake of starter chicks were 83% and 87% lower respectively than the N R C (1994) standard for the first 3 weeks o f age. The much lower C P contents in the starter diets and perhaps the reduction in feed intake by the molds reduced the growth performance o f birds fed the experimental starter diets. In an attempt to meet their energy and protein needs, birds receiving the experimental starter feeds, on average, consumed 3% more feed than birds fed the control starter diet. During the starter period (0-3 weeks), weight gain improved with the increase of dietary threonine. Moreover, feed/gain ratio improved significantly as dietary threonine reached 92% of the N R C (1994) estimated requirements. This indicated that broiler chicks at 0-3 weeks of age required at least 0.74% (or 4.04% of CP) dietary threonine to maximize feed efficiency. This is in good agreement with the 0.73% of the diet (4.13% of CP) reported earlier (Chapter III). Several other studies also reported similar requirement values for threonine. For instance, Thomas et al. (1992) reported that male and female broilers aged 0-3 weeks need 0.77% and 0.71% o f dietary threonine (average is 0.74% of the diet). On the other hand, 0.72% of threonine 104 was required to maximize weight gain for broiler chicks up to 14 days o f age (Rangel-Lugo et al, 1994). Studies conducted by Holsheimer et al. (1994) indicate that, when low protein corn-soybean meal diets supplemented with A A were fed to male and female broiler chicks until 3 weeks of age, improvements in gain and feed/gain ratio were obtained when dietary threonine content was increased to 0.725% of the diet. K i d d et al. (1996) found that threonine levels ranging from 92% (0.736% of the diet) to 112% (0.896% of the diet) o f the N R C (1994) recommendations failed to improve weight gain of 1-21 day old broiler chicks, indicating that broiler chicks required not more than 0.736% of dietary threonine. More recently, Yamazaki et al. (1997a) reported a requirement value o f 0.65% digestible threonine or 0.74% total threonine for 1-3 weeks starting period. However, Rhone Poulenc (Rhodimet Nutrition Guide, 1993), Austic (1994) and the N R C (1994) recommended a higher requirement value of 0-3 week old broilers for threonine (0.80% of diet), whereas Leeson and Summers (1997) suggested a lower level o f 0.70% of the diet. Robbins (1987) clearly showed that when expressed as a percentage o f the diet, the estimated threonine requirement increased as dietary C P increased. However, when expressed as a percentage o f protein, the estimated threonine requirements remained constant relative to dietary protein content. This was confirmed by Austic and Rangel-Lugo (1989) and Rangel-Lugo et al. (1994), who found that the requirement for threonine is dependent on the C P level o f the diet, and requirement values o f 0.63% to 0.77% were reported by these researchers. When expressed as a % of C P , the estimated requirement value found in the present study for threonine is higher than that o f Austic and Rangel-Lugo (1989) and Rangel-Lugo et al. (1994), who found that the threonine requirement ranges from 2.72% to 3.70% of C P . However, Holsheimer et al. (1994) reported a value o f 3.95% of C P , which is close to the estimated value found in the current study. Data from several recent studies, from the previous experiment (Chapter III) and from the present experiment indicate that the N R C (1994) may have overestimated the requirements of 0-3 week old broilers for threonine. Broiler chicks (0-3 weeks old) fed the 113% tryptophan diet gained significantly more (P < 0.05) weight than the one fed the lower level (102% of N R C (1994) recommendation). Increasing the level of tryptophan to 125% of the N R C (1994) recommendation did not further improve the weight gain. It seems that the N R C (1994) has underestimated the requirement for tryptophan. The current study indicated that 0.23% of tryptophan is required in the diet or 1.22% of C P for optimal weight gain. However, this is higher than the value (0.19% of diet) obtained in 105 the first study (Chapter III). The reasons for the discrepancy are unclear. Using purified diets containing five graded levels o f tryptophan, K i m et al. (1997) found that 1-3 week old broiler chicks required 0.173% of dietary tryptophan. Rogers and Pesti (1990) reported that the requirement of the broiler chicks aged 7 to 21 days for tryptophan was estimated to be 0.80% of C P for growing chick. This equates 0.184% of the diet with 23% C P . On the other hand, Leeson and Summers (1997) recommendation for tryptophan (0.20% of the diet) agreed with that o f the N R C (1994). Several recent studies supported the results of the present study that 0-3 week old broiler chicks need more than 0.20% of dietary tryptophan. In an experiment conducted by Steinhart and Kirchgessner (1984), the birds fed a diet containing 0.22% tryptophan gave the best results with regard to weight gain, feed intake and feed efficiency. Han et al. (1991) carried out studies to determine requirements for digestible histidine and tryptophan in 8 to 22 day old chicks fed a histidine and tryptophan deficient intact protein diet containing 25% C P and 3,200 kcal A M E V k g . They found that the requirement for digestible tryptophan was 0.20% of the diet (or 0.80% of C P ) for maximal weight gain and feed efficiency. This indicates that a total tryptophan level of 0.22% in the diet is necessary for broiler chicks fed a 23% C P corn soybean meal diet. Thomas et al. (1992) proposed a dietary tryptophan requirement value o f 0.225%, whereas the Rhodimet Nutrition Guide (1993) and Austic (1994) recommended values o f 0.23% and 0.24%, respectively. When expressed as a % o f C P , the estimated requirement obtained in the present study is higher than most o f the reported values and only in agreement with that o f Abebe and Morris (1990) and Austic (1994). Data from the previous experiment (Chapter III) suggested a lower requirement value o f broiler chicks for tryptophan (0.19% of the diet) and agreed with that recommended by the N R C (1994). However, data from the present experiment and from several recent studies indicate that the N R C (1994) may have underestimated the requirements o f 0-3 week old broilers for tryptophan. Broilers fed a control grower diet ate significantly more and gained significantly more weight than the broilers fed the experimental grower diets. The inferior growth performance o f the birds fed the experimental grower diets might be related to the poorer growth performance o f these birds during the 0-3 week study period. Although feed intake and weight gain of 0-3 week old broiler chicks fed the experimental starter diets were reduced by mold and low dietary protein content, birds fed the experimental grower diets, on average, consumed 9.3% more feed and gained 23% more weight than the N R C (1994) estimation. Moreover, the overall weight gain 106 (0-6 week) o f birds fed the experimental diets was 10% higher than the N R C (1994) estimation. The result o f the present study indicated that body weight gain o f 3-6 week old broilers did not increase and feed/gain ratios did not improve as dietary threonine and tryptophan increased beyond 90% and 96% of the N R C (1994) recommendations, respectively. This is equal to 0.67% of the diet or 3.4% o f C P for threonine and 0.17% of the diet or 0.89% of C P for tryptophan. Only a limited number o f studies have been conducted to evaluate the requirement of 3-6 week old broilers for threonine. Recently, Thomas et al. (1992) reported that the performance o f male broilers aged 3-6 weeks receiving 0.61% threonine i n the diet was similar to those fed higher threonine levels. Leeson and Summers (1997) recommended a value o f 0.60% of the diet, whereas Yamazaki et al. (1997a) suggested a value o f 0.54% digestible threonine for 3-6 week old broiler chicks. A higher value of 0.70% of the diet has been reported by the Rhodimet Nutrition Guide (1993) and Webel et al. (1996). On the other hand, K i d d and Kerr (1997) found that weight gain and feed efficiency of broilers (30-42 day o f age) did not further improve as the dietary threonine level increased beyond 0.65% of the diet. Penz et al. (1997) recommended 0.68% and 0.70% of dietary threonine for maximizing weight gain and feed efficiency, respectively. Interestingly, when the requirement value was expressed as a % o f C P , the estimated requirement for threonine (3.4% of CP) obtained in the present study was lower than that o f K i d d (1996) but close to the Penz et al. (1997) estimation. Data from several recent studies and from the present experiment indicate that the N R C (1994) may have overestimated the requirements o f 3-6 week old broilers for threonine. For tryptophan, comparison with other studies is difficult because not much data on tryptophan requirement are available for broilers beyond 3 weeks o f age. The earliest reported by Hunchar and Thomas (1976) found that 4-7 week old broilers required 0.176% and 0.167% of dietary tryptophan for maximum growth and optimum feed efficiency, respectively. Us ing the ideal protein concept, Baker's (1997) estimation for tryptophan requirement was 0.15% of digestible tryptophan for 21 to 42 day old male broilers. The tryptophan requirement value suggested by Leeson and Summers (1997) (0.17% of diet or 0.85% o f CP) is similar to the finding obtained in the present study. The Rhodimet Nutrition Guide (1993), however, recommended a higher level o f tryptophan (0.20% of diet) for 3-6 week old broilers. A value as low as 0.131% was reported by Hurwitz et al. (1978). However, data from Hurwitz et al. (1978) were derived from a computer model that has not been vigorously examined. Data from several 107 recent studies and from the present experiment indicate that the N R C (1994) recommended tryptophan requirement (0.18% of diet) for 3-6 week old broilers is valid. For the layer study, performance o f the layers (egg production, mean egg weight, daily egg mass and feed conversion ratio) during the production study were not affected by different dietary treatment. These results suggested that the threonine and tryptophan levels in layer diets should not be targeted at more than 448 mg of threonine/hen/day and 152 mg of tryptophan/hen/day for 42-50 week old laying hens. Limited data were available concerning the threonine and tryptophan requirements of laying hens. Using diets consisting mainly of corn, wheat and milo, Yamazaki et al. (1997b) reported that the requirement o f laying hens at 32-42 weeks o f age for digestible threonine was 329 mg/hen/day. Ishibashi et al. (1998) recommendations for threonine were 455 and 462 mg/hen/day for egg mass and feed efficiency, respectively. This is in good agreement with the N R C (1994) estimate for the threonine requirement o f laying hens (470 mg/hen/day). On the other hand, Coon (1998), Rhodimet Nutrition Guide (1993), Huyghebaert and Butler (1991) and Leeson and Summers (1997) recommended values of 495, 517, 522 and 627 mg/hen/day, respectively. These recent data indicate that laying hens should have a daily threonine intake at the N R C (1994) recommended level or higher. Results from this experiment showed that laying hens consuming 152 mg o f tryptophan per hen daily could maintain a high level of egg production. However, a further increase in tryptophan intake failed to increase the level of egg production. This is i n close agreement with the recommendations o f Leeson and Summers (1997) and the N R C (1994). Leeson and Summers (1997) recommendation for tryptophan is 154 mg/hen/day, whereas the N R C (1994) recommendation is 160 mg/hen/day. A value as low as 122 mg/hen/day was reported by Coon (1998) , whereas 165 mg/hen/day was recommended by the Rhodimet Nutrition Guide (1993). In another study, Jensen et al. (1990) conducted four experiments and found that the requirement o f laying hens for tryptophan ranges from 95 mg to 168 mg/hen/day. Data from these recent studies and from the present experiment indicate that the N R C (1994) recommended tryptophan requirement for laying hens is valid. One of the most important factors affecting the utilization o f dietary protein is the balance o f A A i n the feed. The closer the A A composition o f the diet matches the requirement for maintenance and growth, the less protein the animal needs and wastes. It is wel l known that reduced protein diets improve protein utilization by minimizing the excesses o f A A and result in 108 the reduction o f N excretion (Waldroup et al, 1976; Fancher and Jensen, 1989; Blai r et al, 1999). The results of the present study confirm our previous findings that reducing dietary C P appears to be a practical way o f reducing N excretion (Blair et al, 1999). Based on the findings o f the balance study, N output over the broiler starter period (0-21 days) would be about 112 g/(8 birds) with the reduced protein starter diets used in this study and would be 175 g/(8 birds) with the commercial starter diets used in this study. This represents a 36% reduction in the N output in the excreta. N o trend was observed with different threonine and tryptophan levels in the first N balance study. Nitrogen output over the grower period (22-42 days) would be about 45 g/bird with the reduced protein grower diets used in this study and would be 59 g/bird with the commercial grower diets used in this study. This represents a 25% reduction in N output in the excreta. Increasing the level of dietary tryptophan resulted in a decrease in excreta output, and an increase in the N content of excreta. N o trend was observed with dietary threonine level. When compared to the control broiler diets, the amount o f N retained was increased by 16% and 4.5% in the starter and grower periods, respectively, indicating better N utilization. Further reductions in the C P content of layer diets to about 12% in the experimental layer diets resulted in 46% lower excretion of N per day than layers fed the commercial layer diet, but the lower dietary C P gave a slightly reduced egg production. Layers fed the experimental diets also excreted on average 18% less excreta than layers fed the commercial diet. The excreta excreted by layers fed the experimental diets also contained 34% less N than that of layers fed the control diet. Nitrogen retained as a percentage o f intake was 13% higher with reduced-CP layer diets than with the control layer diet. There was a trend for N retention to decrease with increased dietary threonine, and the N output to decrease with increased tryptophan. The results o f the N balance studies are in agreement with the conclusions o f F E F A N A (1992), Moran et al. (1992), Summers (1993), Deschepper and De Groote (1995) and Ibrahim (1997) that by reducing the C P o f a diet, N excretion could also be significantly reduced and this provides an option for poultry producers to minimize N pollution from the farm. Conclusions Findings in the present studies indicate that when formulating diets for 0-3 week old broilers, dietary levels of threonine and tryptophan should be targeted at 0.74% o f the diet (4.04% of CP) and 0.23% of the diet (1.22% of CP) , respectively. For 3-6 week old broilers, 109 dietary levels o f threonine and tryptophan should be targeted at 0.67% o f the diet (3.20% of CP) and 0.17% of the diet (0.89% of CP) , respectively. Laying hens aged 42-50 week old, as indicated by the present study should be targeted at a daily intake o f 448 mg threonine/hen and 152 mg tryptophan/hen. The results o f this trial confirm our previous findings that reducing dietary C P improves the utilization of protein and appears to be a practical way o f reducing N excretion. The results of the balance studies clearly showed that crystalline A A supplementation of the reduced-protein diets improved the A A balance in the diet, hence improving the protein utilization efficiency and resulting in reduced N content in the excreta. Results from this and other studies also indicated that the N R C (1994) has overestimated the requirements o f threonine in 0-3 week and 3-6 week old broilers and underestimated tryptophan requirements o f 0-3 week old broiler chicks. On the other hand, the N R C (1994) recommendations o f threonine and tryptophan for layers and tryptophan for 3-6 week old broilers are supported by a number o f recently reported values. These results are valuable to poultry nutritionists for feed formulation and have economic implications with regard to the supplementation of reduced protein diets with feed-grade threonine and tryptophan. Clearly, more research is necessary to determine the requirement data for threonine and tryptophan in the future, particularly for laying hens and broilers during the growing period beyond 21 days o f age. References Abebe, S., and T. R. Morris , 1990. Effects o f protein concentration on responses to dietary tryptophan by chicks. Br . Poult. Sci . 31:267-272. Andrews, R. P., and N . A . Baldar, 1985. Amino acid analysis o f feed constituents. V o l . 32 no. 2. Science Tools. Association o f Official Analytical Chemists, 1984. Official methods o f analysis. 14 t h ed. Assoc. Off. Ana l . Chem. Washington, D C . Austic, R. E . , 1994. Update on amino acid requirements and ratios for broilers. Pages 114-120 in: Proceedings o f the Maryland Nutrition Conference, College Park, M D . Austic, R. E . , and M . Rangel-Lugo, 1989. Studies on the threonine requirement of broiler chicks. Pages 136-143 in: Proceedings Cornell Nutrition Conference for Feed Manufacturers, East Syracuse, N . Y . Baker, D . H . , 1997. Pages 1-24 in: Ideal amino acid profiles for swine and poultry and their applications in feed formulation. Biokyowa Publishing Co. , St. Louis, M O . 110 Bartov, I., N . Paster, and N . Lisker, 1982. The nutritional value o f moldy grains for broiler chicks. Poultry Sci . 61:2247-2254. Beasley, J . N . , L . D . Blalock, T. S. Nelson, and G . E . Templeton, 1980. The effect o f feeding corn molded with Penicillium lanosum to broiler chicks. Poultry Sci . 59:708-713. Blair , R. , J. P. Jacob, S. Ibrahim, and P. Wang, 1999. A quantitative assessment o f the use of reduced protein diets supplemented with amino acids to improve nitrogen utilization and reduce nitrogen pollution from broilers and layers. J. App l . Poultry Res. 8:25-47. Coon, C , 1998. Amino acid requirements of commercial laying hens. Pages 70-75 in: Proceedings o f 6 t h Asian Pacific Poultry Congress, Nagoya, Japan. Davis, A . J., and R. E . Austic, 1994. Dietary amino acid balance and metabolism o f the limiting amino acid. Pages 70-81 in: Proceedings of the Cornell Nutrition Conference for Feed Manufacturers. Rochester, N Y . Deschepper, K . and G . De Groote, 1995. Effect o f dietary protein, essential and non-essential amino acids on the performance and carcase composition of male broiler chickens. Br . Poult. Sci . 36:229-245. Estrin, B . , and W . S. Brammell, 1968. Determination of phosphorus in fruits and fruit products by a spectrophotometric molybdovanadate method and by the official gravimetric quinoline molybdate fertilizer method. J. Assoc. Off. Anal . Chem. 52:865-870. Fancher, B . I., and L . S. Jensen, 1989. Influence on performance o f three- to six-week-old broilers o f varying dietary protein contents with supplementation o f essential amino acid requirements. Poultry Sc i . 68:113-123. Federation Europeenne des Fabricants d'Adjuvants pour la Nutrition Animale, 1992. Improvement of the environment: possibilities for the reduction of nitrogen and phosphorus pollution caused by animal production. F E F A N A , Belgium. Fritz, J. C , P. B . Misl ivec , G . W . Pla, B . N . Harrison, C . E . Weeks, and J. G . Dantzman, 1973. Toxicogenicity o f moldy feed for young chicks. Poultry Sci . 52:1523-1530. Han, Y . , H . Suzuki, and D . H . Baker, 1991. Histidine and tryptophan requirement of growing chicks. Poultry Sci . 70:2148-2153. Holsheimer, J. P., P. F . G . Vereijken and J. B . Schutte, 1994. Response o f broiler chicks to threonine-supplemented diets to 4 weeks o f age. Br . Poult. Sci . 35:551-562. Hugh, T. E . , and S. Moore, 1972. Determination o f the tryptophan content o f proteins by ion exchange chromatography of alkaline hydrolysates. J. B i o l . Chem. 247:2828-2834. Hunchar, J. G . , and O. P. Thomas, 1976. The tryptophan requirement o f male and female broilers during the 4-7 week period. Poultry Sci . 55:379-383. I l l Hurwitz, S., D . Sklan, and I. Bartov, 1978. New formal approaches to determination o f energy and amino acid requirements of chicks. Poultry Sci . 57:197-205. Huyghebaert, G . , and E . A . Butler, 1991. Optimum threonine requirement o f laying hens. Br . Poult. Sci . 32:575-582. Ibrahim, S. B . , 1997. Modified poultry diets: A n approach to sustainable animal production. Ph.D. Thesis, Univ . o f British Columbia, Vancouver, Canada. Ishibashi, T., Y . Ogawa, T. Itoh, S. Fujimura, K . Koide, and R. Watanabe, 1998. Threonine requirements o f laying hens. Poultry Sci . 77:998-1002. Jensen, L . S., V . M . Calderon, and C . X . Mendonca, Jr., 1990. Response to tryptophan o f laying hens fed practical diets varying in protein concentration. Poultry Sci . 69:1956-1965. Jones, A . D . , H . S. Hitchcock and G . H . Jones, 1981. Determination o f tryptophan in feeds and feed ingredients by high-performance liquid chromatography. Analyst 106:968-973. K i d d , M . T., 1996. L-threonine for poultry. K y o w a Hakko Technical Review-8. Nutri-Quest, Inc. Chesterfield, M O , U S A . K i d d , M . T., and B . J. Kerr, 1997. Threonine responses in commercial broilers at 30 to 42 days. J. A p p l . Poultry Res. 6:362-367. K i d d , M . T., B . J. Kerr, J. D . Firman, and S. D . Bol ing, 1996. Growth and carcass characteristics o f broilers fed low-protein, threonine-supplemented diets. J. App l . Poultry Res. 5:180-190. K i m , J. H . , W . T. Cho, I. S. Shin, C. J. Yang, and In K . Han, 1997. Partition of amino acids requirement for maintenance and growth o f broiler. III. Tryptophan. A J A S 10:284-288. Leeson, S., and J. D . Summers, 1997. Commercial poultry nutrition. Leeson, S., and J. D . Summers ed. 2 n d edition. University Books, Guelph, Ontario, Canada. Macleod, M . G . , 1997. Effects of amino acid balance and energy:protein ratio on energy and nitrogen metabolism in male broiler chickens. Br . Poult. Sci . 38:405-411. Moore, S. J., 1963. On the determination of cysteine as cysteic acid. J. B i o l . Chem. 238:235-237. Moran, JR., E . T., R. D . Bushong, and S. F . B i l g i l i , 1992. Reducing dietary crude protein for broilers while satisfying amino acid requirements by least-cost formulation: L i v e performance, litter composition, and yield of fast-food carcass cuts at six weeks. Poultry Sci . 71:1687-1694. National Research Council , 1994. Nutrient Requirements o f Poultry. 9 t h rev. ed. National Academy Press, Washington, D C . Penz, A . M . Jr., G . L . Colnago, and L . S. Jensen. 1997. Threonine supplementation o f practical diets for 3- to 6-wk-old broilers. J. App l . Poultry Res. 6:355-361. 112 Rangel-Lugo, M , C . L . Su, and R. E . Austic, 1994. Threonine requirement and threonine imbalance in broiler chickens. Poult. Sci . 73:670-681. Rhodimet Nutrition Guide, 1993. Feed ingredients formulation in digestible amino acids, 2 n d edition 1993 Rhone-Poulenc Animal Nutrition. Robbins, K . R., 1987. Threonine requirement of the broiler chick as affected by protein level and source. Poultry Sci . 66:1531-1534. Rogers, S. R. , and G . M . Pesti, 1990. The influence o f dietary tryptophan on broiler chick growth and l ipid metabolism as mediated by dietary protein levels. Poultry Sci . 69:746-756. S A S Institute, 1996. SAS® User's Guide: Statistics. Version 6 Edition. S A S Institute Inc., Cary, N C . Sharby, T. F. , G . E . Templeton, J. N . Beasley, and E . L . Stephenson, 1973. Toxicity resulting from feeding experimentally molded corn to broiler chicks. Poultry Sci . 52:1007-1014. Snedecor, G . W. , and W . G . Cochran, 1980. Statistical methods. 8 t h ed. Iowa Press, Ames, Iowa. Spackman, D . H . , W . H . Stein and S. Moore, 1958. Automatic recording apparatus for use i n the chromatography o f amino acids. Ana l . Chem. 30:1190-1206. Steinhart, H . , and M . Kirchgessner, 1984. Investigations on the requirement o f tryptophan for broilers. Arch . Gefiiigelkd. 48:150-155. Summers, J. D . , 1993. Reducing nitrogen excretion o f the laying hen by feeding lower crude protein diets. Poultry Sci . 72:1473-1478. Thomas, O. P., M . Farran, and C . B . Tamplin, 1992. Broiler nutrition update: Threonine requirement for 3-6 week-old broilers. Pages 45-53 in: Proceedings Maryland Nutrition Conference, College Park, M D . Waldroup, P. W. , R. J., Mitchel l , J. R. Dayne, and K . R. Hazen, 1976. Performance o f chicks fed diets formulated to minimize excess levels of essential amino acids. Poultry Sci . 55:243-253. Webel, D . M . , S. R. Fernandez, C . M . Parsons, and D . H . Baker, 1996. Digestible threonine requirement o f broiler chickens during the period three to six and six to eight weeks posthatching. Poultry Sci . 75:1253-1257. Woodham, A . A . , and P. S. Deans, 1975. Amino acid requirements o f growing chickens. Br . Poult. Sci . 16:269-287. Yamazaki , M . , Y . Oka, H . Murakami, M . Takemase, M . Ando, and M . Yamazaki , 1997a. Available threonine requirement of broiler chickens at two growing stages. Jpn. Poult. Sci . 34:45-51. 113 Yamazaki, M . , H . Ohguchi, H . Murakami, M . Takemase, S. Hijikuro, M . Ando, and M . Yamazaki, 1997b. Available threonine requirement of laying hens. Jpn. Poult. Sci. 34:52-57. 114 CHAPTER V Evaluation and Enhancement of Palm Kernel Cake as a Poultry Feedstuff Summary Several in vitro and in vivo experiments were conducted to investigate the nutritive value o f palm kernel cake ( P K C ) for poultry and the effect o f enzyme supplementation on its nutrient quality. Palm kernel cake samples were collected from various palm kernel crushers in Malaysia. A l l samples were subjected to proximate analysis and amino acid ( A A ) analysis. The physical structure o f palm kernel and P K C were studied under a scanning electron microscope. Apparent metabolizable energy (AME) and nitrogen corrected true metabolizable energy (TMEn) values o f P K C were determined using Sibbald's (1986) precision-fed procedure. True A A digestibility ( T A A D ) o f P K C was determined using cecectomized cockerels. Several enzymes supplied by All tech Inc. were used for the enzyme studies. The effects o f pretreatment o f P K C with the most effective enzyme were also investigated. Intact and cecectomized cockerels were employed in the study o f nutrient digestibility. On average, P K C was found to contain about 17% crude protein, 15% crude fiber, 8% ether extract, 5% ash, 0.40% calcium and 0.77% phosphorus On a dry matter basis. Screw-pressed P K C contained higher amounts o f residual o i l , gross energy and acid detergent lignin than solvent-extracted P K C . Scanning electron micrographs o f the palm kernel revealed the rectangular honeycomb-like cell walls. The content and the digestibility o f most A A in the screw-pressed P K C were significantly lower (P < 0.05) and this probably was attributed to the formation of Mail lard products during the high temperature o i l extraction process. When compared to the N R C (1994) A A requirements for poultry, P K C was limiting in lysine, sulfur A A , threonine, isoleucine, valine and histidine. Apparent metabolizable energy and TME„ o f P K C were 1,492 kcal/kg and 1,800 kcal/kg, respectively. All tech PKCase (mannanase, cc-galactosidase and protease) was the most effective enzyme for P K C saccharification. PKCase supplementation significantly increased (P < 0.05) the release of reducing sugars in P K C and soybean meal by 26.8% - 67.4% and 20% - 30%, respectively. Even though the crude fiber content o f pretreated P K C was reduced, pretreatment o f P K C with PKCase was not justified i n terms o f its effects on animal production. PKCase supplementation significantly increased (P < 0.05) the A M E and TMEn o f P K C in both intact and cecectomized cockerels, however, 115 cecectomy significantly reduced (P < 0.05) the A M E and TME„ o f P K C . It was concluded that PKCase has great potential in increasing the performance of birds fed PKC-based diets. K e y words: palm kernel cake, amino acids, A M E , TMEn, enzymes, PKCase Introduction Based on the United Nations (1998) estimates, the world population w i l l reach 8.9 bi l l ion in 2050. Meanwhile, agricultural production is declining or stabilizing in many areas. This indicates that competition for food resources between animals and humans w i l l increase steadily in the near future. In fact, Duke (1996) stated that the use o f conventional feedstuffs such as corn and soybean meal might be impractical 20 years from now. The continued increase in population and loss o f farm land (for roads, buildings, etc.) may force farmers to save better quality foods for humans and poultry producers would have to feed lower quality diets to poultry. This scenario is very likely to happen especially in the developing countries, where most o f the human population is found. Most developing countries produce an abundant amount o f non-conventional feed ingredients and by-products such as rice bran, peanut meal, rapeseed meal and palm kernel cake ( P K C ) . In 1996, the production of palm o i l was second largest in the world following soybean o i l ( P O R L A , 1997). Palm kernel cake is a by-product o f the o i l palm industry and about 1.4 mi l l ion tonnes were produced in Malaysia alone in 1996 ( P O R L A , 1997). Not much research has been done on P K C regarding its use as poultry feed. From a number o f available reports (Nwokolo et al, 1977; Yeong, 1985; Onwudike, 1986; Siew, 1989), the nutrient composition o f P K C seems to be quite variable, especially its o i l , protein and energy contents. There are also great discrepancies in the reported digestibility of amino acids ( A A ) in P K C (Nwokolo et al, 1976; Onwudike, 1986; Yeong, 1983). The variation in A A digestibility values might be due to different varieties o f o i l palm and methods o f processing palm kernel o i l . Processing o f P K C involves much heat and pressure to break the kernel and extract as much o i l as possible. Thus, there are possibilities for the formation o f Mail lard compounds between the reducing sugars and A A in P K C , particularly lysine (Mauron, 1981; Hurrell and Finot, 1985). Knowledge of the A A composition of feed ingredients is useful but usually inadequate, because the amounts o f A A that are digestible and available to the animals are often much lower 116 than the quantity contained in the ingredient. This is especially true for less digestible non-conventional feed ingredients such as P K C . A s a result, it is necessary to know both the concentration and digestibility o f A A in feedstuffs in order to formulate diets that w i l l meet the animals' A A requirements most efficiently. However, the main criticism o f digestibility or balance studies concerns the effects o f the hindgut microflora on A A excretion. Consequently, several researchers have proposed the use o f cecectomized cockerels for determining A A digestibility o f feedstuffs (Parsons, 1985, 1986; Johns et al, 1986; Green et al, 1987). The problems o f low availability of energy and A A should be resolved by obtaining accurate values for metabolizable energy and A A digestibility and then using these values in dietary formulation to compensate for any deficiencies in P K C . Although the beneficial aspects o f exogenous enzyme addition to cereal-based poultry diets are wel l documented (Bedford and Classen, 1992; Campbell and Bedford, 1992; Cheeson, 1993; Grimes et al, 1997), the supplementation of exogenous enzymes to improve the digestion o f non-conventional feedstuffs such as P K C is not well established. Recently, several studies have indicated that enzymes with mannanase activity could partly break down the non-starch polysaccharide (mannans) o f P K C , hence improving its nutritive quality (Dusterhoft et al, 1993a,b,c; Daud et al, 1997). Therefore, the objectives o f this study were to determine the physical and chemical characteristics o f Malaysian P K C as a poultry feedstuff and to determine whether its nutritive value could be improved with enzyme supplementation. Materials & Methods Collection and Analyses of Palm Kernel Cake Samples Samples of P K C were collected from palm kernel crushers in various states in Malaysia, namely Johor (six samples), Penang (one sample), Sabah (two samples) and Selangor (four-samples). Nine samples were obtained from screw-press plants, while four were obtained from a solvent extraction plant (from the same plant over 4 months). Proximate analyses for crude protein (CP), crude fiber, ether extract, ash and calcium were carried out following the procedures outlined by the Association of Official Analytical Chemists ( A O A C , 1984). Phosphorus was measured using a spectrophotometric method (Estrin and Brammell , 1968). Gross energy was measured with an adiabatic oxygen bomb calorimeter (Parr, U S A ) . Neutral 117 detergent fibre (NDF) , acid detergent fiber ( A D F ) and acid detergent lignin ( A D L ) were analyzed according to Robertson and V a n Soest (1981). A l l o f the samples were analyzed in triplicate. Palm kernel cake samples were sent to Rhone Poulenc's laboratory in Commentry, France, for A A analysis according to the procedure outlined by Green et al. (1987). Amino acid analyses were conducted in duplicate with a Beckman Mult icrom B 4255 A A analyzer, after 24 h hydrolysis with 6 N hydrochloric acid. Before the analysis o f the sulphur-containing A A , a performic acid oxidation treatment was employed to prevent destruction o f methionine, cysteine and cystine during acid hydrolysis. For tryptophan, alkaline hydrolysis with sodium hydroxide was followed by high-pressure liquid chromatography with external calibration. Feed and excreta samples that were obtained from the digestibility trials were analyzed in triplicate for crude protein, A D F , N D F and A D L . Glycine was omitted in all calculations o f A A digestibility due to the breakdown o f uric acid to glycine during acid hydrolysis o f excreta (Soares et al., 1971). Scanning Electron Microscopy of Palm Kernel and Palm Kernel Cake Fresh palm kernels were cut into 0.5 cm x 0.5 cm cubes. They contained a high amount of o i l , making it difficult to study the palm kernel structure under a scanning electron microscope. A s a result, samples were immersed in petroleum ether for either 10 s or 10 min to extract the o i l . Later, the samples were fixed in 4% glutaraldehyde in 0.1 M sodium cacodylate buffer for 24 h and post-fixed in buffered 1% osmium tetroxide at 4 °C for 2 h. They were then dehydrated in an acetone series (10 min, in 35%, 50%, 75%, 95% and 15 min in 100% with three changes) and critical point dried ( B A L - T E C C P D 030, Critical Point Dryer, Switzerland). They were then mounted on 12 mm x 5 mm aluminum stubs (Agar Scientific, U K ) and coated with 20 nm gold (Polaron Equipment Ltd. S E M Coating Unit E5100, England). Solvent-extracted P K C was also prepared in the above manner for viewing under an electron microscope, except it was not treated with petroleum ether. Samples were examined with a J E O L 6400 Scanning Microscope (Japan) operated at 15 k V . 118 Metabol izable Energy and A m i n o A c i d Digestibi l i ty of P a l m Ke rne l C a k e for Poul t ry Three PKC samples obtained from three of the largest palm kernel crushers in Malaysia were selected for the study (Huplee, Lee and Premium). Both Huplee and Lee were obtained from plants using the screw-press technique for oil extraction, whereas Premium was obtained from a plant using the solvent-extract technique. Both the nitrogen corrected true metabolizable energy (TMEn) and the true amino acid digestibility (TAAD) were determined according to Sibbald's procedure (Sibbald, 1986). Briefly, all birds used in the experiment were fasted for 48 h to remove all digesta in the gastro-intestinal tract. Six cockerels were assigned to each treatment. Thirty grams of test feedstuff was given to each bird via crop intubation. A similar number of cockerels of each type were fasted throughout the experimental period to measure endogenous excretion. A plastic tray was placed under each cage and excreta were collected quantitatively for 48 h. Birds had free access to clean water. Thirty-week-old intact Lohmann Brown cockerels were used for the TME„ study. The excreta samples were dried at 60 °C, re-weighed (dry weight) and ground to pass through a 1 mm mesh screen. Gross energy was measured with an adiabatic oxygen bomb calorimeter. Total N content in the dry excreta was analyzed according to the AO AC (1984) procedure. A correction was made for nitrogen retention, which was either positive or negative. It was done to bring the TMEn data to a basis of nitrogen equilibrium. A correction factor of 8.73 kcal/g of nitrogen was used (Titus et al, 1959). However, 30-week-old cecectomized Lohmann Brown cockerels were used for the TAAD study to minimize the effect of microbial fermentation on AA digestibility. Cecectomy was performed on cockerels when they werel6-week-old according to the procedure of Parsons (1985). All cockerels were given 14 weeks to recover from surgery prior to being used in the experiments. Examination of the cockerels at the end of the experiment indicated that little or no cecal growth had occurred in the cecectomized cockerels. Excreta from two cockerels were pooled and freeze-dried and reweighed. All dried excreta and PKC samples were ground to pass through a 0.5-mm screen mesh and analyzed in duplicate for AA as described above. 119 Selection of Suitable Enzymes for P a l m Ke rne l Cake Sacchari f icat ion Cellulase, pentosanase, P-glucanase, protease and PKCase were obtained from Alltech Inc., (Kentucky, USA). PKCase is an enzyme mixture that contains 107,000 U/g of alpha-galactosidase activity, 2,300 HUT/g of protease activity (HUT = hemoglobin unit on the tyrosine base) and 12,081 U/g of mannanase activity. Manannase activity was determined by the procedure outlined by Araujo and Ward (1990), using locust bean galactomannan as the substrate. The mannanase assay mixture contained 0.5 ml of 1% (w/v) galactomannan, prepared in 0.1 M sodium acetate buffer, pH 5.8, 0.4 ml of 0.1 M sodium acetate buffer and 0.1 ml of enzyme solution (0.05 g PKCase in 250 ml deionized water). The reaction mixture was incubated at 50 °C for 30 min. Reducing sugars produced were determined as mannose reducing equivalents by using the dinitrosalicylic acid (DNS) method of Miller et al. (1960). One unit (U) of mannanase activity is defined as the amount producing 1 mol of product per min. The mannanase assay was also carried out in quadruplet at various temperatures to determine the optimum temperature for PKCase. In order to determine the effect of pH on PKCase's mannanase activity, assays were carried out in quadruplet at various pHs (glycine HCL buffer for pH 2.02; sodium acetate buffer for pH 3.6 to 5.8; phosphate buffer for pH 6.0 to 8.0) under the optimum temperature. To study the effect of different enzymes (cellulase, pentosanase, P-glucanase, protease and PKCase) on solvent-extracted PKC, an enzyme was added to 10 g of PKC at 2kg/t in 50 ml of 0.1 M phosphate buffer solution (pH 6.0). The mixture was incubated in a shaking water bath at 40 °C for 3 h. After incubation, the solution was centrifuged at 1500 revolutions per min for 5 min, then 200 \x\ of the supernatant solution was mixed with 1.80 ml of phosphate buffer and 3.0 ml of DNS solution in a screw-cap test tube. Test tubes were put into boiling water for exactly 5 min and then cooled for 5 min in cold water. Absorbance was read against the blank at 540 nm using a Hitachi U-2000 spectrophotometer (Kyoto, Japan). Standard mannose solutions with different concentrations were also prepared and a standard curve was prepared for every test. There were three replications for each enzyme. Combinations of various enzymes were also tested to investigate the possible interactions among enzymes. Since PKCase was found to be the most effective enzyme for the saccharification of PKC, a second experiment with PKCase was then carried out simulating the gastro-intestinal 120 tract conditions (pH 5.5 in crop; pH 3.0 in proventriculus; pH 6.5 in small intestine) in chickens according to Tervila-Wilo et al. (1996). The procedures were similar to those discussed above, except that solvent-extracted PKC was incubated with or without PKCase and exposed to different digestion stages (acidic condition with pepsin (Merck, 7179) and alkaline condition with pancreatin (Merck, 7133)). The treatments were as follows: Control = PKC incubated in phosphate buffer solution (40°C, pH 6.0, 3 h); PKCase control = PKC incubated with PKCase in phosphate buffer solution (40°C, pH 6.0, 3 h); Simulated control = PKC incubated under gastro-intestinal tract conditions (40°C, 3 h); Simulated PKCase = PKC incubated with PKCase under gastro-intestinal tract conditions (40°C, 3 h). There were three replications for each treatment. Another experiment was carried out to determine the effect of PKCase on different PKC samples (screw-pressed samples: Lee and Huplee; and solvent-extracted samples: Pre2, Pre3 and Pre4) and other feed ingredients namely corn and soybean meal. PKCase was added at either 1 kg/t or 2 kg/t. Five grams of samples were mixed with enzyme in 50 ml of phosphate buffer solution and incubated at 40°C for 3 h. The solution was then measured for reducing sugars released (Miller et al, 1960). There were five replications for each sample. Solvent-extracted PKC (Premium) was also incubated with PKCase to study the effects of enzyme pretreatment on its nutritive quality. Five hundred grams of PKC were mixed with PKCase (at 1 kg/t) in 2 L of distilled water in duplicate. The mixture was left at room temperature (30 °C) for 24 h. The wet samples were then dried in a 60°C force-draft oven until they reached a constant weight; they were labeled as PretPKC and analyzed for proximate constituents as above. Effect of P K C a s e on the Digestibi l i ty of Nutrients in P a l m Kerne l C a k e Both cecectomized (24 birds) and intact Lohmann Brown cockerels (18 birds) used in the TMEn and TAAD study above were also used in this study to determine the effect of PKCase on the digestibility of energy, ADF and NDF in solvent-extracted PKC (Premium and PretPKC). Eighteen cecectomized cockerels were assigned to Premium, Premium + lkg/t PKCase or PretPKC (six cockerels per sample). Another 12 cockerels (six intact and six cecectomized) were fasted for endogenous excreta collection. Twelve intact cockerels were assigned to either Premium or Premium + lkg/t PKCase (six cockerels per sample). The digestibility of PretPKC for intact cockerels was not determined because not enough samples were available. 121 Calculat ions T M E n (kcal/kg), and T A A D (%) were calculated as followed (Sibbald, 1979,1986): T M E n / kg o f feed = [(Fj x G E f ) - ( E x G E e ) - (NR x K ) + ( F E m + U E e ) ] / Fj Where, Fj = feed intake, g G E f = gross energy o f feed, kcal/kg E = excreta output, g G E e = gross energy of excreta, kcal/kg F E m + U E e = metabolic fecal and endogenous urinary energy (fasted birds), kcal/kg N R = nitrogen retention, i.e. nitrogen input minus nitrogen output, g K = is a constant (8.73 kcal/g o f retained nitrogen) The A M E assay was not conducted in this experiment. However, it could be estimated by the theoretical equation outlined by McNab (1990): A M E (kcal/kg) = T M E - ( E E L / Food intake) where, E E L is the endogenous energy loss (energy excreted by fasted birds, kcal/kg) T A A D (%) = 100 x {[(AA in feed - A A in excreta) + A A excreted by fasted bird] / A A in feed} Statistical Analyses This was a completely randomized design experiment. Data were subjected to A N O V A (SAS Institute, 1996) and treatment means separated using Tukey's multiple range tests (Snedecor and Cochran, 1980). 122 Results Col lect ion and Analyses of P a l m Kerne l Cake Samples The results o f the chemical analysis are presented in Table 5.1. N o significant differences in dry matter, C P , crude fiber, ash, calcium, phosphorus, N D F , A D F and nitrogen free extract contents were detected between the P K C from plants because o f the different methods of extraction except for ether extract, gross energy and A D L . Palm kernel cake obtained from solvent extraction plants contained significantly lower (P < 0.05) amounts o f o i l residue (3.29% versus 8.93%), gross energy (4,504 kcal/kg versus 4,718 kcal/kg), and A D L (7.94% versus 10.26%) when compared to P K C from screw-press plants. Most A A except alanine, histidine and tryptophan were significantly lower (P < 0.05) in the screw-pressed than in the solvent-extracted P K C (Table 5.2). However, when expressed as a percentage of C P , most A A except alanine, histidine, methionine, total sulfur A A and tryptophan were significantly lower (P < 0.05) in the screw-pressed than in the solvent-extracted P K C . Table 5.1 Nutr ient composit ion of Ma lays ian pa lm kernel cake 1 ' 2 ( D M basis) Nutr ients M e a n ± S.D. Range Dry matter, % 93.76 ± 2 . 1 4 91.38-96.31 Crude protein, % 16.58 ± 0 . 4 9 15.73 -17.19 Crude fiber, % 14.61 ± 1.23 12.47 -16.09 Ether extract2, % 8.03 ± 2 . 1 2 1.43 -11.39 Gross energy 2, kcal/kg 4,688 ± 122 4 ,422-4 ,843 Ash , % 4.91 ± 0.36 4.26 - 5.56 Calcium, % 0.41 ± 0 . 1 3 0.25 - 0.62 Phosphorus, % 0.77 ± 0 . 1 0 0.64 - 0.92 Neutral detergent fiber, % 70.07 ± 2.07 67.95 - 74.25 A c i d detergent fiber, % 43.08 ± 1.96 40.33 - 45.65 A c i d detergent l ignin 2 , % 9.51 ± 1 . 3 5 6.38-11.57 Hemicellulose 3 , % 26.98 ± 2.39 22.57 - 30.46 Nitrogen free extract, % 49.32 ± 2.77 45.12-54.37 Data represent mean of three replications of 13 palm kernel cakes samples (nine screw-pressed palm kernel cakes and four solvent-extracted palm kernel cakes); ± standard deviation. 2 Comparison between solvent-extracted and screw-pressed palm kernel cakes was not significant for most nutrients except ether extract, gross energy and acid detergent lignin. 3 Neutral detergent fiber - acid detergent fiber. 123 O *33 -«-> o M o-cu M cu Id-I o (A <U M V M cu CZJ .CA "CA CS XI CU es CU fl u CU es fl cu > fl es fl CU fl CU CU es o fl 1 es 13 fl es a "3 -M o M CU T3 S M cu fl v „ bX) o Z CN ID CU I ' H 3 M CU es s CA CA CU M a cu u cu IC/5 fl cu > Io g d <=> +i +; Ivo ^ CN ^ d ° +i +i i n . o c n d ° +1 +1 V O ^ CN 5 V O CN 00 CN CN m O 00 o i—< CN O N O i-H rt i n o 1-H CN »-H CN N O 1-H O N O N O 1-H O N o CN r-H o CN d o d d d d d d d d d d d d d d d d d +1 +1 V O 00 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 cn vo rt r~; <n 00 00 00 O N rt O N O N O N 1—1 N O vq O N cn CN 00 00 O N i n cn O N O CN N O oo oo CN <n 1-H i-H CN CN i-H d CN* d rt 1-H cn rt cn rt cn vd CN cn 1—1 JS oo CN d +1 00 rt CN OO d +1 m CN d +1 cn VO 2 ^ CN rt » CN t - g 00 o H © d ° d O N t-" V O O N © © © +1 +1 +, O N m c~- oo CN —I cn vo o o d d O N O N o d +1 rt 00 O N 00 d CN CN O JS JS JS CN N O O d d © d +1 +1 +1 +1 O N O N cn o cn rt cn d rt cn JS JS JS JS JS JS rt O N O 00 O rt 1-H . - H 1-H »—i d d d © © d d +1 +1 +1 +1 +1 +1 +1 i n 00 O N o CN oo rt f-H 00 cn O i n 00 rt cn rt cn N O CN cn « a CN N a R a a m R R a a R rt rt rt ON CN CN cn NO in O rt m O O O © o o o O o d d d d d d d o d d d d d +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 VO 00 . - H © o CN vo cn ON i n r-- CN q q cn ! cn cn i—t rt CN r- ON cn rt oo 1-H cn rt cn <n cn NO CN cn CN d +1 o i n O N »-H d +1 in V O o rt rt O CN O CN o CN O CN o o cn o cn 1-H CN O cn o cn o rt O cn o rt O CN O cn o rt O d d d d d d d d d d d d d d d d d d d +1 +1 +1 +1 +l +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 rt CN cn O N <n rt rt o cn V O i-H 00 rt V O c~-vo oo oo CN i n O N V O cn V O oo in <n O N O N cn rt cn vo m CN i-H d d d d d d d CN d d d d d d d d d JS JS JS JS JS JS JS JS JS JS JS CN f—i JS JS JS JS JS rt J—< cn CN 1-H <—1 CN Y-H (—) CN CN CN CN rt CN rt CN cn O 1-H O O O O o O i—i O O O o O O o d d d d d d d o d d d d d d d d d d +1 +1 +1 +1 +1 +1 +l +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +l VO cn i n o i n o vo m o CN vq ON cn CN CN CN 00 rt rt cn i—i rt d vo 00 i n vo i n ON rt VO 1-H 1-H d d d d d d CN d d d d d d d d R a R R R R a R R R a rt R R R R R in CN cn CN CN i—1 CN (—\ cn rt CN cn cn cn m CN cn o t—f o O O o O W o O o <—' o o o O o d d d d d d d d d d d d d d d d d d +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 cn i-H o o CN 00 i-H CN cn i n cn VO vq VO oo i n <n VO cn i-H i n i n cn i-H i n r- q <n oo m o rt VO i-H CN d d d d d o d cn d d d d d d d rt O d +1 rt CN d rt O d +1 CN d .c CA >N O CL) fl 2 CD O H 00 D I 8 *2 U c CA „ , + •§ PH 53) CD fl " f l CD O •9 3 (2. " CD fl • a o CD CD fl CD fl fl O CD CA u, U H P H o CD C •c CD ,_ H w O X I O + fl CD c cu fl CD o < > CD .C fl CD O CA CD O fl <D (D . g 'CA I H CD fl ^ CD ^ . g fl "jj fD to PH HI a o •fl '> u •O •a U I to +1 CD •a u a a a. •a CO CA CO CO ^ i CO hH o CO CO m O O V OH a CO -3 >s cd o i f l '3 .60 CO co i-i CS O a o co \j3 a CO || M r l co .S! 1-i M « 3^ co 2 co Oc £H 2 ii -b V S « t3 ^ t l o x CO CO 3 3 p CO 60 CO II .s o VH •a a i i "eo & o CO co co •S & 60 3 rt M l-l HH rt a D. fo co V H rt (fo •3 53 u co O CO &"*-> 1  Q H • rt co 124 Scanning Elect ron Microscopy of P a l m Kerne l and Pa lm Kerne l C a k e The scanning electron micrographs (Figures 5.1, 5.2) show that palm kernel is made up of oi ly rectangular honeycomb-like cell walls. Unfortunately, it was difficult to identify and differentiate the different physical structure of P K C in the electron micrograph (Figure 5.3). Fresh palm kernels consist of white oily flesh. However, P K C samples collected in this study were brown in color. In general, P K C samples obtained from solvent extraction plants had a light brown color, while samples obtained from screw-press plants had a medium to dark brown color. Palm kernel cake samples for the proximate analysis were obtained from palm kernel crushers using different o i l extraction methods (solvent extraction and screw-press extraction). Metabol izable Energy and Amino A c i d Digestibi l i ty of P a l m Kerne l C a k e for Poul t ry N o significant differences (P > 0.05) were found in the A M E and TMEn values even though Huplee and Lee contained higher o i l residue and gross energy than Premium (Table 5.3). When metabolizable energy values (AME and TMEn) were expressed as percentages o f GE, the values were constant for all samples. The digestibility coefficients for six A A were found to be significantly different (P < 0.05) among Lee, Huplee and Premium (Table 5.4). The A A digestibility coefficients for arginine, glutamic acid plus glutamine were significantly lower (P < 0.05) in Lee. The A A digestibility coefficients for lysine and cystine were significantly higher (P < 0.05) in Premium, but tryptophan digestibility was significantly lower (P < 0.05) in Premium. There were no significant differences in tryptophan digestibility between Lee and Premium or between Lee and Huplee. On the other hand, the A A digestibility coefficient for histidine was significantly lower (P < 0.05) in Huplee. However, no significant difference in histidine digestibility was detected between Lee and Premium. For the overall A A digestibility coefficient, it was significantly higher for Premium when compared to Lee (Table 5.4). But no significantly differences i n overall A A digestibility coefficient were found between Premium and Huplee or Lee and Huplee. 125 P a l m kernel o i l C e l l wa l l covered wi th palm kernel oi l F igure 5.1 C e l l w a l l structures (700 x, 15 k V ) of palm kernel (extracted wi th petroleum ether for 10 s) under a scanning electron microscope showing honeycomb-like pa lm kernel cell walls covered wi th o i l . 126 Oil-free cell wa l l Figure 5.2 C e l l w a l l structures (100 x, 15 k V ) of palm kernel (extracted wi th petroleum ether for 10 min) under a scanning electron microscope showing oil-free cell walls. Figure 5.3 P a l m kernel cake under a scanning electron microscope (3,000 x, 15 k V ) . Table 5.3 The effect of different amounts of o i l residue (ether extract) in pa lm kernel cake ( P K C ) on its metabolizable energy values ( D M basis + S.D.) W P K C E E % of D M GE kcal/kg TMEn kcal/kg A M E kcal/kg A M E (% of GE) TMEn (% of GE) Huplee 7.29 ± 0.05 a 4,628 ± 6 a 1,816 ± 9 0 1,531 ± 9 2 33 39 Lee 8.35 ± 0.04 a 4,764 ± 1 9 8 1,820 ± 102 1,492 ± 9 8 31 38 Premium 1 bb = ether e: 3.04 ± 0.02 b ctract; Lib = gross 4,422 ± 18 b energy; 1Mb = 1,762 ± 9 7 nitrogen corrected 1,452 ± 9 1 true metabolizab 33 le enerev: A M b 40 = arroarent metabolizable energy; Data represent mean of six cockerels; ± standard deviation. 2 Huplee and Lee were screw-pressed PKC; Premium was solvent-extracted PKC. "''Treatment means with different superscripts within a row are significantly different at P < 0.05. 128 Table 5.4 T rue amino acid digestibil ity (%) of samples of pa lm kernel cake (Mean ± S.D.) 1 Lee Huplee Premium Mean Processing method Screw-press Screw-press Solvent Color of samples Dark brown Brown Light brown -A m i n o acids Asp + Asn 2 52.8 ±3.5 59.5 ±6 .1 61.1 ± 1.4 57.8 ±5 .3 Arginine 80.9 ± 1.2 b 83.1 ± 0 . 6 a 82.8 ± 0.7 a 82.3 ± 1.3 Lysine 38.8 ± 5.3 b 42.0 ± 3.7 b 50.4 ± 1.5 a 43.8 ± 6.2 Methionine 69.7 ± 1.1 72.5 ± 0.7 73.2 ±3 .2 71.8 ±2 .4 Cystine 33.9 ± 3.3 b 32.6 ± 6.2 b 49.7 ± 10.0 a 38.7 ± 10.2 Threonine 53.5 + 4.6 59.6 ±12.6 58.4 ±1 .4 57.2 ± 7.3 Tryptophan 56.6 ± 2.1 a b 59.9 ± 3.7 a 51.3 ± 5 . 7 " 55.9 ±5 .2 Serine 65.2 ±4 .8 68.1 ±5 .4 69.2 ± 1.7 67.5 ±4 .1 Glu + G l n 2 63.9 ± 2.5 b 74.5 ± 1.2 a 74.1 ± 1.1 8 70.8 ± 5.4 Proline 56.4 ± 3.9 57.6 ±4.1 63.5 ± 3.0 59.2 ±4 .6 Alanine 39.2 ± 6.0 40.8 ± 9.0 48.1 ±6 .2 42.7 ± 7.5 Valine 62.4 ± 3.7 68.3 ±3.2 67.7 ± 2.2 66.1 ±3 .9 Isoleucine 64.6 ± 3.6 68.8 ± 0.3 66.9 ± 3.7 66.8 ±3 .1 Leucine 66.5 ± 2.5 70.1 ± 1.2 69.1 ±2 .9 68.6 ±2 .6 Tyrosine 70.2 ± 3.5 73.5 ± 1.3 71.7 ±0 .3 71.8 ±2 .4 Phenyalanine 71.8 ±3 .2 74.6 ± 1.4 72.6 ± 2.8 73.0 ±2 .6 Histidine 59.0 ± 1.0 a b 54.0 ± 6 . 9 " 64.2 ± 2.4 a 59.1 ±5 .7 Mean 59.2 ± 2.7 b 62.3 ± 2.6 a b 64.4 ± 2.4 a 62.0 ±3 .3 1 Data represent mean of three replications of two cecectomized cockerels; ± standard deviation. 1 Asp = aspartic acid; Asn = asparagine; Glu = glutamic acid; Gin = glutamine. a ' Treatment means with different superscripts within a row are significantly different at P < 0.05. 129 Selection of Suitable Enzymes for P a l m Kerne l Cake Sacchari f icat ion PKCase was found to contain 12,081 U / g of mannanase activity. The optimum temperature and p H for the mannanase activity were 60 °C and 5.0, respectively (Figures 5.4 and 5.5). All tech enzymes such as cellulase and PKCase significantly increased (P < 0.05) the release o f reducing sugars from P K C (Table 5.5). PKCase released the highest amount o f reducing sugars from P K C , and combining PKCase with other enzymes did not yield a higher level of reducing sugars than PKCase alone. The fact that PKCase activity was maintained under the gastro-intestinal conditions (Table 5.6) indicates that PKCase still yielded significant amounts of reducing sugars under these conditions. PKCase significantly increased (P < 0.05) the release o f reducing sugars from various P K C samples and soybean meal (Tables 5.7 and 5.8). However, PKCase failed to increase the release of reducing sugars from corn. Pretreatment of P K C with PKCase at 30 °C did not affect most of the nutrient components o f P K C (Table 5.9). However, the crude fiber level was significantly reduced (P < 0.05) by PKCase pretreatment. Drying wet pretreated P K C samples is a time consuming and energy-driven process. In addition, some of the pretreated samples became infested with molds during the drying processes. Effect of P K C a s e on the Digestibi l i ty of Nutrients in P a l m Kerne l Cake PKCase supplementation significantly increased (P < 0.05) metabolizable energy (AME and TMEn) o f P K C in both intact and cecectomized birds (Table 5.10). Cecectomy significantly reduced (P < 0.05) the A M E and TME„ contents of P K C . However, PKCase supplementation and pretreatment alleviated the negative effect. Cecectomy also significantly reduced true D M retention (Table 5.10). While cecectomy did not have any effect on N D F retention, it improved A D F retention significantly (P < 0.05). % 131 C M ° » * a s § 5 fe QJ > P i u WD _4. x P x T cP o o o o o o o o o o o 3> OH % ' A ^ I A i p E 9 S B I I E U U E U I 3 A i ; B p ^ [ 132 Table 5.5 Release of reducing sugars (expressed as mannose equivalents) f rom solvent-extracted pa lm kernel cake ( P K C ) after incubation with different A l l tech enzymes. . ..^ , , N Mean reducing sugars released All tech enzyme(s) n . c ^ I (umol mannose per ml) + S.D. Control 5.81 ± 0.17 c Cellulase 8.25 ± 0.22 b Pentosanase 6.02 ± 0 .19 c Protease 5.98 ± 0 .15 c P-glucanase + cellulase 8.52 ± 0.15 b P K C a s e 2 13.34 ± 0 .35 a PKCase + cellulase 13.53 ± 0.10" PKCase + pentosanase 13.32 ± 0 . 1 6 PKCase + protease 13.30 ± 0 . 1 8 PKCase + p-glucanase + cellulase 13.64 ± 0.15 1 Each value represents the mean of three replicates; ± standard deviation. 2 Alltech, Inc. (Kentucky, USA). It contains mannanase, ct-galactosidase and protease activity. a , b ' c Means with different superscripts differ significantly (P < 0.05) a a a Table 5.6 Effect of incubat ing solvent-extracted pa lm kernel cake ( P K C ) with Al l tech P K C a s e under gastro-intestinal (GI) tract conditions \ Treatments 2 Mean reducing sugars released (limol mannose per ml) ± S .D. 3 Control 5.81 ± 0 .36 d PKCase control 11.95 ± 0 .29 c Simulated control 15.26 ± 0.53 b Simulated PKCase 20.80 ± 0.17 a 1 PKCase is an enzyme mixture designed by Alltech, Inc. (Kentucky, USA) especially for PKC. It contains mannanase, cc-galactosidase and protease activity. 2 Control = PKC incubated in phosphate buffer solution (40°C, pH 6.0, 3 h); PKCase control = PKC incubated with PKCase in phosphate buffer solution (40°C, pH 6.0, 3 h); Simulated control = PKC incubated under GI tract conditions (40°C, 3 h); Simulated PKCase = PKC incubated with PKCase under GI tract conditions (40°C, 3 h). 3 Each value represents the mean of three replicates; ± standard deviation. a' b' c' d Means with different superscripts differ significantly (P < 0.05). 133 Table 5.7 The effect of incubating several pa lm kernel cake ( P K C ) samples wi th Al l tech P K C a s e ! . P K C samples Reducing sugars released (umol mannose/ml) Enzyme effect (p.mol mannose/ml) Enzyme effect % increase Screw-pressed PKC Huplee Huplee + lkg/t PKCase 6.51 ± 0 . 1 5 b 9.91 ± 0 . 7 8 a 3.40 ± 0.78 52% Lee Lee + lkg/t PKCase 5.27 ± 0.03 b 8.82 ± 0 .28 a 3.55 ± 0.28 67% Solvent-extracted PKC Pre2 Pre2 + lkg/t PKCase 9.82 ± 0.12 b 12.45 ± 0 . 1 1 a 2.63 ± 0 . 1 1 27% Pre3 Pre3 + lkg/t PKCase 4.45 ± 0.01 b 6.61 ± 0 . 3 1 a 2.18 ± 0 . 3 1 49% Pre4 Pre4 + lkg/t PKCase 6.04 ± 0.12 b 9.32 ± 0.37 a 3.28 ± 0 . 3 7 54% PKCase is an enzyme mixture designed by Alltech, Inc. (Kentucky, USA) especially for PKC. It contains mannanase, a-galactosidase and protease activity; Mean of five replicates, ± standard deviation. a ' b Means in the same row and same PKC samples with different superscripts differ significantly (P < 0.05) 134 Table 5.8 The effect of incubat ing corn, soybean kernel cake ( P K C ) wi th Al l tech P K C a s e ! . meal ( S B M ) and solvent-extracted pa lm Feed ingredient Reducing sugars released (umol mannose/ml) Enzyme effect (umol mannose/ml) Enzyme effect % increase Corn 9.62 ± 0.27 - -Corn + lkg/t PKCase 9.49 ± 0 . 1 4 0.00 0% Corn + 2kg/t PKCase 9.58 ± 0 . 1 9 0.00 0% S B M 3.05 ± 0.09 c _ S B M + lkg/t PKCase 3.66 ± 0.09 b 0.61 ± 0.09 20% S B M + 2kg/t PKCase 3.98 ± 0.07 8 0.92 ± 0.07 30% P K C 4.42 ± 0.08 c _ P K C + lkg/t PKCase 6.63 ± 0.25 b 2.20 ± 0.25 50% P K C + 2kg/t PKCase 7.37 ± 0 . 3 7 8 2.94 ± 0.37 67% PKCase is an enzyme mixture designed by Alltech, Inc. (Kentucky, USA) especially for PKC. It contains mannanase, a-galactosidase and protease activity; Mean of five replicates, ± standard deviation. a ' b Means in the same row and same PKC samples with different superscripts differ significantly (P < 0.05) Table 5.9 Prox imate analysis of untreated pa lm kernel cake ( P K C ) and PKCase- t rea ted pa lm kernel cake 1 , 2 . Sample A s h C P E E G E (kcal/kg) C F Untreated P K C (% D M ) 5.84 ± 0.04 17.19 ± 0 . 4 6 5.49 ± 0 . 1 1 4,591 ± 16 12.6 ± 0 . 2 8 PKCase-treated P K C (% D M ) 5.70 ± 0.09 17.21 ± 0.33 5.37 ± 0.06 4,611 ± 18 10.9 ± 0 . 1 b Solvent-extracted PKC was incubated with or without lkg/t of PKCase under 30 °C for 24 h. PKCase is an enzyme mixture designed by Alltech, Inc. (Kentucky, USA) especially for PKC. It contains mannanase, a-galactosidase and protease activity; values represent mean of six analyses (± standard deviation) CP = crude protein; EE = ether extract; GE = gross energy; CF = crude fiber. a ' b Means in the same column with different superscripts differ significantly (P < 0.05) 135 Table 5.10 The effect of enzyme (PKCase) supplementation on nitrogen corrected true metabolizable energy ( T M E „ ) , true dry matter (DM) retention, neutral detergent f iber (NDF) and acid detergent fiber ( A D F ) retention *. Solvent-extracted Palm kernel cake A M E (kcal/kg) TMEn (kcal/kg) True D M retention, % NDF retention, % A D F retention, % Normal birds Premium (P) 1,438 b 1,929 b 37.64 8 17.74 4 . 1 3 b P + lkg/t PKCase 1,624 a 2,115 a 38.17" 20.59 4.03 b Cecectomized birds Premium (P) 1,039" 1,703 c 26.33 c 17.97 5.05" P + lkg/t PKCase 1,269 c 1,933 b 32.27 b 19.94 5.29" Pre tPKC 2 1,225 c 1,853 b 25.33 c 18.12 5.46" Overall mean 1,322 1,908 32.18 19.94 5.29 Pooled S E M 3 40 28 1.05 0.81 0.21 Data represent mean of six cockerels 2 Solvent-extracted PKC was incubated with lkg/t of PKCase under 30 °C for 24 h. PKCase is an enzyme mixture designed by Alltech, Inc. (Kentucky, USA) especially for PKC. It contains mannanase, a-galactosidase and protease activity. 3 Standard error of the mean a ' b , c Means in the same column with different superscripts differ significantly (P < 0.05) 136 D I S C U S S I O N Palm kernel cake samples used in this study contained a high dry matter content, a moderate amount o f protein, a ratio of calcium to phosphorus of about 1:2 and a high level of crude fiber, N D F and A D F . Electron micrographs reveal that palm kernels contained a large amount of o i l , hence the efficiency of the o i l extraction process w i l l have a major impact on the amounts o f o i l residue in P K C . The high values o f A D F , N D F and A D L indicate that P K C is comprised mainly o f cell wal l material and this is confirmed by the appearance o f the palm kernel under the electron microscope. The cell walls in P K C are mainly made up o f complex carbohydrates such as mannans (Dusterhoft and Voragen, 1991; Dusterhoft et al, 1992; Daud and Jarvis, 1992). Poultry do not possess the enzyme (mannanase) required to hydrolyze mannans in P K C . Therefore, the crystalline and insoluble mannans are not likely to be depolymerized under the digestive tract conditions in fowl (Daud and Jarvis, 1992). Thus, the potential o f feed enzyme (with mannanase activity) supplementation o f PKC-based diets remains promising. It is very likely that during the extraction of o i l from palm kernels that some o f the cell wal l structures observed in the electron scanning micrographs w i l l be distorted and damaged. This should facilitate the release o f some o f the nutrients within the cells. However, it is also likely that large amounts o f nutrients w i l l remain trapped within the cells and be unavailable to the animals. The high fiber content, the lack o f mannanase to break down mannans and the likelihood o f entrapment o f nutrients in the cells could be the reasons for the poorer performance o f birds fed PKC-based diets as reported by Ahmad (1982), Longe (1984) and Osei and A m o (1987). The gross energy content of P K C is dependent on the amount o f o i l residue, which is dependent on the efficiency of palm kernel o i l extraction. Solvent extraction plants are more efficient in extracting o i l and this is reflected in the lower o i l content and energy level o f its P K C . It was interesting to note that P K C from solvent extraction plants contained significantly lower amounts o f A D L . I believe that this is not due to different varieties of o i l palm used. Due to the nature o f o i l extraction conditions in the screw-press plants, palm kernels are exposed to extremely high temperature and pressure, which is l ikely the reason for the darker color of the screw-pressed P K C . The reactions o f proteins with reducing sugars (Maillard or browning reactions) are perhaps the most common cause o f nutritional damage to proteins during high 137 temperature and pressure food processing (Hurrell and Finot, 1985). It is l ikely that the reactions between proteins and reducing sugars (Maillard reactions) during the o i l extraction process led to the formation of heat-damaged products that are more resistant to acid digestion in the A D L procedure, leading to a higher A D L content in screw-pressed P K C . The heat-damaged protein, in turn, might also be more resistant to acid hydrolysis during A A analyses and may have reduced the release o f individual A A and their recovery. This might be the reason for the lower quantity o f most A A in screw-pressed P K C . When compared with corn or soybean meal, the ratio o f A A in P K C in relation to the N R C (1994) requirement was far from balanced (Tables 5.11, 5.12 and 5.13). The order o f limiting A A (in descending order) in P K C is cystine, lysine, threonine, methionine, isoleucine and histidine for starter broiler chicks (0-3 week old). On the other hand, corn is limiting only in lysine, tryptophan and arginine whereas soybean meal is limiting only i n the sulfur A A (Table 5.11) . For grower broiler chicks (3-6 week old), P K C is limiting in lysine, cystine, threonine, isoleucine, histidine and methionine (in descending order). Corn is limiting in lysine, tryptophan, arginine, threonine and isoleucine whereas soybean meal is limiting only in the sulfur A A (Table 5.12) . For the laying hens, P K C is limiting in cystine, lysine, isoleucine, tryptophan, methionine, threonine and valine. Corn, on the other hand, is limiting only in lysine, tryptophan, isoleucine and arginine whereas soybean meal is limiting only in the sulfur A A (Table 5.13). It is important to note that P K C contains a high level o f arginine (approximately 200% of N R C (1994) requirement) and a low level of lysine. Special attention should be paid to the adverse arginine : lysine ratio during feed formulation because excess arginine has been shown to depress growth o f chicks fed a lysine-deficient diet and this adverse effect was reversed by supplementary lysine ( D ' M e l l o and Lewis, 1970). Soybean meal protein has a better A A balance than P K C protein. A s a result, it is not very likely that P K C can replace much soybean meal in feed formulation. Palm kernel cake could replace corn to a greater extent i f o i l could be brought in to offset the low metabolizable energy of P K C . The failure o f P K C to substantially replace soybean meal in feed formulation has been demonstrated in the studies o f Ahmad (1982), Yeong and Mukherjee (1983) and Onifade and Babatunde (1998). With the commercial availability o f crystalline A A such as lysine, methionine, threonine and tryptophan, the quality o f P K C could be greatly improved. 138 Table 5.11 A m i n o acid composition (% of C P ) of pa lm kernel cake ( P K C ) , corn and soybean meal ( S B M , 4 4 % C P ) in relation to the N R C (1994) requirements for the growth of 0-3 week o ld b ro i l e rs 1 (90% D M ) . Amino acids N R C (1994) P K C (% of N R C ) Corn (% of N R C ) S B M (% of N R C ) Arginine 5.43 10.67(197%) 4.57 (84%) 7.29(134%) Lysine 4.78 2.47 (52%) 3.13 (65%) 6.23 (130%) Methionine 2.17 1.68 (78%) 2.17(100%) 1.44 (66%) Cystine 1.74 0.88 (51%) 2.17(125%) 1.53 (88%) Threonine 3.48 2.65 (76%) 3.49 (100%) 3.99(115%) Tryptophan 0.87 0.89 (102%) 0.73 (83%) 1.71 (197%) Valine 3.91 4.47(114%) 4.82 (123%) 4.80 (123%) Isoleucine 3.48 3.02 (87%) 3.49 (100%) 4.54(130%) Leucine 5.22 5.48 (105%) 12.07(231%) 7.86(151%) Histidine 1.52 1.37 (90%) 2.77(182%) 2.71 (179%) Phenylalanine 3.13 3.49(112%) 4.57 (146%) 5.01 (160%) 'Values for Corn and SBM were obtained from the National Research Council (NRC, 1994) Table 5.12 A m i n o acid composit ion (% of C P ) of pa lm kernel cake ( P K C ) , corn and soybean meal ( S B M , 4 4 % C P ) in relation to the N R C (1994) requirements for the growth of 3-6 week old b ro i l e rs 1 (90% D M ) . Amino acids N R C (1994) P K C (% of N R C ) Corn (% of N R C ) S B M (% of N R C ) Arginine 5.50 10.67 (194%) 4.57 (83%) 7.29 (132%) Lysine 5.00 2.47 (49%) 3.13 (63%) 6.23 (125%) Methionine 1.90 1.68 (89%) 2.17(114%) 1.44 (76%) Cystine 1.70 0.88 (52%) 2.17(128%) 1.53 (90%) Threonine 3.70 2.65 (72%) 3.49 (94%) 3.99(108%) Tryptophan 0.90 0.89 (99%) 0.73 (81%) 1.71 (190%) Valine 4.10 4.47 (109%) 4.82(117%) 4.80(117%) Isoleucine 3.65 3.02 (83%) 3.49 (96%) 4.54 (124%) Leucine 5.45 5.48 (101%) 12.07 (221%) 7.86 (144%) Histidine 1.60 1.37(86%) 2.77 (173%) 2.71 (170%) Phenylalanine 3.25 3.49 (107%) 4.57 (141%) 5.01 (154%) Values for Corn and SBM were obtained from the National Research Council (NRC, 1994) 139 Table 5.13 A m i n o acid composit ion (% of C P ) of pa lm kernel cake ( P K C ) , corn and soybean meal ( S B M , 4 4 % C P ) in relation to the N R C (1994) requirements for laying h e n s 1 (90% D M ) . Amino acids N R C (1994) P K C (% of N R C ) Corn (% of N R C ) S B M (% of N R C ) Arginine 4.67 10.67 (229%) 4.57 (98%) 7.29 (156%) Lysine 4.60 2.47(54%) 3.13 (68%) 6.23 (136%) Methionine 2.00 1.68 (84%) 2.17 (108%) 1.44(72%) Cystine 1.87 0.88 (47%) 2.17(116%) 1.53 (82%) Threonine 3.13 2.65 (85%) 3.49(111%) 3.99 (127%) Tryptophan 1.07 0.89 (83%) 0.73 (68%) 1.71 (160%) Valine 4.67 4.47 (96%) 4.82 (103%) 4.80 (103%) Isoleucine 4.33 3.02 (70%) 3.49 (81%) 4.54 (105%) Leucine 5.47 5.48 (100%) 12.07 (221%) 7.86(144%) Histidine 1.13 1.37(121%) 2.77 (245%) 2.71 (240%) Phenylalanine 3.13 3.49(112%) 4.57 (146%) 5.01 (160%) Values for Corn and SBM were obtained from the National Research Council (NRC, 1994). Requirement values were based on 100 g intake per hen per day. The Mail lard reactions may also lead to the formation o f inter- and intra-molecular cross-linkages within P K C proteins, which reduces the overall protein digestibility and the bioavailability o f all A A . The lysine molecule is destroyed during advanced Mail lard reactions, as are other A A such as tryptophan, methionine and cystine, presumably through reacting with active intermediate compounds such as dicarbonyls and aldehydes (Finot et al, 1982; Hurrell et al, 1983). These cross-linkages also reduce the rate o f protein digestion by preventing enzyme penetration or by masking the sites o f enzyme attack (Hurrell and Finot, 1985). This may explain the significantly lower digestibilities of some A A in screw-pressed P K C (Table 5.4). The relative amounts o f individual A A i n P K C and their digestibility values found i n this study are in close agreement with those o f Yeong (1983). However, the mean A A digestibility coefficient found in the present study was lower than those reported by Nwokolo et al. (1976) and Onwudike (1986). The average digestibility o f A A in P K C in the present study was 61.7% compared to 48%, 64.4%, 84.5% and 83.3% reported by Vilarino et al. (1996), Yeong (1983), Nwokolo et al. (1976) and Onwudike (1986), respectively. It was also interesting to find that the lysine digestibility in P K C was approximately 90% as reported by Nwokolo et al. (1976) and Onwudike (1986), whereas the values o f 58.6%, 26% and 43.8% were reported by Yeong (1983), Vilarino et al. (1996) and the present study, respectively. The reasons for these 140 discrepancies are not clear. However, the A A digestibility values determined in the present study were derived from cecectomized cockerels, whereas those reported by others were obtained using intact cockerels. Johns et al. (1986) have clearly demonstrated that an excreta digestibility technique gave appreciably higher A A digestibility values when heat-damaged meat and bone meal was fed to intact rather than to cecectomized cockerels. Screw-pressed P K C was used in the studies conducted by Nwokolo et al. (1976), Onwudike (1986) and Vilarino et al. (1996), whereas solvent-extracted P K C was used by Yeong (1983). Both types o f P K C were used in the present study and both were found to have lower A A digestibility than that reported by Nwokolo et al. (1976) and Onwudike (1986). In the present study, solvent-extracted P K C was found to contain significantly higher levels of digestible A A than screw-pressed P K C . Probably screw-pressed P K C was exposed to a higher temperature during the o i l extraction process. Due to the harsh environment during the processing of palm kernels and the susceptibility o f most A A to heat damage, I feel that the A A digestibility values reported by Nwokolo et al. (1976) and Onwudike (1986) were somewhat overestimated. The poor A A digestibility in P K C is likely to be attributed to protein entrapment within the cells as well as the high temperatures used during the o i l extraction process. The present study found that only about 33% and 40% of gross energy was available as A M E and TMEn respectively to poultry. This is one of the main constraints in using P K C for poultry. Siew (1989) reported that solvent extracted P K C from Malaysia contained about 1,480 kcal/kg A M E and 1,760 kcal/kg TME„, while Rhone Poulenc (Rhodimet Nutrition Guide, 1993) reported an A M E value of 1,340 kcal/kg. These values agree with the findings o f the present study. However, much higher values for A M E have been reported by several researchers, ranging from 1,963 kcal/kg to 3,000 kcal/kg (Nwokolo et al, 1977; Ngoupayou, 1984; Nwokolo, 1986; Onwudike, 1986; Panigrahi and Powell , 1991; Vilarino et al, 1996). Some o f the A M E values reported were somewhat higher than expected. For instance, Ngoupayou (1984) reported an A M E value o f 3,000 kcal/kg for P K C with 8% ether extract, whereas an A M E value o f 2,153 kcal/kg was reported by Nwokolo (1986) for P K C with only 2.09% ether extract. In another study, Nwokolo et al. (1977) reported an A M E value of 2,796 kcal/kg for P K C containing 43.7% A D F , 21.1%) A D L and 4,680 kcal/kg of gross energy. These A M E values were even higher than the TMEn values reported in the present study. It is not clear how broilers in the study conducted by Nwokolo et al. (1977) could metabolize about 60% o f the gross energy in P K C when it contained such a high level of fiber and lignin. 141 Knowledge o f the polysaccharide composition o f targeted feed ingredients is one of the most important factors for the successful employment o f feed enzymes. Recently, the polysaccharide components of P K C have been characterized by Dusterhoft and Voragen (1991), Dusterhoft et al. (1992) and Daud and Jarvis (1992). Palm kernel cake was found to contain mainly linear mannans (polymer o f mannose), moderate amounts o f cellulose, and small amounts o f other polysaccharides. A s a result, it is not surprising to find that PKCase (containing mannanase, protease and a-galactosidase) is the most effective enzyme mixture for breaking down the mannans in P K C . Other enzymes such as P-glucanase, pentosanase and protease were not effective in breaking down the cell walls o f P K C . Cellulase worked fairly wel l with P K C , however it was still inferior to PKCase. These findings reflect the polysaccharide composition of P K C . A synergistic reaction between cellulase and PKCase was not observed in this study. This is in agreement with the conclusions of Dusterhoft et al. (1993b) and Daud et al. (1997) that neither mannan degradation enhances cellulose hydrolysis nor cellulose degradation enhances mannan hydrolysis. The determined optimum p H and temperature for mannanase activity (in PKCase) were in agreement with the study o f Mendoza et al. (1994), who found that the optimum p H and temperature ranged from 5.0 to 6.0 and 50 °C to 60 °C, respectively, for a crude enzyme excreted by a mannan-utilizing bacterium. PKCase worked very wel l under the p H conditions o f the gastro-intestinal tract (Figure 5.5). However, only 40% of the optimum enzyme activity was attainable under the 40 °C temperature found in fowl. This implies that a further increase in temperature would be necessary for greater improvements in the saccharification of P K C . Wi th the exception of one sample, up to 50% more reducing sugars were released from various P K C samples with enzyme treatment. This indicates that PKCase acts consistently across various P K C samples. This is an important feature for the prediction o f the improvement in animal performance when P K C from various sources is used. A s expected, PKCase did not have any effect on the release of reducing sugars from corn because starch was the only major polysaccharide present. On the other hand, the beneficial effect o f PKCase on soybean meal was expected. This is because PKCase also contained a-galactosidase activity that could break down the non-starch polysaccharides (mainly polymers o f galactose) o f soybean meal. Even though not measured, protease activity present in PKCase should also have a beneficial effect on protein and A A digestion in P K C , corn and soybean meal. In addition, using twice the amount o f PKCase 142 further increased the breakdown o f cell walls of P K C and soybean meal. However, the amount of reducing sugars released was not proportional to the quantity of PKCase used. Most grains and seeds contain endogenous enzymes required for the breakdown o f storage polysaccharides during germination. Soaking and wetting barley and wheat have been shown to improve the performance o f broiler chicks fed diets containing these pretreated ingredients and this has been attributed to the activation o f enzymes in the grain (Svihus et al, 1997; Yasar and Forbes, 1997). Enzyme pretreatment o f wheat has also been shown to improve broiler performance by reducing the gut's viscosity, and increasing the digestibility o f protein, fat, ash, as wel l as the energy (Svihus et al, 1997). However, pretreatment o f P K C with PKCase did not have a major effect on most of the nutrients. This is perhaps due to low enzyme activities at the room temperature o f 30 °C. Crude fiber content in the pretreated P K C was significantly (P < 0.05) reduced by 1.7%. Even though this reduction was most l ikely due to the effect o f the enzymes, it is also possible that natural fungus originally present on the P K C samples might have helped the digestion o f fiber during the 24 h incubation period. The reduction in fiber content was associated with an increase in A M E and TMEn values measured with cecectomized cockerels. Data in the present study clearly showed that PKCase is capable o f increasing the A M E and TME„ values o f P K C , the magnitude of increase being even greater with cecectomized cockerels. The A M E and TMEn values of P K C were increased by 13% and 9.6% respectively by PKCase in normal intact cockerels. Wi th cecectomized cockerels, it went up by 22% and 13.5%, respectively. Without PKCase supplementation, the A M E and TME„ values o f P K C were reduced significantly when measured in cecectomized birds, indicating that the ceca might be taking part in the digestion o f fiber. Thornburn and Wil lcox (1965) investigated the role of ceca in the digestion of crude fiber using intact and cecectomized birds. They found that the overall dry matter digestibility o f four feeds (whole wheat, whole oats, whole barley and mixed feed in pellet form) was reduced by cecectomy. This is in agreement with the findings presented in here. However, Thornburn and Wil lcox (1965) also stated that even though cellulose digestibility was reduced in individual birds after cecectomy, the reduction was not always apparent. This is because the avian rectum had microflora capable o f degrading fiber as Duke et al (1984) reported that cecectomized turkeys still degraded 7% o f dietary cellulose. Because o f the presence o f ceca in intact cockerels, it was expected that A D F and N D F digestibility values for intact cockerels might be higher than that o f cecectomized cockerels, i f 143 ceca are involved in the digestion of fiber. However, this was not observed in this study. Perhaps the ceca is not involved in fiber digestion as Mattocks (1971) found that no fiber entered the ceca in domestic goose and concluded that it seems unlikely that ceca contribute to the feed's utilization by cellulose digestion. Researchers from Japan also concluded that ceca do not play a significant part in crude fiber digestion in domesticated birds (Nakahiro and Isshiki, 1975). Nevertheless, ceca have a definite role in the wi ld state that they can allow birds to conserve water, nitrogen and energy (McNab, 1973). On the other hand a study conducted by Savory (1992) discovered that degradation o f cellulose by the intestinal microflora does occur normally in (conditioned) fowls. Other researchers (Gasaway, 1976 and Duke et al, 1984) emphasized that the extent to which measurable cellulose digestion in birds takes place depends on preconditioning with high fiber diets. When not used for the metabolizable energy study, cockerels were fed low-fiber broiler diets (therefore, not preconditioned with high fiber diets) in the present study. This might partly explain the non-significant difference in N D F digestion between the cecectomized and intact cockerels. However, it does not explain why the digestion of A D F was higher in cecectomized birds. Conclusions The results o f the present study showed that P K C contained a moderate quantity of most nutrients. The digestibility of A A and metabolizable energy is influenced by the method of o i l extraction. The A A in P K C are not well balanced to meet poultry requirements and several A A are limiting. However, crystalline A A could be used to improve P K C protein quality. Due to the low digestibility o f A A in P K C , formulating poultry diets containing P K C on a digestible A A basis should be an improvement over formulating on a total A A basis. There are indications that the low digestibility o f A A in P K C is attributed to protein entrapment within cells as wel l as to products formed due to the high temperatures used during the o i l extraction process. Similarly, the high fiber content and the lack o f appropriate enzymes for breaking it down could have led to the low A M E and TMEn contents in P K C . PKCase (mannanase, a-galactosidase and protease) was found to be very effective in breaking down the polysaccharide component o f P K C . PKCase supplementation increased the A M E and TME„ o f P K C in both intact and cecectomized cockerels. However, cecectomy reduced the A M E and TME„ o f P K C . Pretreatment o f P K C with 144 enzymes did not seem to be justified due to the high cost o f drying after treatment. In addition, there are risks o f mold infestation during the drying processes. Given its beneficial effects on P K C , further studies investigating the effects of PKCase on broiler and layer performance are warranted. References Ahmad, M . Y . , 1982. The feeding value o f palm kernel cake for broilers. M A R D I Res. B u l l . 10:120-126. Araujo, A . , and O. P. Ward, 1990. Extracellular mannanases and galactanases from selected fungi. J. Industial Microbiol . 6:171-178. Association o f Official Analytical Chemists, 1984. Official methods o f analysis. 14 t h ed. Assoc. Off. Ana l . Chem. Washington, D C . Bedford, M . R., and H . L . Classen, 1992. Reduction of intestinal viscosity through manipulation o f dietary rye and pentosanase concentration is effected through changes in the carbohydrate composition o f the intestinal aqueous phase and results in improved growth rate and food conversion efficiency o f broiler chicks. J. Nutr. 122:560-569. Campbell, G . L . and M . R. Bedford, 1992. Enzyme applications for monogastric feeds: A review. Can. J. A n i m . Sci . 72:449-466. Cheeson, A . , 1993. Feed enzymes. A n i m . Feed Sci . Technol. 45:65-79. Daud, M . J. , and M . C . Jarvis, 1992. Mannan of palm kernel. Phytochem. 31:463-464. Daud, M . J. , N . Samad, and S. Rasool, 1997. Specific commercial enzymes for nutritive value improvement of palm kernel cake for poultry diets. Pages 137-138 in: 19 t h M S A P Annual Conference. Y . W . Ho , M . Z . Saad, F . Y . Chin, I. Zulk i f l i , and H . K . Wong ed. Johor Bahru, Johor, Malaysia. D ' M e l l o , J. P. F . , and D . Lewis, 1970. Amino acid interactions in chick nutrition. III. Interdependence in amino acid requirements. Br . Poult. Sci . 11:367-385. Duke, G . E . , 1996. Practical advancements in digestive physiology and futuristic research needs in poultry. J. App l . Poultry Res. 5:82-85. Duke, G . E . , E . Eccleston, S. Kirkwood, C. F . Louis and H . P. Bedbury, 1984. Cellulose digestion by domestic turkeys fed low or high fiber diets. J. Nutr. 114:95-102. 145 Dusterhoft, E . M . and A . G . J. Voragen, 1991. Non-starch polysaccharides from Sunflower (Helianthus annuus) Mea l and Palm Kernel (Elaeis guineenis) M e a l - Preparation o f cell wal l material and extraction of polysaccharide fractions. J. Sci . Food Agric . 55:411-422. Dusterhoft, E . M . , M . A . Posthumus, and A . G . J. Voragen, 1992. Non-starch polysaccharides from Sunflower (Helianthus annuus) Mea l and Palm Kernel (Elaeis guineenis) Mea l -Investigation o f the structure of major polysaccharide. J. Sci . Food Agr ic . 59:151-160. Dusterhoft, E . M . , F . M . , Engels, and A . G . F . Voragen, 1993a. Parameters affecting the enzymic hydrolysis o f oil-seed meals, lignocellulosic by-products o f the food industry. Bioresource Technol. 44:39-46. Dusterhoft, E . M . , A . W . Bonte, and A . G . J. Voragen, 1993b. Solubilisation of non-starch polysaccharides from oil-seed meals by polysaccharides - degrading enzymes. J. Sc i . Agric . 63:211-220. Dusterhoft, E . M . , A . W . Bonte, J. C. Venekamp, and A . G . J. Voragen, 1993c. The role of fungal polysaccharidases in the hydrolysis o f cell wall materials from sunflower and palm-kernel meals. Wor ld J. Microbiology and Biotechnology 9:544-554. Estrin, B . , and W . S. Brammell, 1968. Determination of phosphorus in fruits and fruit products by a spectrophotometric molybdovanadate method and by the official gravimetric quinoline molybdate fertilizer method. J. Assoc. Off. Anal . Chem. 52:865-870. Finot, P. A . , E . Magnenat, G . Guignard, and R. F . Hurrell, 1982. The behaviour o f tryptophan during "early" and "advanced" Mail lard reactions. Int. J. Nutr. Res. 52:226. Gasaway, W . C , 1976. Cellulose digestion and metabolism by captive rock ptarmigan. Comp. Biochem. Physiol . 54A:179-182. Green, S., S. L . Bertrand, M . J. C . Duron, and R. Mail lard, 1987. Digestibilities o f amino acids in soyabean, sunflower and groundnut meals, determined with intact and cecectomized cockerels. Br . Poult. Sc i . 28:643-652. Grimes, J. L . , P. R. Ferket, and A . N . Crouch, 1997. Enzyme supplementation o f broiler and turkey diets to enhance wheat utilization. Pages 131-139 in: Proceedings o f All tech's 13 t h Annual Symposium, Biotechnology in the feed industry. T. P. Lyons and K . A . Jacques, ed. All tech, Inc. Kentucky, U S A . Nottingham University Press. Hurrell , R . F. , and P. A . Finot, 1985. Effects of food processing on protein digestibility and amino acid availability. Pages 233-246 in: Digestibility and Amino A c i d Availabil i ty in Cereals and Oilseeds. Finley, J. W . and Hopkins, D . T., ed. American Association of Cereal Chemistry, Inc. Hurrell, R . F . , P. A . Finot and J. E . Ford, 1983. Storage o f mi lk powders under adverse conditions. 1. Losses of lysine and of other essential amino acids as determined by chemical and microbiological methods. Br . J. Nutr. 49:343-354. 1 4 6 Johns, D . C , C . K . L o w , J. R. Sedcoles, and K . A . C . James, 1986. Determination o f amino acid digestibility using caecectomized and intact adult cockerels. Br . Poult. Sci . 27:451-461. Longe, O. G . , 1984. Effects o f increasing the fiber content of a layer diet. Br . Poult. Sci . 25:187-193. Mattocks, J. G . M . , 1971. Some aspects o f the problems of cellulose digestion and caecal function in the domestic goose. N . Sc. Thesis, Univ . o f Bath. Mauron, J. , 1981. The Mail lard reaction in food: a critical review from nutritional standpoint. Prog. Food Nutr. Sci . 5:5-35. M c N a b , J. M . , 1973. The avian caeca: A review. World 's Poult. Sci . J. 29:251-263. McNab , J. M . , 1990. Apparent and true metabolizable energy of poultry diets. Pages 41-54 in: Feedstuff Evaluation. J. Wiseman and D . J. A . Cole, ed. Butterworths, U . K . Mendoza, N . S., M . Ara i , T. Kawaguchi, F . S. Cubol, E . G . Panerio, T. Yoshida, and L . M . Joson, 1994. Isolation o f mannan-utilizing bacteria and the culture conditions for mannanase production. World J. Microbiol . Biotechnol. 10:51-54. Mi l l e r , G , R . B lum, W . Glennon, and A . Burton, 1960. Measurement o f Carboxymethylcellulase activity. Ana l . Biochem. 2:127-132. Nakahiro, Y . , and Y . Isshiki, 1975. Effect o f cecal ligation on digestibility o f crude fiber, cellulose and pentosan in chickens. Jpn. Poult. Sci . 12:138-140. National Research Council , 1994. Nutrient Requirements of Poultry. 9 t h rev. ed. National Academy Press, Washington, D C . Ngoupayou, Ngou J. D . , 1984. Nutritional value o f palm kernel cake in broiler diets. Poult. Sci . 63 (Suppl. 1):155-156. (Abstr.) Nwokolo , E . , 1986. A comparison of metabolizable energy content o f eight common feed ingredients determined with young guinea fowls (keets) and pullet chicks. A n i m . Feed Sci . Technol. 15:1-6. Nwokolo, E . N . , D . B . Bragg, and W . D . Kitts, 1976. The availability o f amino acids from palm kernel, soybean, cotton seed and rapeseed meal for the growing chick. Poult. Sci . 55:2300-2304. Nwokolo, E . N . , D . B . Bragg, and H . S. Saben, 1977. A nutritive evaluation o f palm kernel meal for use in poultry rations. Trop. Sci . 19:147-154. Onifade, A . A . , and G . M . Babatunde, 1998. Comparison o f the utilization o f palm kernel meal, brewers' dried grains and maize offal by broiler chicks. Br . Poult. Sci . 39:245-250. 147 Onwudike, O. C , 1986. Palm kernel as a feed for poultry. 1. Composition o f palm kernel meal and availability o f its amino acids to chicks. A n i m . Feed Sci . Technol. 16:179-186. Osei, S. A . , and J. A m o , 1987. Research note: palm kernel cake as a broiler feed ingredient. Poult. Sci . 66:1870-1873. Panigrahi, S., and C. J. Powell , 1991. Effects of high rates of inclusion o f palm kernel meal in broiler chick diets. A n i m . Feed Sci . Technol. 34:37-47. Parsons, C . M . , 1985. Influence o f cecectomy on digestibility o f amino acids by roosters fed distillers' dried grains with solubles. J. Agric . Sci . Camb. 104:469-472. Parsons, C . M . , 1986. Determination o f digestible and available amino acids i n meat meal using conventional and cecectomized cockerels or chick growth assays. Br . J. Nutr. 56:227-240. P O R L A , 1997. Porla palm o i l statistics. 16 t h ed. Palm O i l Registration and Licensing Authority. Ministry o f Primary Industries, Malaysia. Rhodimet Nutrition Guide, 1993. Feed ingredients formulation in digestible amino acids, 2 n d edition 1993 Rhone-Poulenc Animal Nutrition. Robertson, J. B . , and P. J. Van Soest, 1981. The detergent system of analysis and its application to human foods. Pages 123-158 in: The Analysis o f Dietary Fibre i n Food. W.P .T . James and O. Theander, ed. Marcel Dekker, Inc., New York, N Y . S A S Institute, 1996. SAS® User's Guide: Statistics. Version 6 Edition. S A S Institute Inc., Cary, N C . Savory, C . J. , 1992. Enzyme supplementation, degradation and metabolism o f three U - 1 4 C -labelled cell wal l substrates in the fowl. Br . J. Nutr. 67:91-102. Sibbald, I. R. , 1979. A bioassay for available amino acids and true metabolizable energy in feedingstuffs. Poultry Sci . 58:668-673. Sibbald, I. R. , 1986. The T. M . E . system of feed evaluation: methodology, feed composition data and bibliography. Tech. B u l l . 1986-4E, Res. Branch, Agriculture Canada, Ottawa, Ont. Canada. Siew, W . L . , 1989. Characteristics and uses o f Malaysian palm kernel cake. P O R I M Techno. Palm O i l Res. Inst. Malaysia. No . 14. Snedecor, G . W. , and W . G . Cochran, 1980. Statistical methods. 8 t h ed. Iowa Press, Ames, Iowa. Soares, J. H . , D . Mi l le r , N . Firtz, and M . Sanders, 1971. Some factors affecting the biological availability o f amino acids in fish protein. Poultry Sci . 50:1134-1143. 148 Svihus, B . , R. K . Newman, and C . W . Newman, 1997. Effect o f soaking, germinating, and enzyme treatment o f whole barley on nutritional value and digestive tract parameters o f broiler chickens. Br . Poult. Sci . 38:390-396. Tervila-Wilo, A . , T. Parkkonen, A . Morgan, M . Hopeakoski-Nurminen, K . Poutanen, P. Heikkinen, and K . Autio, 1996. In vitro digestion of wheat microstructure with xylanase and cellulase from Trichoderma reesei. J. Cereal Sci . 24: 215-225. Thornburn, C . C . and J. S. Wilcox, 1965. The caeca o f the domestic fowl and digestion o f the crude fiber complex. I. Digestibility trials with normal and cecectomized birds. B r . Poult. Sci . 6:23-31. Titus, H . W. , A . L . Mehring, Jr., D . Johnson, Jr., L . L . Nesbitt, and T. Tomas, 1959. A n evaluation o f M C F (micro-cel-fat), a new type of fat product. Poultry Sci . 38:1114-1119. United Nations, 1998. Revision of the world population: Estimates and Projections. www.popin.org/pop 1998/1 .htm. Vilar ino, M . , F . Rudeaux, A . Leon, and M . Picard, 1996. Metabolizable energy and digestible amino acids contents in cockerels of two o i l extracted by-products: palm kernel meals (Elaeis guineensis) and germ-bran extracted maize (Zea mays). Revue d Elevage et de Medecine Veterinaire des Pays Tropicaux. 49:229-234. Yasar, S., and J. M . Forbes, 1997. Effects of wetting and enzyme supplementation o f wheat-based foods on performance and gut responses o f broiler chickens. Br . Poult. Sci . 38 (Suppl.):S43-S44. Yeong, S. W. , 1983. Amino acid availability of palm kernel cake, palm o i l sludge and sludge fermented product (Prolima) in studies with chickens. M A R D I Res. B u l l . , 11:84-88. Yeong, S. W. , 1985. Palm oi l by-products as feeds for poultry. Pages 175-186 in: Proc. Natl . Symp. On o i l palm by-products for agro-based industries, 5-6 November, 1985. Malaysia. Yeong, S. W. , and T. K . Mukherjee, 1983. The effect of palm oi l supplementation in palm kernel cake-based diets on the performance of broiler chickens. M A R D I Res. B u l l . , 11:378-384. 149 C H A P T E R V I Effects of Dietary Inclusion of P a l m Kerne l Cake and Enzyme Supplementat ion on Bro i le r and Laye r Performance Summary A total o f 400 day-old female broiler chicks and 392 twenty eight weeks old laying hens was used to study the effects of dietary inclusion of solvent-extracted palm kernel cake ( P K C ) and enzyme (mixture o f mannanase, a-galactosidase and protease) supplementation o f P K C on broiler and layer performance. In the 6 week broiler study, a corn-soybean meal control diet and 20% P K C diets with or without enzyme supplementation (1 or 2 kg/t) were used. Palm kernel cake-based diets were included in either the starter phase, or grower phase or in both phases. In the 8 week layer study, two levels o f P K C (12.5% and 25%) and three levels o f enzymes (0, 1 or 2 kg/t) were arranged in a factorial manner to form six experimental P K C diets. A corn-soybean meal layer diet was used as the control. Enzyme supplementation improved the digestibility of PKC-based diets and the performance o f broilers and layers fed PKC-based diets. The broiler study found that 20% P K C could be used during the broiler starter phase or the broiler grower phase but not during both the starter and grower phases unless supplemental enzyme was used. Mortality due to heat stress was significantly higher (P < 0.05) in broilers fed PKC-based grower diets without enzyme supplementation. Broilers fed P K C diets with or without enzyme supplementation deposited significantly more (P < 0.05) abdominal fat than controls. Dietary inclusion o f 12.5% and 25% P K C in layer diets did not adversely affect mean egg production or daily egg mass. However, layers consumed significantly more (P < 0.05) PKC-based diets and had significantly poorer (P < 0.05) F C R than controls. Enzyme supplementation o f PKC-based layer diets decreased feed intake and F C R significantly (P < 0.05). Dietary inclusi6n o f P K C or enzyme did not affect eggshell quality, but egg yolk color became significantly paler (P < 0.05) when layers were fed the 25% P K C diet. Parametric linear programming was used to investigate the price at which P K C (with or without lkg/t enzyme) could be used by a computerized feed formulation program and at what level of inclusion. It was found that the use of P K C in poultry diets is dependent on the metabolizable energy of the diet, the price o f P K C , and the A M E value 150 of P K C . Because o f its low nutrient contents, P K C is more suited for use in the lower energy layer diets than in the higher energy broiler diets. K e y words: Palm kernel cake, enzyme, broiler, layer, feed formulation Introduction A s the world's human population continues to increase and the acres o f farmland continue to decrease, the use o f feedstuffs that are not directly available to humans for food is increasingly important. Similar concerns have been raised by Berepubo et al. (1995), who emphasized that in most developing countries, especially Nigeria, feed resources for livestock and poultry production have become increasingly expensive because o f the increasing competition between humans and domesticated animals for scarce grains and protein feeds. More recently, Sheldon (1998) has also emphasized the importance o f developing alternative and cheaper feed ingredients in order to avoid competition with those essential for humans. From this standpoint, it is therefore logical to look for alternative feeds that are not consumable by humans but are acceptable to farm animals. Palm o i l has become a major vegetable o i l in the past few decades and many developing countries including Malaysia have devoted a huge amount o f farmland for o i l palm (Elaeis guineensis Jacq.) plantations ( P O R L A , 1997). One of the most important by-products o f palm o i l production is palm kernel cake (PKC) . Even though large quantities o f P K C are available for feed, the use of P K C in the feed industry is mainly limited to the ruminant sector. Palm kernel cake is not widely used in the poultry industry because o f its high fiber and low energy contents. Furthermore, information regarding the nutrient composition o f P K C is limited and there are great discrepancies in the reported values. Although the subject of using P K C in poultry diets has been studied by several researchers, the recommended levels o f inclusion seem to vary from one study to another. Onifade and Babatunde (1998) found that broiler growth performance was depressed in birds fed diets containing as little as 10% P K C when compared to a corn-soybean meal diet. However, studies conducted by Ahmad (1982) and Garcia and Gernat (1998) indicated that dietary inclusion of 10% P K C did not reduce growth o f broiler chicks when compared with a corn-soybean meal control diet. Osei and A m o (1987), on the other hand, reported that inclusion o f P K C at 12.5% and 15% of the diet reduced growth and feed efficiency. 151 Even though Longe (1984) reported poorer egg production for layers fed 20% P K C , other authors found that 20% P K C could be used in broiler and layer diets without reducing performance (Yeong, 1980; Hutagalung, 1980; Yeong and Mukherjee, 1983; Ngoupayou, 1984). Sti l l other researchers have reported that levels as high as 40% for layers and 50% for broilers could be used without reducing performance (Onwudike, 1988; Panigrahi and Powell , 1991). The reasons for these wide variations in reported inclusion levels are not clear. Perhaps, they are caused by the variety o f o i l palm used in different countries, methods o f processing palm kernel o i l , age o f the birds, and duration and design o f the experiments. Recently, several studies have identified the potential o f using exogenous enzymes to degrade the non-starch polysaccharides o f P K C (Dusterhoft et al, 1993a,b,c; Daud et al, 1997). Previous studies (Chapter V ) also found that an enzyme mixture (PKCase) from Alltech, Inc. (Kentucky, U S A ) has the ability to increase the metabolizable energy of P K C . Therefore, the objectives o f the following experiments were to determine the effects o f different inclusion levels o f P K C and enzyme supplementation on broiler and laying hen performance under hot and humid tropical conditions. Materials & Methods A 6 week broiler study and an 8 week layer study were conducted. True dry matter digestibility ( T D M D ) , nitrogen corrected true metabolizable energy (TMEn) and apparent metabolizable energy (AME) of broiler and layer diets were determined in a digestibility study. Palm kernel cake obtained from a solvent extraction plant was used throughout the study. A n enzyme mixture (Alltech PKCase) containing mannanase, a-galactosidase and protease activities was used in the broiler and layer studies. Bro i l e r G r o w t h Study A total o f 400 female day-old broiler chicks (Hubbard) was used in the study. The chicks were vaccinated against Marek's disease at the hatchery and against Newcastle disease at 7 and 21 days o f age during the experimental period. Birds were housed in battery brooders (0.9 m 152 wide x 1.2 m length x 0.5 m height) in an open-sided building at the Poultry Research Unit at the Universiti Putra Malaysia, Malaysia. A l l chicks were wing-banded at one day o f age and were randomly distributed to 40 battery pens (10 birds per pen). Birds received 24 h o f light daily and had free access to feed and water. Individual body weights of the birds were measured at day old and then weekly until 6 weeks of age. Feed intake was also measured weekly. Max imum and minimum temperatures within the building were measured daily. Da i ly mortalities were recorded and the carcasses were sent to the veterinary pathology laboratory at the Universiti Putra Malaysia for post-mortem examination. On the last day o f the experiment, five birds from each dietary treatment were sacrificed for the measurement o f abdominal fat. Abdominal fat was removed from the gizzard to the cloaca following the procedures outlined by Cahaner et al. (1986). In order to investigate whether broilers fed diets with different level o f fiber would result in a higher body temperature, eight birds from each dietary treatment group were also randomly selected and measured for rectal temperature in the afternoon (1:00 p.m.) during the last day o f the experiment. Bro i l e r Dietary Treatment A total o f four starter diets (SI , S2, S3, S4) and four grower diets ( G I , G 2 , G 3 , G4) were used in the study. Diets SI and G I were the control diets that did not contain P K C or enzymes, while the rest o f the diets contained 20% P K C with or without enzyme supplementation. The compositions o f the starter and grower diets are shown in Tables 6.1 and 6.2, respectively. The levels o f nutrients in the diets were formulated according to the N R C (1994) recommendation for broilers.lt was also the objective of this study to determine whether feeding chicks diets containing P K C at different phases would yield different results. Three different phases were used: Starter phase = P K C was included only in the starter diets; Grower phase = P K C was included only in the grower diets; Both phases = P K C was included in both starter (0-3 weeks) and grower (3-6 weeks) diets. The enzyme (PKCase) was included in the P K C diets at three levels (0, 1, 2 kg/t). Three phases and three levels o f the enzyme were arranged in a factorial manner to yield nine dietary treatments (Table 6.3). Control starter and grower diets were used in the control dietary treatment. A s a result, there was a total o f ten dietary treatments in the study. There were four replications o f ten birds per dietary treatment. 153 Table 6.1 Nutr ient composit ion of broi ler starter (0-3 week) diets. Diet S I D i e t S 2 Die t S3 D i e t S 4 Ingredients (%) C o n t r o l starter P K C starter P K C starter + I E 1 P K C star ter + 2 E 1 Corn 53.89 29.28 29.18 29.08 Soybean meal 36.19 34.18 34.18 34.18 Fishmeal 3.00 3.00 3.00 3.00 Palm kernel cake2 (PKC) 0.00 20.00 20.00 20.00 Palm oil 3.73 10.44 10.44 10.44 Choline chloride (60%) 0.25 0.25 0.25 0.25 Vit-min premix 3 0.10 0.10 0.10 0.10 Salt (NaCI) 0.20 0.21 0.21 0.21 Antioxidant4 0.01 0.02 0.02 0.02 PKCase 5 - - 0.10 0.20 DL-methionine 0.18 0.19 0.19 0.19 Limestone 1.30 1.18 1.18 1.18 Dicalcium phosphate 1.15 1.15 1.15 1.15 Total 100.00 100.00 100.00 100.00 Calculated composition (% air-dry unless otherwise indicated) Nutrients AME, kcal/kg 3,000 3,000 3,000 3,000 Crude protein 21.30 21.37 21.37 21.37 Arginine 1.45 1.73 1.73 1.73 Lysine 1.25 1.25 1.25 1.25 Methionine 0.50 0.50 0.50 0.50 Methionine + cystine 0.90 0.90 0.90 0.90 Tryptophan 0.32 0.32 0.32 0.32 Threonine 0.85 0.86 0.86 0.86 Crude fat 6.31 12.70 12.70 12.70 Crude fiber 3.80 5.76 5.76 5.76 Calcium 1.02 1.03 1.03 1.03 Available phosphorus 0.45 0.45 0.45 0.45 Determined composition (% air-dry basis otherwise indicated) Nutrients Dry matter 90.02 91.22 90.73 91.00 AME, kcal/kg 3,176 3,094 3,257 3,189 Crude protein 21.95 22.30 22.23 21.87 Crude fat 6.09 11.90 11.38 12.30 Crude fiber 4.01 6.11 6.06 5.94 Ash 6.08 6.13 6.55 6.37 Calcium 1.00 1.10 1.05 1.12 Total phosphorus 0.70 0.75 0.77 0.74 1 Levels of enzyme. 1E = 1 kg enzyme per tonne of feed; 2E = 2 kg enzyme per tonne of feed. 1 Solvent-extracted palm kernel cake. 3 Supplied per kg diet: Fe 100 mg; Mn 110 mg; Cu 20 mg; Zn 100 mg; I 2 mg; Se 0.2 mg; Co 0.6 mg; santoquin 0.6 mg; vitamin A 6,667 IU; vitamin D 1,000 IU; vitamin E 23 IU; vitamin K3 1.33 mg; cobalamin 0.03 mg; thiamin 0.83 mg; riboflavin 2 mg; folic acid 0.33 mg; biotin 0.03 mg; pantothenic acid 3.75 mg; niacin 23.3 mg; pyridoxine 1.33 mg. 4FRA® OX Dry, Franklin Products International B. V. 5 Enzyme mixture, Alltech Inc. USA: it contains mannanase, a-galactosidase and protease activity. 154 Table 6.2 Nutrient composition of broi ler grower (3-6 week) diets. DietGl Diet G2 Diet G3 Diet G4 Ingredients (%) Control Grower PKC Grower PKC Grower + IE 1 PKC Grower + 2E 1 Corn 60.30 35.96 35.85 35.76 Soybean meal 31.86 29.62 29.62 29.62 Fishmeal 3.00 3.00 3.00 3.00 Palm kernel cake2 (PKC) 0.00 20.00 20.00 20.00 Palm oil 2.44 9.11 9.11 9.11 Choline Chloride (60%) 0.20 0.20 0.20 0.20 Vit-min premix 3 0.10 0.10 0.10 0.10 Salt (NaCl) 0.10 0.12 0.12 0.12 Antioxidant4 0.01 0.02 0.02 0.02 PKCase 5 - - 0.10 0.20 DL-methionine 0.033 0.048 0.048 0.048 Limestone 1.30 1.18 1.18 1.18 Dicalcium phosphate 0.65 0.65 0.65 0.65 Total 100.00 100.00 100.00 100.00 Calculated composition (% air-dry unless otherwise indicated) Nutrients AME, kcal/kg 3,000 3,000 3,000 3,000 Crude protein 20.00 20.00 20.00 20.00 Arginine 1.34 1.61 1.61 1.61 Lysine 1.15 1.14 1.14 1.14 Methionine 0.38 0.38 0.38 0.38 Methionine + cystine 0.72 0.72 0.72 0.72 Tryptophan 0.29 0.29 0.29 0.29 Threonine 0.80 0.80 0.80 0.80 Crude fat 5.22 11.58 11.58 11.58 Crude fiber 3.65 5.59 5.59 5.59 Calcium 0.90 0.91 0.91 0.91 Available phosphorus 0.35 0.35 0.35 0.35 Determined composition (% air-dry unless otherwise indicated) Nutrients Dry matter 89.87 91.21 91.28 91.24 AME, kcal/kg 3,146 2,958 3,155 3,029 Crude protein 19.74 20.21 19.72 19.99 Crude fat 5.52 10.55 10.32 10.20 Crude fiber 3.14 5.02 5.13 5.09 Ash 5.39 6.15 5.89 5.71 Calcium 0.92 0.88 0.90 0.92 Total phosphorus 0.59 0.64 0.69 0.66 1 Levels of enzyme. IE = 1 kg enzyme per tonne of feed; 2E = 2 kg enzyme per tonne of feed. 1 Solvent-extracted palm kernel cake 3 Supplied per kg diet: Fe 100 mg; Mn 110 mg; Cu 20 mg; Zn 100 mg; I 2 mg; Se 0.2 mg; Co 0.6 mg; santoquin 0.6 mg; vitamin A 6,667 1U; vitamin D 1,000 IU; vitamin E 23 IU; vitamin K3 1.33 mg; cobalamin 0.03 mg; thiamin 0.83 mg; riboflavin 2 mg; folic acid 0.33 mg; biotin 0.03 mg; pantothenic acid 3.75 mg; niacin 23.3 mg; pyridoxine 1.33 mg. 'FRA® OX Dry, Franklin Products International B. V. 5 Enzyme mixture, Alltech Inc. USA: it contains mannanase, a-galactosidase and protease activity. 155 Table 6.3 Arrangement of dietary treatments for the broi ler s t u d y 1 0 kg/t enzyme 1 kg/t enzyme (IE) 2 kg/t enzyme (2E) Both 20% P K C starter (S2) 20% P K C grower (G2) Treatment 2 20% P K C starter + I E (S3) 20% P K C grower + I E (G3) Treatment 5 20% P K C starter + 2E (S4) 20% P K C grower + 2E (G4) Treatment 8 Starter 20% P K C starter (S2) Control grower (GI) Treatment 3 20% P K C starter + I E (S3) Control grower (GI) Treatment 6 20% P K C starter + 2E (S4) Control grower (GI) Treatment 9 Grower i—— Control starter (SI) 20% P K C grower (G2) Treatment 4 Control starter (SI) 20% P K C grower + I E (G3) Treatment 7 Control starter (SI) 20% P K C grower + 2E (G4) Treatment 10 1* 1 1 Treatment 1 (control group) = control starter and control grower diets Digestibi l i ty of Bro i le r Diets True dry matter digestibility ( T D M D ) , apparent metabolizable energy (AME) and nitrogen corrected true metabolizable energy (TMEn) o f all starter and grower diets were determined using Sibbald's procedures (1986) as outlined in Chapter V and six cockerels were assigned to each diet. L a y e r Product ion Study A total o f 392 Lohmann Brown laying hens (28 weeks old) was used in this 8 weeks study. A l l birds were housed in single-bird wire cages (0.3 m wide x 0.4 m length x 0.4 m height); eight layers fed from a single trough, constituted a replicate. There were seven replications for each diet. A total of seven rows in an open-sided laying house was used and the birds received 16 h o f light per day. It was considered that rows closer to the open side might experience higher temperatures. A s a result, a randomized block design was used for the study in which the seven diets were randomly assigned to each row (or block). Max imum and minimum temperatures were recorded daily in at least two locations within the building. Feed intake per replicate was determined weekly. Eggs were collected once daily (10:30 a.m.) and egg production was recorded. Eggs collected were classified as normal, cracked, deformed-shell, soft-shell or no shell. A l l eggs produced during a 2-day period (Tuesday and Wednesday) every week were individually weighed. Egg specific gravity was measured once weekly (Wednesday) on all eggs collected on a single day. The saline solutions for egg specific gravity measurements were in increments of 0.005 and ranged from 1.060 to 1.100. Before every measurement, specific 156 gravity solutions were verified with a hydrometer ( Z E A L , England), and water or salt was added as required. During the last day o f the experiment, four eggs from each replicate were broken and measured for yolk color with a Roche's Y o l k Color Fan. L a y e r Dietary Treatments A total o f seven layer diets was used in this study. Diet L l was set as control diet (without P K C and PKCase) and fed to layers in the control group. Two levels o f P K C (12.5 and 25%) and three levels o f enzyme (PKCase) were arranged in a factorial manner to yield another six diets (Table 6.4). Diets L 2 - L 7 were fed to layers in the experimental groups. The compositions o f the diets are shown in Table 6.5. Most of the nutrients recommended by the N R C (1994) for brown egg layers are derived from study with white egg layers. Therefore, the N R C (1994) recommendations for brown egg layers might not be accurate. A s a result, the nutrient levels recommended by the Lohmann Brown breeder company was followed during the formulation o f the layer diets. Table 6.4 Ar rangement of dietary treatments for the layer s t u d y 1 0 kg/t enzyme 1 kg/t enzyme ( IE) 2 kg/t enzyme (2E) 12.5% P K C 12.5% P K C Diet L 2 12.5% P K C + I E Diet L 3 12.5% P K C + 2E Diet L 4 25% P K C 25% P K C Diet L5 25% P K C + I E Diet L 6 25% P K C + 2E Diet L 7 Diet L l = control corn-soybean meal diet without palm kernel cake (PKC) or enzyme supplementation Digestibi l i ty of Laye r Diets True dry matter digestibility, A M E and TMEn o f al l layer diets were determined using Sibbald's procedures (1986) as outlined in Chapter V and six cockerels were assigned to each diet. 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C J •3 .8 I b oo C N cn m rj- m - ^ 0 d ^ O S C N - H V O 00 rf oo S S l l 1 2 o d .CS CJ •§ O S O S C N _ , V Q °°. °° © s O S 0 0 _ J 00 C N rH ^ C N © °°. © 2 V O V O 5 S «3 3 IP-a CO o „ r * O H Q p u cj 2 S u u In CJ r O i s i> S u o rCl u. CO rS a. 1 ° - a >13 CA .SP g rS "2 l r l c/> c 159 Economica l Analyses of Diets In order to determine the cost of using corn, soybean meal, P K C , P K C + lkg/t enzyme and palm o i l to supply energy and protein, the concentrations o f energy and protein per unit of price for these ingredients were calculated. Since the enzyme (PKCase) is not currently available at the commercial market, the price o f P K C + I E was set to be equal to P K C without enzyme. Parametric linear programming was used to investigate the price and the level o f inclusion at which P K C (with or without lkg/t enzyme) would be used by a computerized feed formulation program (The B r i l l Corporation, Georgia 30092, U S A ) . The current prices (Ringgit Malaysia, R M ) o f commonly used ingredients were obtained from a local feedmill in Malaysia. The price of P K C was changed from R M O to R M 7 0 0 per tonne to investigate the effect o f the price o f P K C on its inclusion rate. In order to reflect the practical situation, the maximum level o f o i l allowed in the diet was set at 5%. A s determined in the previous study (Chapter V ) , A M E values o f 1,438 kcal/kg and 1,624 kcal/kg were assigned to P K C and P K C + lkg/t enzyme, respectively. Nutrient requirements of broiler starters, broiler growers and layers were set according to the N R C (1994) recommendations. Since P K C has a low A M E content, it is also the objective o f this study to determine whether more P K C could be used in a low A M E poultry diet (containing 200 kcal/kg less than the N R C (1994) recommended A M E level) than in a high A M E poultry diet (containing the N R C (1994) recommended A M E level). Analyses of Samples Feed and excreta samples were treated and analyzed as in Chapters I V and V . Statist ical Analyses The broiler study was a factorial experiment in a completely randomized design whereas the layer study was a factorial experiment in a completely randomized block design. A l l data generated for the experimental diets were subjected to analysis o f variance ( A N O V A ) by using the General Linear Models ( G L M ) procedure o f SAS® software (SAS Institute, 1996). I f treatments were found to be significantly different, Tukey's multiple range test (Snedecor and 160 Cochran, 1980) was used to determine the statistical significance among treatment least-square means. The results obtained for birds fed the control diet were compared to those from birds fed the experimental diets using a one-way A N O V A by using the General Linear Models ( G L M ) procedure o f SAS® software (SAS Institute, 1996). I f treatments were found to be significantly different, the Bonferroni (Dunn) T test (SAS Institute, 1996) was used to determine the statistical significance between treatment least-square means. The Bonferroni (Dunn) T test was used because it w i l l avoid detecting random differences when comparing a large group o f treatments (ten dietary treatments in the broiler study and seven dietary treatments in the layer study). Mortality was subjected to Chi-Square analysis. Results Broiler Study During the 0-3 week of the broiler study, inclusion o f 20% P K C in the starter diet significantly reduced (P < 0.05) the body weights of the 3 week old broiler chicks (Table 6.6). Enzyme supplementation of the PKC-based starter diets improved body weighty and it was significant (P < 0.05) when the enzyme level was 2kg/t. There were no significant differences (P > 0.05) in 3 week body weight between birds fed control starter diets and PKC-based diets containing 2kg/t o f enzyme. Body weight gain (0-3 weeks) was significantly lower (P < 0.05) in birds fed PKC-based diets without enzyme supplementation than the other diets. Birds fed control starter diets and PKC-based starter diets containing 2kg/t o f enzyme gained significantly more (P < 0.05) weight than birds fed PKC-based starter diets containing 0 or lkg/t o f enzyme. Enzyme supplementation of PKC-based diets significantly improved (P < 0.05) body weight gain at 3 weeks o f age (Table 6.6). Feed intake and feed conversion ratio (FCR) were significantly lower (P < 0.05) in birds fed the control diet (SI). Enzyme supplementation o f the PKC-based diets (S3 and S4) did not reduce feed consumption but significantly reduced (P < 0.05) the F C R of birds fed diet S4 (Table 6.6) During the 3-6 week o f the broiler study, body weight gain was significantly lower (P < 0.05) for birds fed PKC-based grower diets without enzyme supplementation than for birds fed the control grower diet (GI) (Table 6.7). Again, enzyme supplementation alleviated the poorer 161 performance o f PKC- fed birds. There were no significant differences in body weight gain (3-6 weeks) between birds fed the control grower diets and enzyme supplemented PKC-based grower diets (Table 6.7). Feed intake was significantly higher (P < 0.05) for birds fed enzyme supplemented PKC-based grower diets than for those fed the control grower diets. Feed conversion ratio was significantly lower (P < 0.05) for birds fed the control grower diets than for those fed the other grower diets. Table 6.6 The effects of dietary inclusion of solvent-extracted pa lm kernel cake ( P K C ) and enzyme supplementation on live body weight (g), weight gain (g), feed intake (g) and feed conversion ratio ( F C R ) of female broi lers at 3 weeks of age. Starter diet Body wt. Body wt. Weight gain Feed intake (g) F C R WeekO Week 3 Week 0-3 Week 0-3 Week 0-3 Control (SI) 44 714" 6 7 0 8 1090 b 1.62 c 20% P K C (S2) 44 677 b 633 c 1143" 1.81 a 20% P K C + I E 1 (S3) 43 691 b 648 b 1145" 1.77 a b 20% P K C + 2E (S4) 43 710" 666" 1156" 1.74" Overall mean 43 698 654 1134 1.74 Pooled S E M 2 0.2 2.7 2.7 8.2 0.02 An enzyme mixture from Alltech Inc., Kentucky, USA. It contains mannanase, a-galactosidase and protease activity; IE = lkg/t and 2E = 2kg/t 2 Pooled standard error of the mean; data represent mean of four replications of 10 birds. abcTreatment means with different superscripts within a column are significantly different at P < 0.05. Table 6.7 The effects of dietary inclusion of solvent-extracted pa lm kernel cake ( P K C ) and enzyme supplementation on live body weight (g), weight gain (g), feed intake (g) and feed conversion ratio ( F C R ) of female broi lers at 6 weeks of age. Grower diet Body wt. Weight gain Feed intake (g) F C R Week 6 Week 3-6 Week 3-6 Week 3-6 Control (GI) 1 8 3 1 a 1132 8 2511 b 2.22 b 20% P K C (G2) 1771 b 1084 b 2591 8 b 2.41 8 20% P K C + I E 1 (G3) 1822 8 1117 a b 2627 8 2.35" 20% P K C + 2E (G4) 1831 8 1 1 1 6 a b 2629 8 2.37 8 Overall mean 1814 1112 2590 2.34 Pooled S E M 2 6.9 5.9 18.1 0.02 An enzyme mixture from Alltech Inc., Kentucky, USA. It contains mannanase, a-galactosidase and protease activity; IE = lkg/t and 2E = 2kg/t 2 Pooled standard error of the mean; data represent mean of four replications of 10 birds. abcTreatment means with different superscripts within a column are significantly different at P < 0.05. 162 When the whole experimental period was considered (0-6 weeks), no significant differences in 0-6 week weight gain and 0-6 week feed intake were found among birds in dietary treatments 1-10 (Table 6.8). However, birds in dietary treatments 2 and 5 (fed 20% P K C diets throughout with no or 1 kg enzyme per tonne) had significantly poorer (P < 0.05) F C R than birds in dietary treatment 1 (control). N o significant differences (P > 0.05) in F C R were found among birds in dietary treatments 2-10 (Table 6.8). When the overall data (dietary treatments 2-10) were analyzed as a factorial experiment, overall weight gain (0-6 week) o f the birds was not influenced by dietary inclusion o f P K C at different phases (both, starter, grower) (Table 6.9). However, enzyme supplementation (1 or 2 kg/t) significantly increased (P < 0.05) weight gain o f birds fed PKC-based diets. There was no significant effect o f the inclusion o f P K C during different phases and enzyme supplementation on feed intake. However, the trend with enzyme supplementation was for increased feed intake and decreased F C R with increased amount o f enzymes (Table 6.9). Using PKC-based diets during the starter or grower phase significantly reduced (P < 0.05) F C R than when PKC-based diets were used i n both phases. For the broiler starter diets, true dry matter retention was significantly higher (P < 0.05) with the control starter diet (Table 6.10) than with the other starter diets. When compared to birds fed the PKC-based diet without enzyme supplementation, adding enzyme at 1 or 2 kg/t to PKC-based diets significantly improved (P < 0.05) the true dry matter retention o f birds. Both A M E and TMEn o f PKC-based diets were significantly higher (P < 0.05) with lkg/t enzyme supplementation (Table 6.10) than without enzyme supplementation. Increasing the levels o f enzyme in the PKC-based starter diets from 1 kg/t to 2 kg/t did not further increase its A M E and TME„ values. For the broiler grower diets, true dry matter retention was significantly higher (P < 0.05) with the control grower diet (Table 6.11) than with the other PKC-based diets. Even though the A M E and TME„ of the control grower diets (GI) , and enzyme supplemented PKC-based grower diets (G3 and G4) were not significantly different, the A M E and TME„ o f the control grower diet and enzyme supplemented (lkg/t) PKC-based diet were significantly higher (P < 0.05) than PKC-based grower diet without enzyme supplementation (Table 6.11). 163 Table 6.8 Effect of dietary inclusion of solvent-extracted pa lm kernel cake ( P K C ) and enzyme supplementation on female broi lers overal l performance (weight gain, feed intake and feed conversion ratio (FCR) ) dur ing the 6 week experimental per iod. Dietary Treatment Arrangement of diets Weight gain (g) Feed intake (g) F C R Control 1820 3609 1.97 P K C Starter P K C Grower 1727 3771 2.19 P K C Starter Control Grower 1759 3608 2.05 ab Control Starter P K C Grower 1728 3622 2.10 ab P K C Starter + lkg/t PKCase 1 P K C Grower + lkg/t PKCase 1766 3739 2.12 P K C Starter + lkg/t PKCase Control Grower 1765 3614 2.05 ab Control Starter P K C Grower + lkg/t PKCase 1792 3742 2.09 ab P K C Starter + 2kg/t PKCase P K C Grower + 2kg/t PKCase 1819 3787 2.10 ab P K C Starter + 2kg/t PKCase Control Grower 1810 3707 2.05 ab 10 Control Starter P K C Grower + 2kg/t PKCase 1754 3662 2.09 ab Overall mean Pooled S E M 2 1774 6.9 3688 21.3 2.08 0.01 An enzyme mixture from Alltech Inc., Kentucky, USA. It contains mannanase, a-galactosidase and protease activity; 1E = lkg/t and 2E = 2kg/t Pooled standard error of the mean; data represent mean of four replications of 10 birds. ab, Treatment means with different superscripts within a column are significantly different at P < 0.05. 164 Table 6.9 Factor ia l compar ison: Effect of dietary inclusion of solvent-extracted pa lm kernel cake ( P K C ) at different phases and enzyme supplementation on female broi lers overal l performance (weight gain, feed intake and feed conversion ratio ( F C R ) ) dur ing the 6 week experimental per iod. Treatment Weight gain (g) Feed intake (g) F C R P h a s e 7 Both 1770 3766 2 .14 a Starter 1778 3643 2 . 0 5 b Grower 1758 3675 2 . 0 9 b E n z y m e 2 Okg/t 1739 b 3667 2.11 l k g / t 1774" 3698 2.09 2 kg/t 1796 8 3719 2.08 Overall mean 1769 3695 2.09 Pooled S E M 3 7.3 22.8 0.01 Factorial effects 4 Phase N S N S *** Enzyme *** N S N S Phase x Enzyme N S N S N S 1 Both = PKC was included in both the starter and grower diets; Starter = PKC was included only in the starter diets; Grower = PKC was included only in the grower diets. 2 An enzyme mixture from Alltech Inc., Kentucky, USA. It contains mannanase, a-galactosidase and protease activity. 3 Pooled standard error of the mean; data represent mean of four replications of 10 birds. 4 NS = not significant (P > 0.05) or * * * = significant at P < 0.05 a b Treatment means with different superscripts within a column for phase or enzyme are significantly different at P <0.05. 165 Table 6.10 The effect of dietary enzyme supplementation on true D M retention and apparent metabolizable energy ( A M E ) and nitrogen corrected true metabolizable energy ( T M E „ ) of bro i ler starter diets \ Starter diets True D M retention, % A M E , kcal/kg TMEn, kcal/kg Control (SI) 74.2 8 3 ,176 8 b 3,669 a b 20% P K C (S2) 6 2 . 6 c 3,094 b 3,580 b 20% P K C + I E 2 (S3) 67 .3" 3,257 8 3,745 8 20% P K C + 2E (S4) 66.4 b 3,189 a b 3,676 a b Overall mean 67.6 3,179 3,667 Pooled SEM 3 1.1 20.2 20.3 DM basis 2 An enzyme mixture from Alltech Inc., Kentucky, USA. It contains mannanase, a-galactosidase and protease activity; 1E = 1 kg/t and 2E = 2kg/t 3 Pooled standard error of the mean; data represent mean of six cockerels. Treatment means with different superscripts within a column are significantly different at P < 0.05. Table 6.11 The effect of dietary enzyme supplementation on true D M retention and apparent metabolizable energy ( A M E ) and nitrogen corrected true metabolizable energy ( T M E „ ) of bro i ler grower diets *. Grower diets True D M retention, % A M E , kcal/kg TMEn, kcal/kg Control (GI) 73.2 8 3,1468 3,639 a 20% P K C (G2) 62.2 c 2,958 b 3,444 b 20% P K C + I E 2 (G3) 66.8 b 3,155" 3,641 8 20% P K C + 2E (G4) 62.6 b c 3,029 a b 3,515 8 b Overall mean 66.1 3,070 3,558 Pooled S E M 3 1.1 29.9 30.1 DM basis 2 An enzyme mixture from Alltech Inc., Kentucky, USA. It contains mannanase, a-galactosidase and protease activity; IE = lkg/t and 2E = 2kg/t 3 Pooled standard error of the mean; data represent mean of six cockerels. Treatment means with different superscripts within a column are significantly different at P < 0.05. 166 Broilers in dietary treatment 1 deposit significantly less (P < 0.05) abdominal fat than broilers in dietary treatments 2-10 (Table 6.12). Factorial analysis revealed that neither inclusion of P K C during different phases nor enzyme supplementation significantly (P > 0.05) affected abdominal fat deposition (Table 6.13). Rectal temperatures of the 6-week-old broilers fed different grower diets were not significantly different (P > 0.05) (Table 6.14). Even though not significantly different, rectal temperature o f birds fed enzyme supplementation P K C diets was numerically lower when compared to the birds fed P K C diet without enzyme supplementation. A total o f 22 broilers was found dead when the temperature inside the building reached 36°C during days 32, 37 and 40 of the experiment. Veterinarians from the post-mortem laboratory concluded that all o f the birds died of heat stress. The data for mortality due to heat stress were subjected to Chi-square analysis. The result of the analysis showed that the birds fed 20% P K C grower diets without enzyme supplementation had significantly higher (P < 0.05) mortality due to heat stress than those fed the control diet (Table 6.15). When birds fed the enzyme supplemented P K C -based grower diets were compared with those fed the control diets, no significant differences (P > 0.05) in mortality due to heat stress were found. Layer Study There were no significant interactions between blocks and diets. Therefore, the data were analyzed as a completely randomized design experiment. When comparing data from layers fed layer diets L 1 - L 7 , no significant (P > 0.05) dietary effects were observed on mean egg production, hen-day egg production or daily egg mass (Table 6.16). Birds fed enzyme supplemented (2kg/t) 25% PKC-based layer diets (L7) produced significantly heavier (P < 0.05) eggs than birds fed diets L l , L 3 , L 4 , L5 and L 6 . On the other hand, birds fed a 25% PKC-based diet (L5) produced significantly smaller (P < 0.05) eggs than the rest o f the birds, except those fed an enzyme supplemented (2kg/t) 12.5% PKC-based diet (L4). Birds fed the control ( L l ) and enzyme-supplemented (lkg/t) 12.5% PKC-based (L3) diets consumed significantly less (P < 0.05) feed than other birds (Table 6.16). Birds fed the 12.5% P K C diets (L2, L 3 and L4) also consumed significantly less (P < 0.05) feed than those fed the 25% P K C diets (L5, L 6 and L7) . The best F C R (feed intake/egg mass) was obtained from birds fed diets L l and L 3 , whereas significantly poorer (P < 0.05) F C R were found in birds fed diets L 2 , L 4 , L 5 , L 6 and L 7 (Table 6.16). 167 Table 6.12 Effect of dietary inclusion of solvent-extracted pa lm kernel cake ( P K C ) and enzyme supplementation on broi ler abdominal fat deposition at week 6. Dietary treatment Arrangement of diets Abdomina l fat (% body weight) I Control 1.02 b 1.60 a P K C Starter P K C Grower P K C Starter Control Grower Control Starter P K C Grower P K C Starter + lkg/t PKCase 1 P K C Grower + lkg/t PKCase P K C Starter + lkg/t PKCase Control Grower Control Starter P K C Grower + lkg/t PKCase P K C Starter + 2kg/t PKCase P K C Grower + 2kg/t PKCase P K C Starter + 2kg/t PKCase Control Grower 1.52' 1.73 1.48 1.41 1.57 1.78 1.62 t n Control Starter „ P K C Grower+ 2kg/t PKCase L 5 y Overall mean 1.51 Pooled S E M 2 0.05 An enzyme mixture from Alltech Inc., Kentucky, USA. It contains mannanase, a-galactosidase and protease activity. 2 Pooled standard error of the mean; data represent mean of five broilers. abTreatment means with different superscripts within a column are significantly different at P < 0.05. 168 Table 6.13 Factor ia l compar ison: Effect of dietary inclusion of solvent-extracted pa lm kernel cake ( P K C ) at different phases and enzyme supplementation on bro i ler abdomina l fat deposition at week 6. Treatment Abdomina l fat (% body weight) P h a s e 1 Both 1.60 Starter 1.49 Grower 1.65 Enzyme 2 Okg/t 1.60 1 kg/t 1.52 2 kg/t 1.62 Overa l l mean 1.58 Pooled S E M 3 0.05 Factorial effects 4 Phase N S Enzyme N S Phase x Enzyme N S Both = PKC was included in both the starter and grower diets; Starter = PKC was included only in the starter diets; Grower = PKC was included only in the grower diets. 2 An enzyme mixture from Alltech Inc., Kentucky, USA. It contains mannanase, a-galactosidase and protease activity. 3 Pooled standard error of the mean; data represent mean of five broilers. 4 NS = not significant (P > 0.05) or *** = significant at P < 0.05 Table 6.14. The effect of broi ler grower diets on six-week-old bro i lers ' rectal temperature. Grower diets Body temperature °C (± S .D . ) 1 Control (GI) 44.22 ± 0.53 20% P K C (G2) 44.49 ± 0.45 20% P K C + I E 2 (G3) 44.41 ± 0.48 20% P K C + 2E (G4) 44.21 ± 0.59 Overall mean 44.31 ± 0 . 5 2 Data represent mean of eight broilers; ± standard deviation. PKC = palm kernel cake. 2 An enzyme mixture from Alltech Inc., Kentucky, USA. It contains mannanase, a-galactosidase and protease activity; IE = lkg/t and 2E = 2kg/t 169 Tab le 6.15 Chi -square analysis: Compar ing the effect of different bro i ler grower diets on broi ler mortal i ty due to heat stress dur ing 3-6 weeks of age l . Diet comparison Signif icant level 2 G I vs G2 *** G I vs G3 N S G I vs G4 N S G I vs G3 + G4 N S G I vs G2 + G3 + G4 N S G2 vs G3 N S G2 vs G4 N S G2 vs G3 + G4 N S G3 vs G4 N S 1 GI = grower diet without palm kernel cake (PKC); G2 = grower diet with 20% PKC; G3 = grower diet with 20% PKC + lkg/t Alltech PKCase; G4 = grower diet with 20% PKC + 2kg/t Alltech PKCase. PKCase is an enzyme mixture from Alltech Inc., Kentucky, USA. It contains mannanase, a-galactosidase and protease activity. 2 *** = significant at P < 0.05; NS = not significant or P > 0.05. 170 CU fl o fl o fl cu E n , n . s cn CU & & fl <u •a fl es cu w "33 fl s_ -S4 CS a TJ cu cu cs u X CU z fl cu O cn c/j ^ CM CU O CU fl £ .2 oo S cu fl ° ••• co b s CS -fl CU on • ~ fl •a tt-l © cs «2 t t - i u © £ cu CU CU X! H cu OH fl _ « H S cs a .o VO 3 u PH •o VO ON CD O • a t r r H H <D r P £ 3 60^ cu —^^  >. CO •a S 5 S .fl 00 '55 , si ^ 00 00 W § 2 6 » fl a 8 OH J3 co w in a © a vq a in in 00 ON in o T - H o fl o l - H T - H T - H 00 o 00 o T - H m o fl o in ON o 00 vq © m T - H m ©' in O <n © in © m T - H m o m u JB ca u •a w •a u •D a T - H T - H vq ON o CO T - H in m UO m in fl m fl m fl m in m in m tH u o. <U (D due icat 8 a u >H CO 00 00 W cu ) H in ON u •o w u a a u -O T - H VO CN fl O o o O CN T - H »—i CN CN CN CN CN CN i> q i - H - - < co vq CN CO CN CN CN ON ON ON ON ON Ov o T - H fl i - H CN c ' co co *n l - H i - H i - H i - H i - H i - H fl fl fl fl fl fl 1 o U co k-H d + PH PH \=> NO in in CN CN W CN ^ + u e> ^ u PH ^ CN m * ^ CN w W T-H CN + + u u PH PH in in CN CN 00 i - H o o CN O ea H o HJ <! p-1H M n 171 Factorial analysis (diets L2-L7) revealed that the levels o f P K C or enzymes have no significant effect on mean egg production, hen-day egg production, egg weight or egg mass (Table 6.17). Significantly more (P < 0.05) feed was consumed by birds fed the 25% P K C diet than with the 12.5% P K C diet. When compared with layers fed P K C diets without supplemental enzyme, enzyme supplementation at lkg/t significantly reduced (P < 0.05) feed intake o f birds fed P K C diets. However a significant difference in feed intake was not observed in layers fed P K C diets supplemented with either lkg/t or 2kg/t of enzyme (Table 6.17). A significant interaction was found between P K C level and enzyme level for egg weight and F C R , the interactions being shown in Figures 6.1 and 6.2 for egg weight and in Figures 6.3 and 6.4 for F C R . Inconsistent results were found for egg weight (Figures 6.1 and 6.2). Egg weight was significantly reduced with enzyme supplementation when the 12.5% P K C diet was fed. However, the reverse was true for the 25% P K C diet (Figure 6.1). When no enzyme was used, birds fed the 12.5% P K C diet produced significantly heavier (P < 0.05) eggs than those fed the 25% P K C diet (Figure 6.2). N o significant effect in egg weight was found at the lkg/ t enzyme level but birds fed the 25% P K C diet produced significantly heavier (P < 0.05) eggs than those fed 12.5% P K C diets with the 2kg/t enzyme level (Figure 6.2). For F C R (Figures 6.3 and 6.4), no effect o f the enzyme was found at 12.5% P K C , although there was a trend for better F C R with enzyme supplementation (Figure 6.3). Enzyme supplementation, on the other hand, significantly improved (P < 0.05) the F C R o f birds fed 25% P K C diets (Figure 6.3). Birds fed 25% P K C diets had significantly poorer (P < 0.05) F C R at 0 and 1 kg/t enzyme levels. However, the differences were alleviated at 2kg/t enzyme level (Figure 6.4). When comparing birds fed diets L 1 - L 7 , no significant difference was found in eggshell quality among diets (Table 6.18). Egg yolk color was significantly paler (P < 0.05) for eggs produced by birds fed 25% PKC-based diets (L5, L 6 and L7). Factorial analysis (comparing diets L 2 - L 7 ) revealed that the levels of P K C and enzyme supplementation had no significant effect on eggshell quality (Table 6.19). However, egg yolk color was significantly paler (P < 0.05) for birds fed 25% P K C diets. 172 S & a CU TJ a u ON cu cs cu "cu a cu a eu TJ cu -w cu « s-•*•> X eu a *> - * . j - CA O ^ t/3 CU CU © It I t «] cu 3 > "3 ° C c« •H a .2 M cu fi ° Z v> O cu cu ,CU U := 5 CU 03 2 S H «2 c « o O" •S3 fl CM CU s S 5 eu S 5 « a o a w .— .2 NO S CU ,S CO [H CB U •a t •4-» *0 1) r P 60 ^ 60 60 cu —^' JZ? co 3 a 6 0 ^ w .5? o i f r-l CU DH T> CU <L> «rJ* o CTj CU O "EH rH OH 60 60 W 8 JS a O rH CN CN 2 PH 00 0 0 o © IO IT) H © in in in in C N ro C N C N OS OS r—< r-H . u u in O CN m r-H CN a ja ja •ct h CO H © q CN CN CN JS a ja J3 R 00 ro r H © SO ro OS 00 in NO © o o O o r—I r H r-H r H r H m so C N O O -H' m m m os q C N u-i IO m m m OS T-H 00 H CN CN OS ON ON CN ro so "d-2 4^—' -4-J S so ~5b 6o •» M M M 2 © rH (N O rH H O CN O NO NO ^ o o o <n m in CN o CN © CO CN ON ro es 5 ^ a | H cu > O m 0 0 -o r - H *o -O PH * * * * * GO oo 0 0 oo KJ .53 PH 0 0 0 0 0 0 0 0 0 0 T T . 4 U <4H § s s 60 o fl o in m m in in m v d ^ i n ^ ^ ^ c S 1 0 in in in in ( § ) ; q § p v\ § 8 g ( § ) ; q § i 3 A \ § § a 175 n i o M i O ' - ' i n c N i o O N i o CN <~S CN ^ CN <=> ^ ^ °9 CN CN CN 1—{ i—1 0 I 1 B J U O I S J 9 A U O 0 p 9 9 j [ fl u fl <u •> fl o ON fl mo fl A o (X) fl u S-5 p f l H WD r V PH CN I PH IT) CN r - H I • V Ch a CUD a o CA •c « a. E o •o a a v 2 61) >> cu s E? fl r~ C O CN in CN CN CN CN "I CN CN i n o CN CN in os i n oo O i ; B J U0ISJ9AU03 p 9 9 J 177 Table 6.18 The effects of dietary inclusion of solvent-extracted pa lm kernel cake ( P K C ) and enzyme supplementation on the mean egg quali ty over 8 weeks f rom 28 weeks of age. Layer diets Egg shell specific gravity Egg yolk co lor 1 Control ( L l ) 1.091 3 .9 a 12.5% P K C (L2) 1.091 3.9 a 12.5% P K C + I E 2 (L3) 1.092 3.7 8 12.5% P K C + 2 E ( L 4 ) 1.091 3.8" 25% P K C (L5) 1.091 2.3 b 25% P K C + I E (L6) 1.092 2.4 b 25% P K C + 2E (L7) 1.092 2.4 b Overall mean 1.091 3.2 Pooled S E M 3 i . . . . . . . i 0.0001 0.1 ' Measured with Roche yolk color fan;1 An enzyme mixture from Alltech Inc., Kentucky, USA. It contains mannanase, a-galactosidase and protease activity; IE = lkg/t and 2E = 2kg/t 3 Pooled standard error of the mean; data represent mean of seven replications of eight layers 'Treatment means with different superscripts within a column are significantly different at P < 0.05. Table 6.19 Factor ia l Compar ison : The effects of dietary inclusion of solvent-extracted pa lm kernel cake ( P K C ) and enzyme supplementation on the mean egg qual i ty over 8 weeks f rom 28 weeks of age. Factor Egg shell specific gravity Egg yolk color P K C 12.5% 1.092 3 .8 a 25.0% 1.091 2.3 b E n z y m e 2 Okg/t 1.091 3.1 l k g / t 1.092 3.0 2 kg/t 1.091 3.1 Overall mean 1.091 3.1 Pooled S E M 3 0.0001 0.1 Factorial effects 4 P K C N S *** Enzyme N S N S P K C X Enzyme N S N S ' Measured with Roche yolk color fan;1 An enzyme mixture from Alltech Inc., Kentucky, USA. It contains mannanase, a-galactosidase and protease activity. 3 Pooled standard error of the mean; data represent mean of seven replications of eight layers 4 NS = not significant (P > 0.05) or *** = significant at P < 0.05; abTreatment means with different superscripts within a column for PKC or enzyme are significantly different at P < 0.05. 178 There were no significant differences in true dry matter retention between birds fed the control layer diet ( L l ) and enzyme-supplemented P K C diets (L3, L 4 , L 6 or L7) (Table 6.20). Birds fed a 25% PKC-based layer diet (L5) had a significantly lower (P < 0.05) true dry matter retention than those fed the control layer diet ( L l ) or enzyme-supplemented P K C diets (L3, L 4 , L 6 or L7) . The control diet ( L l ) , 12.5% P K C diet (L2) and 25% P K C diet (L5) had lower A M E and TMEn values when compared to 25% P K C diet supplemented with 2 kg/t enzyme. Enzyme-supplemented P K C diets (L3, L 4 , L 6 and L7) had significantly higher A M E and TME„ values than P K C diets with no enzyme supplementation (L2 and L5) (Table 6.20). Factorial analysis (diets L2-L7) found that birds fed diets containing 25% P K C retained significantly less (P < 0.05) dry matter than those fed the 12.5% P K C diets (Table 6.21). Enzyme supplementation significantly increased (P < 0.05) true dry matter retention in birds fed P K C diets. The levels of P K C did not significantly influence A M E and TME„ of the diets, but adding the enzyme significantly increased (P < 0.05) the A M E and TME„ values of P K C diets. Table 6.20 Apparen t and nitrogen corrected true metabolizable energy 1 ( A M E and T M E n ) and true dry matter retention of layer diets. Layer diet True dry matter retention (%) A M E kcal/kg TMEn kcal/kg Control ( L l ) 71.4 8 2,889 b c 3,382 b c 12.5% P K C (L2) 62.9 b c 2,742 £ 3,226 c 12.5% P K C + IE 2 (L3) 69.6" 3,020 a b 3,509 a b 12.5% P K C + 2E (L4) 68.1 8 b 2,983 a b 3,469 8 b 25% P K C (L5) 56.7c 2,756 c 3,240 c 25% P K C + IE (L6) 65.2 8 b 2,973 a b 3,458 a b 25% P K C + 2E (L7) 66.2 8 b 3,101 8 3,586 a Overall mean 65.7 2,923 3,410 Pooled S E M 3 0.9 24 24 ' Dry matter basis 2 An enzyme mixture from Alltech Inc., Kentucky, USA. It contains mannanase, a-galactosidase and protease activity; IE = lkg/t and 2E = 2kg/t 3 Pooled standard error of the mean; data represent mean of six cockerels. abcTreatment means with different superscripts within a column are significantly different at P < 0.05. 179 Table 6.21 Factorial comparison: Apparent and nitrogen corrected true metabolizable energy1 (AME and TME„) and true dry matter retention of layer diets. True dry matter AME TMEn retention (%) kcal/kg kcal/kg Palm kernel cake 12.5% 66.9 8 2,915 3,401 25.0% 62.7 b 2,943 3,428 Enzyme Okg/t 59.8 b 2,749 b 3,233 b lkg/t 67.4" 2,996" 3,483" 2 kg/t 67.2" 3,042" 3,527 8 Overall mean 64.8 2,929 3,415 Pooled SEM 3 0.8 26 26 Factorial effects 4 Palm kernel cake (PKC) *** NS NS Enzyme *** *** *** PKC X Enzyme NS NS NS Dry matter basis 2 An enzyme mixture from Alltech Inc., Kentucky, USA. It contains mannanase, a-galactosidase and protease activity. 3 Pooled standard error of the mean; data represent mean of six cockerels. 4 NS = not significant (P > 0.05) or *** = significant at P < 0.05 ""Treatment means with different superscripts within a column for palm kernel cake or enzyme are significantly different at P < 0.05. Economical Analyses of Diets The relative costs of energy and protein for corn, soybean meal, PKC, PKC + lkg/t enzyme and palm oil are presented in Table 6.22. Based on the prevailing prices in Malaysia, corn supplies the cheapest source of energy, while soybean meals supply the cheapest source of protein. Even though PKC contains lower AME than soybean meal, it is much cheaper than soybean meal. As a result, PKC provides a cheaper source of energy than soybean meal. On the other hand, PKC contains a moderate amount of protein and costs less per tonne than corn, making it a cheaper protein source than corn. Parametric linear programming revealed that the computerized feed formulation program was not willing to use any level of PKC or PKC + lkg/t enzyme in the high AME (3,200 kcal/kg) broiler starter and high AME (3,200 kcal/kg) broiler grower diets even when the prices of both PKC and PKC + lkg/t enzyme were set at zero (Table 180 6.23). For low A M E (3,000 kcal/kg) diets the feed formulation program still rejected P K C in the starter diet. However, the computerized feed formulation program was wi l l ing to include 5.0 to 5.7% o f P K C + lkg/t enzyme into the low A M E starter diet when the price was less than RM31/tonne. The feed formulation program included 6% P K C into the low A M E grower diet when the price of P K C was less than RM700/tonne. On the other hand, a higher level (6.4% -12.4%) o f P K C + lkg/t enzyme was used in the low A M E grower diet (Table 6.23). For layer diets, the computerized feed formulation program used 21% and 8% P K C in the low (2,700 kcal/kg) and high (2,900 kcal/kg) A M E layer diets respectively, when the price of P K C was less than RM700/tonne (Table 6.23). The level of inclusion o f P K C + lkg/t enzyme in the low A M E layer diet ranged from 23% to 29%. Lower levels (10% to 16%) o f P K C + lkg/t enzyme were used in the high A M E layer diet (Table 6.23). Table 6.22 The relative costs of apparent metabolizable energy ( A M E ) and protein (CP) in corn , soybean meal ( S B M ) , solvent-extracted pa lm P K C a s e ( P K C + I E ) and pa lm oi l u . kernel cake ( P K C ) , P K C + l kg / t Ingredients Cost per tonne ( R M ) A M E J (kcal/kg) C P J (%) Cost per Mea l ( R M ) Cost per kg C P ( R M ) Corn 786 3,350 8.5 0.23 9.25 S B M 1,071 2,220 44 0.48 2.43 P K C 520 1,452 15 0.36 3.47 P K C + I E 520 1,624 15 0.32 3.47 Palm o i l 2500 8,430 0 0.30 -RM = Ringgit Malaysia 2 PKCase is an enzyme mixture obtained from Alltech Inc., Kentucky, USA. It contains mannanase, a-galactosidase and protease activity. This enzyme is not currently available at the commercial market, therefore the cost of PKC + IE was assigned to be equal to PKC. The above analysis shows that the enzyme would have to be priced at no more than RM61.6/kg for it to be economic for commercial use. 3 The AME and CP values for corn and SBM were obtained from the NRC (1994) for poultry, whereas the values for PKC and PKC + IE were determined in the previous study (Chapter V). The AME value for palm oil was obtained from Yeong and Mukherjee (1983). 181 Table 6.23 Evaluat ion of the effect of the price of pa lm kernel cake ( P K C ) and P K C + lkg / t P K C a s e ( P K C + I E ) on its inclusion rate in low and high apparent metabolizable energy ( A M E ) broi ler (starter and grower) and layer diets by parametr ic l inear p r o g r a m m i n g 1 , 2 . Starter (23% CP) A M E (kcal/kg) 3,000 3,000 3,000 3,200 Price of P K C P K C (RM/tonne) % inclusion Price o f P K C + I E (RM/tonne) 0 0 0 - 2 2 2 3 - 3 1 >31 0 P K C + I E % inclusion 5.7 5.0 0 0 Grower (20% CP) A M E (kcal/kg) 3,000 3,000 3,000 3,200 0 - 7 0 0 0 - 2 2 2 3 - 3 1 32 - 700 0 12.4 11.7 6.4 0 Layer (15% CP) A M E (kcal/kg) 2,700 2,700 2,700 2,900 2,900 2,900 0 - 7 0 0 0 - 7 0 0 21 0 - 2 4 2 5 - 3 1 32 - 700 0 - 6 6 6 7 - 7 4 75 - 700 29 28 23 16 15 10 RM = Ringgit Malaysia. For broiler diets, low AME = 3,000 kcal/kg and high AME = 3,200 kcal/kg. For layer diets, low AME = 2,700 kcal/kg and high AME = 2,800 kcal/kg. Solvent-extracted PKC was used.2 An enzyme mixture from Alltech Inc., Kentucky, USA. It contains mannanase, a-galactosidase and protease activity. Discussion U p to now, no published study has been conducted to investigate the effects o f using P K C during different phases of the broiler production cycle or on the effects o f enzyme supplementation o f PKC-based diets on poultry performance. The broiler study clearly showed that weight gains for 0-3 weeks and 3-6 weeks were significantly reduced when 20% P K C was incorporated into the starter and grower diets, respectively, even though the diets were 182 formulated to contain similar levels of nutrients. The birds fed 20% P K C starter or grower diets also consumed more feed and had a poorer F C R than those fed the control diet. The reasons for the poorer growth performance included a reduction in dry matter retention and in the metabolizable energy content o f P K C based diets. True dry matter retention by birds fed the starter and grower diets containing 20% P K C was 15% lower than in birds fed the control diet. Lower P K C amino acid digestibility as indicated in Chapter V could also have retarded growth. The results of the present studies also demonstrate that adding 20% P K C into both broiler starter and broiler grower diets (0-6 week) results in a poorer F C R when compared to birds fed control corn-soybean meal diets (0-6 week). The lower digestibility o f P K C amino acid and energy and the possible entrapment of nutrients within the cells by indigestible cell walls (Chapter V ) could have led to the poorer F C R of birds fed the 20% PKC-based diet from 0-6 weeks of age. However, when 20% P K C was included in the broiler diet during the starter phase, and a corn-soybean meal diet was fed during the grower phase (Case I), or in the broiler diet during the grower phase while a corn-soybean meal diet was fed during the starter phase (Case II), the performance o f the birds at 6 weeks was not jeopardized. Even though broilers gain less weight during 0-3 week of age when P K C based diet was fed, they were able to compensate the lower weight gain by growing faster when corn-soybean meal diet was fed during 3-6 weeks (Case I, compensatory growth). On the other hand (Case II), birds were better able to utilize P K C during the grower phase because o f their better developed digestive tract. This indicates that 20% P K C can be used during the starter or grower phase but not in both the starter and grower phases. This is in agreement with the findings o f several studies that inclusion o f high level o f P K C in broiler diets for 5 weeks or longer led to poorer bird performance (Ahmad, 1982; Garcia and Gernat, 1998 and Onifade and Babatunde, 1998). Ahmad (1982) found that 10% P K C could be included in a broiler diet from 15 to 56 days of age without reducing growth performance, but as P K C level reached 20%, daily weight gain was significantly reduced and feed/gain ratio was increased. This agrees with the finding in the present study that feed/gain ratio was significantly increased when broilers were fed a 20% PKC-based diet instead o f a corn-soybean meal diet from 0-6 week o f age. Ahmad (1982) also stated that the high fiber content o f P K C reduced its nutrient digestibility, especially for protein, and concluded that there was no economic advantage in using P K C over corn and soybean meal. In another study, Garcia and Gernat (1998) found that birds fed corn-soybean meal or 10% P K C diets from 0 to 6 weeks of age had significantly higher 183 body weights, higher carcass weights and better F C R than birds fed diets containing 20% or 30% P K C . Other authors (Onifade and Babatunde, 1998) concluded that adding 10%, 15% or 20% P K C (Nigeria, screw-pressed) into broiler diets from 0 to 35 days o f age reduced weight gain when compared to broilers fed the corn-soybean meal control diet. However, the authors failed to adjust the metabolizable energy o f the diets to that o f the control diet and this might have reduced the birds' performance. These results are contradictory to the findings of Yeong (1980), Hutagalung (1980), Yeong and Mukherjee (1983) and Ngoupayou (1984) stating that broiler growth performance was not jeopardized when the diet contained 20% P K C . Yeong and Mukherjee (1983) found that by adding 9% palm oi l to a 20% P K C diet (which brought the metabolizable energy level to that o f the control diet), growth performance and feed efficiency of the birds fed P K C diets was not different from that o f birds fed the control diet during the 8 week study. In the present study, palm o i l was also added to 20% P K C diets (which brought the metabolizable energy level to that of the control corn-soybean meal diet). Even though weight gain o f the broilers fed different diets was not significantly different, broilers fed the 20% P K C diets were not as efficient (in utilizing feed) as broilers fed the control diets. It is l ikely that birds increased their feed intake to compensate the poor nutrient availability in P K C based diets. During a 4 week feeding trial, Ngoupayou (1984) found that P K C was quite palatable to broiler chicks, and this was confirmed in the present study as feed intake was increased in birds fed PKC-containing diets. But this is in disagreement with Swick and Tan (1997) who reported that P K C is unpalatable to poultry. Given the high fiber content and low digestibility o f protein, amino acids and energy (Chapter V ) in P K C , it is surprising that some studies used more than 20% P K C in broiler diets without adverse effects on growth performance. Nwokolo et al. (1977) concluded that broilers could be fed diets containing up to 30% P K C without any apparent adverse effects on performance. However, this was based only on an 8-day experiment. In another study, Onwudike (1986) found that starter birds were able to utilize 28% screw-pressed P K C without any significant effect on performance (1-7 week o f age). It was interesting to note that the daily weight gain of the birds fed the control diet for the 6 week study period was only 21.2 g, which is equivalent to 890.4 g/42days. This is too low when compared to commercial standards. Perhaps this happened because the control diet was based on com and groundnut cake instead o f corn and soybean meal. Even though the dry matter retention of the diet was 73% in 184 the control group compared to 51% in the 50% PKC group, Panigrahi and Powell (1991) concluded that Malaysian PKC could be incorporated at up to 50% in broiler diets from 0 to 7 weeks of age without depressing growth and feed intake. However, the authors concluded that such diets were uneconomic because of the high inclusion of oil, and they were too oily to be considered practical. The reasons for the discrepancies in dietary inclusion rate of PKC reported in this and other studies are not clear. Perhaps, they are caused by the variety of oil palm used in different countries, methods of processing palm kernel oil, age of the birds, and duration and design of the experiments. The potential of the enzyme (PKCase) for PKC saccharification was demonstrated in the previous study (Chapter V). In the current study, enzyme supplementation significantly increased true dry matter retention, A M E and TMEn in the starter and grower PKC-based diets, leading to a better performance by the birds. The improvement in performance of the broilers fed the enzyme-supplemented PKC-based diets was likely due to the breaking down of the non-starch polysaccharide (mannans) of PKC by mannanase, hence releasing more energy (reducing sugars) and other nutrients which could have been trapped inside the cell. A trend for increased weight gain was observed with increased levels of the enzyme in the PKC-based broiler diet. However, if 20%) PKC was added into both the broiler starter and grower phases (0-6 weeks), 2kg/t of enzyme is required for better FCR. Due to the low AME value of PKC, the addition of PKC into poultry diets inevitably results in the need for greater inclusion of oil to offset the replacement of high-energy corn. High levels of oil in the 20% PKC diets unfortunately also led to a higher abdominal fat deposition. This could be a major drawback for the use of PKC diets in the broiler industry. The effect of diet constituents and environmental temperatures on the animal performance has been the subject of several investigations (Schoenherr et al, 1989 and Black et al, 1993). The interaction between diet and environmental temperature could be attributed to the lower heat increment of dietary fat compared to that of dietary protein and carbohydrates (Black et al, 1993). In the present study, broilers started dying when the temperature inside the building reached 36°C. Mortality due to heat stress was significantly higher in broilers fed the 20% PKC diets without enzyme supplementation (contain 5% crude fiber) than in broilers fed the control corn-soybean meal diets (contain 3% crude fiber). Perhaps, increasing heat production from crude fiber digestion and fermentation led to higher heat load during hot environmental temperature, thus 185 causing the significantly higher mortality due to heat stress in broilers fed P K C grower diets without enzyme supplementation. No significant differences in mortality due to heat stress were detected between broiler fed the control grower and enzyme-supplemented P K C diets. The reduction in mortality due to heat stress by enzyme supplementation perhaps was accomplished via the reduction in dietary crude fiber content as the previous study (Chapter V) found that crude fiber content was significantly reduced in enzyme-treated P K C . Not many published studies on P K C have dealt with laying hens. The present study indicated that laying hens were capable of maintaining production performance when 12.5% or 25% P K C was included in their diets. However, the layers consumed significantly more P K C containing diets in order to offset the poorer digestibility of P K C diets. This led to significantly poorer F C R in PKC-fed groups. A direct relationship between feed intake and the level of P K C was found. It has been long established that birds attempt, as a priority, to consume a certain quantity of feed necessary to meet their energy requirements (Larbier and Leclercq, 1994). In the present study, both the A M E and T M E n contents of the PKC-containing layer diets (L2-L7) were not lower than those of the control corn-soybean meal diet (Ll ) . Therefore, it is not likely that layers consumed significantly more P K C diets to meet their energy needs. Other factors might have overridden the bird's tendency to eat to a given energy level. For instance, Parsons et al. (1984) and Edmonds et al. (1985) found that broilers increased rather than decreased their voluntary feed intake when dietary protein level in a corn-soybean meal diet was reduced from 24% to 16% by replacing soybean meal with corn. Thus, the birds actually consumed more of a low protein-high energy than a high protein-low energy diet. Therefore, the birds appeared to be trying to eat to meet their protein-amino acid needs rather than their energy needs. In the present study, the increase in feed intake of layers fed PKC-containing diets was probably due to the lower amino acid and dry matter digestibility of the PKC-based diets, especially at high levels of PKC. As a result, it is possible that layers consumed more of the PKC-based diets to meet their amino acid needs. Contrary to the findings of the present study, Longe (1984) found that 24-week-old laying hens fed 20% P K C diets produced fewer eggs than laying hens fed a control corn-soybean meal diet. As the calculated daily energy consumption between layers fed the control and 20% P K C diets were not different, it is not clear why layers fed the P K C diet produced fewer eggs (Longe, 1984). Feed intake of layers fed PKC-based diets was significantly higher, in agreement 186 with the finding o f the present study. However, in this case, the layers consumed more of the 20% P K C diet probably to meet their energy needs because the author (Longe, 1984) failed to adjust the A M E of the PKC-containing diet to that o f the control diet,. Interestingly, another study from the same country (Nigeria) found that laying hens could tolerate up to 40% P K C in their diets without adverse effects on feed intake or egg production (Onwudike, 1988). However, layers fed either the control or 40% P K C diets only attained a hen-day production o f 62% and 36.5 g of daily egg mass for 31-week-old laying hens (Onwudike, 1988). It is not clear whether this is a common and acceptable performance found in Nigeria, but it is certainly unacceptable in many parts o f the world including Malaysia. More recently, another study has also reported equivalent performance of laying hens fed either a control or a 40% P K C diet (Panigrahi and Waite, 1998). It was noted that this experiment was carried out in an environment-controlled room in England, it is not sure whether layers fed the 40% P K C would perform equally in hot and humid tropical countries such as Nigeria or Malaysia. Even though the diets in the current study did not contain such high levels o f P K C (25% versus 40% P K C used by Onwudike (1988)), excellent performance (92.2% egg production and 50.7g egg mass) was achieved with layers fed 25% P K C layer diets. The beneficial effects o f enzyme supplementation on true dry matter retention (improved by 12.5%), A M E (improved by 9.8%) and TME„ (improved by 8.4%) were also observed in the present layer study. Enzyme supplementation significantly reduced feed consumption and F C R in the P K C - f e d groups. A positive trend was also found, indicating higher egg production, egg weight and egg mass with enzyme supplementation. The layer study indicates that it is possible to use a 12.5% P K C diet with lkg/t enzyme supplementation with layers since it results in a F C R equal to that obtained with the control diet. Dietary inclusion of P K C or enzyme supplementation did not influence eggshell quality. The mean egg specific gravity o f 1.091 indicates excellent shell quality. Practically, this means that only about 0.7% of the eggs can be expected to crack i f the eggs are run through a commercial egg grading and sizing plant (Holder and Bradford, 1979). Egg yolk color, however, was significantly paler as the dietary level o f P K C reached 25%, in agreement with the findings of Panigrahi and Waite (1998). Therefore, in countries where consumers prefer a darker color yolk, pigmenting agents would have to be added to layer diets containing 25% P K C . 187 Cereals provide most of the energy whereas protein concentrates provide most of the protein (amino acids) in a practical poultry diet. However, the type of cereals and protein concentrates used in poultry feeds depend entirely on their availability and the concentration of energy and protein they supply per unit of price. Even though, the price o f P K C + lkg/t enzyme ( P K C + IE) was set to be equal to the price o f P K C without enzyme, P K C + I E contained 172 kcal/kg more A M E than P K C without enzyme and the extra energy is worth at least RM0.04 per Mea l (Table 6.22). Therefore the enzyme would have to be priced at no more than RM61.6 /kg for it to be economic for commercial use. Parametric linear programming reveals what should be the price o f P K C and P K C + I E for it to be included into a poultry diet at a certain level. For instance, the price o f P K C has to be less than R M 7 0 0 per tonne for a 6% inclusion rate in a 3,000 kcal/kg broiler grower diet. On the other hand, the price o f P K C and enzyme should fall between R M 3 2 and R M 7 0 0 per tonne for a 23% inclusion rate in a 2,700 kcal/kg layer diet. Parametric linear programming revealed that there are three major factors that affect the inclusion rates o f P K C into poultry diets. First of all , the metabolizable energy value assigned to P K C . For instance, in the current study, the computerized feed formulation program chose to use more than P K C in the broiler and layer diets. This is because P K C + I E contains higher A M E and it is less expensive than P K C to supply energy. It should be noted that i f the 5% constraint (maximum) on palm oi l was removed, the feed formulation program would choose to use more palm oi l , since it provided a cheaper source o f energy than P K C or P K C + I E . The second factor is the price o f P K C . Due to its low A M E value, the cost per unit o f energy is very important for its use in commercial feed formulation. When the price o f P K C is low, the cost per kilocalorie is also low, and the lower the cost the more P K C can be used. For example, when the price o f P K C per tonne is below R M 3 1 , 28% - 29% o f P K C + I E is used in the low A M E layer diet. The dietary inclusion rate is reduced to 23% when the price of P K C + I E per tonne exceeds R M 3 1 . The dietary level of A M E is another factor that affects the inclusion rates o f P K C into poultry diets. For instance, more P K C is used in the low A M E (2,700 kcal/kg) layer diets than in the high A M E (2,900 kcal/kg) layer diets. The feed formulation program does not include much P K C or P K C + I E in the high nutrient density broiler starter and grower diets, indicating that P K C is probably more suitable for use in lower energy diets such as layer diets than in broiler diets. 188 The beneficial effect of enzyme (with mannanase activity) on PKC saccharification has been demonstrated by Dusterhoft et al. (1993a,b,c), Daud et al. (1997) and the previous study (Chapter V). However, the effect of adding enzyme to PKC-based diets on poultry performance has not been studied before. The present study indicates that the release of more energy and nutrients from PKC with enzyme supplementation translates into better performance of both broilers and layers in a hot tropical environment. C o n c l u s i o n s The findings in the present study indicate that using 20% PKC in both the starter and grower phases is not recommended, unless supplemental enzyme is used. The lower nutrient digestibility of PKC diets resulted in reduced growth performance when it was included in both the starter and grower phases. There are also higher risks of mortality due to heat stress in hot tropical conditions when PKC-based grower diets are fed, because of a higher heat increment. Broilers fed PKC diets tend to deposit more abdominal fat than broilers fed control diets. Laying hens were able to tolerate 12.5% and 25% PKC in their diets without adversely affecting egg production and egg mass. However, laying hens fed 12.5% and 25% PKC diets consumed significantly more feed and had a poorer FCR than laying hens fed control diets. Enzyme supplementation of PKC-based diets improved growth performance and feed efficiency, of broilers and layers by increasing the DM digestibility and AME value. The diminishing yolk color associated with a high inclusion rate of PKC in layer diets could be a major problem for egg producers in Asian countries where customers prefer a darker yolk color. Parametric linear programming found that PKC was not likely to be used in commercial broiler diets under the current price conditions in Malaysia. However, higher levels of PKC could be incorporated into commercial layer diets. Enzyme supplementation of PKC-based remains promising and future studies should be conducted to evaluate whether a higher level of enzyme-supplemented PKC could be used in poultry diets. Several recent studies (Oyofo et al, 1989; Hinton et al, 1990; Allen et al, 1997) have showed that mannose (from the digestion of PKC) greatly reduced the colonization of salmonella in the gastro-intestinal tract. Further study in this area is also warrant. 189 References Ahmad, M. Y., 1982. The feeding value of palm kernel cake for broilers. MARDI Res. Bull. 10:120-126. Allen, V. M, F. Fernandez, and M .H. Hinton, 1997. Evaluation of the influence of supplementing the diet with mannose or palm kernel meal on salmonella colonisation in poultry. Br. Poult. Sci. 38:485-488. Berepubo, N. A., H. D. Mepba, O. J. Agboola, and R. I. Onianwah, 1995. Inclusion rate and true metabolizable energy of palm kernel cake in broiler chicken diets in a humid tropical environment. J. Appl. Anim. Res. 7:27-34. Black, J. L., B. P. Mullan, M. L. Lorschy, and L. R. Giles, 1993. Lactation in the sow during heat stress. Livestock Prod. Sci. 35:153-170. Cahaner, A., Z. Nitsan, and I. Nir, 1986. Weight and fat content of adipose and nonadipose tissues in broilers selected for or against abdominal adipose tissue. Poultry Sci. 65:215-222. Daud, M. J., N. Samad, and S. Rasool, 1997. Specific commercial enzymes for nutritive value improvement of palm kernel cake for poultry diets. Pages 137-138 in: 19th MSAP Annual Conference. Y. W. Ho, M. Z. Saad, F. Y. Chin, I. Zulkifli, and H. K. Wong ed. Johor Bahru, Johor, Malaysia. Dusterhoft, E. M., F. M., Engels, and A. G. F. Voragen, 1993a. Parameters affecting the enzymic hydrolysis of oil-seed meals, lignocellulosic by-products of the food industry. Bioresource Technol. 44:39-46. Dusterhoft, E. M., A. W. Bonte, and A. G. J. Voragen, 1993b. Solubilisation of non-starch polysaccharides from oil-seed meals by polysaccharides - degrading enzymes. J. Sci. Agric. 63:211-220. Dusterhoft, E. M., A. W. Bonte, J. C. Venekamp, and A. G. J. Voragen, 1993c. The role of fungal polysaccharidases in the hydrolysis of cell wall materials from sunflower and palm-kernel meals. World J. Microbiology and Biotechnology 9:544-554. Edmonds, M. S., C. M. Parsons, and D. H. Baker, 1985. Limiting amino acids in low-protein corn-soybean meal diets fed to growing chicks. Poultry Sci. 64:1519-1526. Garcia, C. A., and A. G. Gernat, 1998. The effect of using different levels of palm kernel meal in broiler diets. Poultry Sci. 77(Suppl. l):44.(Abstr.) Hinton, A., D. E. Corrier, G. E. Spates, J. O. Norman, R. L. Ziprin, R. C. Beier, and J. R. Deloach, 1990. Biological control of Salmonella typhimurium colonisation in young chickens. Avian Dis.34:626-633. 190 Holder, D . P., and M . V . Bradford, 1979. Relationship o f specific gravity o f chicken eggs to number of cracked eggs observed and percent shell. Poultry Sci . 58:250-251. Hutagalung, R. I., 1980. Availabili ty of feedstuffs for farm animals. Proc! Abstr. First As ia -Australasia Animal Science Congress. 40:15.(Abstr.) Larbier, M . , and B . Leclercq, 1994. Nutrition and feeding o f poultry. Nottingham University Press. Loughborough, Leicestershire, England. Longe, O. G . , 1984. Effects o f increasing the fiber content of a layer diet. Br . Poult. Sci . 25:187-193. National Research Council , 1994. Nutrient Requirements of Poultry. 9 t h rev. ed. National Academy Press, Washington, D C . Ngoupayou, Ngou J. D . , 1984. Nutritional value o f palm kernel cake in broiler diets. Poult. Sci . 63 (Suppl. 1):155-156. (Abstr.) Nwokolo, E . N . , and D . B . Bragg, 1977. Influence o f phytic acid and crude fiber on the availability o f minerals from four protein supplements in growing chicks. Can. J. A n i m . Sci . 57:475-477. Onifade, A . A . , and G . M . Babatunde, 1998. Comparison of the utilization of palm kernel meal, brewers' dried grains and maize offal by broiler chicks. Br . Poult. Sci . 39:245-250. Onwudike, O. C , 1986. Palm kernel as a feed for poultry. 3. Replacement o f groundnut cake by palm kernel meal in broiler diets. A n i m . Feed Sci . Technol. 16:195-202. Onwudike, O. C , 1988. Palm kernel as a feed for poultry. 4. Use of palm kernel meal by laying birds. A n i m . Feed Sci . Technol. 20:279-286. Osei, S. A . , and J. A m o , 1987. Research note: palm kernel cake as a broiler feed ingredient. Poult. Sci . 66:1870-1873. Oyofo, B . A . , J. R. Deloach, D . E . Corrier, J. O. Norman, R. L . Ziprin, and H . H . Mollenhauser, 1989. Effects o f carbohydrates on Salmonella typhimurium colonisation in broiler chickens. Av ian Dis . 33:531-534. Panigrahi, S., and C. J. Powell , 1991. Effects of high rates of inclusion o f palm kernel meal in broiler chick diets. A n i m . Feed Sci . Technol. 34:37-47. Panigrahi, S., and B . S. Waite, 1998. Use of rations up to forty per cent palm kernel meal for egg production. Br . Poult. Sci . 39(Suppl.): S37-S38. Parsons, C . M . , M . S. Edmonds, and D . H . Baker, 1984. Influence o f dietary electrolyte balance, energy, and amino acid supplementation on the Monensin response in chicks fed diets varying in protein content. Poultry Sci . 63:2438-2443. 191 P O R L A , 1997. Porla palm oi l statistics. 16 t h ed. Palm O i l Registration and Licensing Authority. Ministry o f Primary Industries, Malaysia. S A S Institute, 1996. SAS® User's Guide: Statistics. Version 6 Edition. S A S Institute Inc., Cary, N C . Schoenherr, W . D . , T. S. Stahly, and G . L . Cromwell , 1989. The effects of dietary fat or fiber addition on yield and composition o f milk from sows housed in a warm or hot environment. J. A n i m . Sci . 67:482-495. Sheldon, B . L . , 1998. Poultry and poultry products as resources for human health and food in the 21 s t century. Pages 1-8 in: Proceedings of 6 t h Asian Pacific Poultry Congress, Nagoya, Japan. Sibbald, I. R. , 1986. The T. M . E . system o f feed evaluation: methodology, feed composition data and bibliography. Tech. B u l l . 1986-4E, Res. Branch, Agriculture Canada, Ottawa, Ont. Canada. Snedecor, G . W . , and W . G . Cochran, 1980. Statistical methods. 8 t h ed. Iowa Press, Ames, Iowa. Swick, R. A . , and P. H . Tan, 1997. Characteristics of protein meals: Considerations in using common asian protein meals. Zootecnica Int. 20(1): 14-25. Yeong, S. W. , 1980. The nutritive value of palm oi l by-products for poultry. Proc. Abstr. First Asia-Australasia Animal Science Congress. 45:17. (Abstr.) Yeong, S. W. , and T. K . Mukherjee, 1983. The effect o f palm oi l supplementation in palm kernel cake-based diets on the performance of broiler chickens. M A R D I Res. B u l l . , 11:378-384. 192 C H A P T E R VII General Conclusions A s the world human population continues to increase, it is crucial to reduce the competition between animal and human for food. Data presented in this thesis have proved that by improving utilization of poultry feedstuffs with supplemental amino acids and enzymes, more feed could be conserved. Hence, more food w i l l be available for human consumption. Protein is a costly item in poultry diets, so maximizing the efficiency of protein and amino acid ( A A ) utilization is economically important. In addition, the high energy cost of uric acid synthesis and excretion implies several nutritional advantages o f balancing the A A composition o f absorbed protein close to the requirement. Unfortunately, valid A A requirement values for broilers and laying hens are not available for most of the essential A A , especially for threonine and tryptophan. A s a result, when formulating poultry diets, the nutritionist must be concerned about the dietary levels of threonine and tryptophan. Under such circumstances, the nutritionist could use the A A profile (0-3 week old broiler chicks) determined in this study. For instance, the recommended ratio of threonine and tryptophan to lysine is 65% and 16%, respectively. I f the nutritionist is formulating a diet with 1.00% digestible lysine, the level o f digestible threonine and tryptophan in the diet should be targeted at 0.65% and 0.16%, respectively. On the other hand, the nutritionist could also use the results from the reduced protein study as a reference. That is, the nutritionist could set the minimum levels of dietary threonine and tryptophan for 0-3 week old broilers at 0.74% o f the diet (or 4.04% of CP) and 0.23% of the diet (or 1.22% of CP) , respectively. Reducing dietary threonine to 0.74% did not depress growth performance or nitrogen retention in the present study and increasing the level o f tryptophan in the diet to 0.23% proved to be beneficial in terms o f growth and nitrogen retention in the present study for 0-3 week old broiler chicks. Results from the present and other recent studies indicate that the N R C (1994) has overestimated the requirements for threonine (Thomas et al, 1992; Holsheimer et al, 1994; Rangel-Lugo et al, 199»4; K i d d et al, 1996; Leeson and Summers, 1997; Yamazaki et al, 1997) and underestimated the requirement for tryptophan (Abebe and 193 Morris , 1 9 9 0 ; Han et al, 1 9 9 1 ; Thomas et al, 1 9 9 2 ; Rhodimet Nutrition Guide, 1 9 9 3 ; Austic, 1 9 9 4 ) for 0 - 3 week old broiler chicks. A s indicated by the N R C ( 1 9 9 4 ) , the A A requirement values o f broilers beyond 3 weeks of age and laying hens are based on limited published data. Since broilers consume the most feed during the second phase of the production cycle, it is important for the nutritionist to know exactly what the A A requirements are for 3 - 6 week old broilers. Based on this study, the level o f dietary threonine and tryptophan for 3 r 6 week old broilers should be targeted at 0 . 6 7 % o f the diet ( 3 . 4 % o f C P ) and 0 . 1 7 % o f the diet ( 0 . 8 9 % o f CP) , respectively. Nutritionists should also be aware that most o f the recently available data (Thomas et al, 1 9 9 2 ; Rhodimet Nutrition Guide, 1 9 9 3 ; Webel et al, 1 9 9 6 ; Baker, 1 9 9 7 ; K i d d and Kerr, 1 9 9 7 ; Penz et al, 1 9 9 7 ; Yamazaki et al, 1 9 9 7 ) point strongly to the fact that the N R C ( 1 9 9 4 ) has overestimated the threonine requirement for 3 - 6 week broilers. However, the N R C ( 1 9 9 4 ) recommendation for tryptophan is well received (Rhodimet Nutrition Guide, 1 9 9 3 ; Baker, 1 9 9 7 ; Leeson and Summers, 1 9 9 7 ) . Laying hens ( 4 2 - 5 0 weeks o f age), as indicated by this study, should be targeted at a daily intake o f 4 4 8 mg threonine/hen and 1 5 2 mg tryptophan/hen. The N R C ( 1 9 9 4 ) recommendations for threonine and tryptophan for laying hens are supported by this and other recent studies (Coon ( 1 9 9 8 ) and Ishibashi et al. ( 1 9 9 8 ) for threonine, and Rhodimet Nutrition Guide ( 1 9 9 3 ) and Leeson and Summers ( 1 9 9 7 ) for tryptophan). The results of the balance studies clearly show that crystalline A A supplementation o f the reduced-protein diets improved the A A balance in the diet, hence improving the protein utilization efficiency and reducing nitrogen content in the excreta. Lowering the C P content o f the experimental diets resulted in a 3 6 % , 2 5 % and 4 6 % reduction in nitrogen output in the excreta o f starting and growing broilers and layers, respectively. In order to improve the utilization o f protein, valid data on the requirements for essential A A are crucial. Furthermore, by improving the efficiency o f protein utilization, less nitrogen w i l l be excreted into the environment. The latter is becoming an important issue in countries with a high concentration of animals and a limited land base for manure disposal. Future studies should concentrate on determining the requirements o f poultry for essential A A other than lysine and methionine, especially for older birds. In addition, further research is needed to more accurately determine the amounts o f digestible A A in feedstuffs and the digestible A A requirements of poultry. This is because the use o f digestible A A values in the formulation of poultry diets is a means o f 194 improving the utilization o f protein sources that are known to be less digestible than soybean meal (Fernandez et al, 1995; Rostagno et al, 1995; Wang and Parsons, 1998). A s the world population continues to increase, improving the utilization o f feed ingredients that are not in direct competition with human food is equally important. Before incorporating palm kernel cake (PKC) into poultry diets, nutritionists should be aware that there are large discrepancies in the reported nutritive quality for P K C and that the reasons for these discrepancies are not clear. Therefore, the results presented in this thesis may reflect only the quality o f P K C obtained from Malaysia, and some o f these findings might not be applied to P K C obtained from another countries. On average, Malaysian P K C was found to contain moderate amounts o f most nutrients (16.6% C P ; 14.6% C F ; 4.9% ash; 0.41% calcium and 0.77% total phosphorus). In general, P K C samples obtained from screw-press extraction plants contained a higher amount of residual o i l (8.93% versus 3.29%), gross energy (4,718 kcal/kg versus 4,504 kcal/kg) and acid detergent lignin (10.26% versus 7.94%) than P K C samples from solvent-extraction plants. In addition, the concentration o f A A , digestibility o f A A and metabolizable energy are dependent on the method o f o i l extraction. Therefore, because o f its higher nutrient digestibility and higher digestible A A contents, poultry nutritionists should try to use P K C obtained from solvent extraction plants. The low digestibility o f P K C A A (62%) indicates that formulating P K C diets using digestible A A values should improve the utilization o f protein and should give a better growth performance over P K C diets formulated using total A A . Nonetheless, future research in this area is necessary. Apparent metabolizable energy ( A M E ) and nitrogen corrected true metabolizable energy (TMEn) values for P K C were found to be low compared with that o f corn. Therefore, the main constraint in using large quantities of P K C in poultry diets is its low energy content. Therefore, when P K C is included in poultry diets, nutritionists should try to bring the energy content o f the diet to the normal standard. Also , future research to further improve the metabolizable energy content of P K C is crucial. In addition, nutritionists should pay special attention to the A A balance in the diet, because P K C contains a high level of arginine and it is also limiting in several A A including lysine and methionine. U p to now, no published study has been conducted to investigate the effects of using P K C during different phases o f the broiler production cycle or on the effects o f enzyme supplementation o f PKC-based diets on poultry performance. A n enzyme mixture (mannanase, a-galactosidase and protease) was found to be very effective in breaking down the non-starch 195 polysaccharides o f P K C . Therefore, nutritionists should consider the use o f supplemental enzymes in PKC-based diets i f it is cost-effective. Results from the broiler trial demonstrated that 20% P K C could be used in the starter phase or grower phase. However, dietary inclusion of 20% P K C in both the starter and grower phases is not recommended unless supplemental enzyme is used. Even though research conducted in other countries was conducted using higher than 20% P K C without reducing broiler performance, the results o f the present study do not recommend the use o f more than 20% Malaysia P K C in broiler diets because o f its low A M E level and low A A digestibility. When PKC-based grower diets are fed to broilers under elevated environmental temperatures (36 °C), broiler producers should pay attention to the temperature and ventilation inside the building, because these birds are more likely to die from heat stress than those fed corn-soybean meal diets. The exact mechanism regarding how PKC-based diets could have induced higher mortality due to heat stress under elevated environmental temperatures is not clear and further research is necessary to investigate the possible reasons. Not many published studies on P K C have dealt with laying hens. In the present study, laying hens were able to tolerate 12.5% and 25% P K C without reducing egg production or egg mass. However, layers fed either the 12.5% or 25% P K C diets consumed significantly more feed and had poorer F C R than layers fed the corn-soybean meal control diet. Poultry producers should be aware that even though P K C did not affect eggshell quality, egg yolk color became significantly paler when 25% PKC-based diets were fed to the layers. Therefore, using a high level o f P K C (25%) is not recommended in countries where consumers prefer a darker yolk color unless a pigmenting agent is used. In addition, because of the lower energy and A A requirements of laying hens compared with broilers, P K C is more suitable for layer diets. Enzyme supplementation o f PKC-based poultry diets looks promising, and further research in this area is warranted. H igh fiber content, poor protein quality and also low metabolizable energy in P K C indicate that it could be a valuable feed ingredient for ruminants. However, with the commercial availability of synthetic amino acids and enzymes it is now possible to improve the nutritive quality o f P K C , thus making it suitable for use as a poultry feed ingredient as indicated by the data presented in this thesis. In addition, it is not clear whether the mannose released by enzyme treatment w i l l be absorbed by the animal, and further research should be conducted to investigate whether the efficiency o f absorption of mannose in the digestive tract is equivalent to that o f glucose. 196 Recently, a few studies have demonstrated that mannose from either a purified source or from the digestion o f P K C could bind salmonella (Izat et al, 1990; A l l en et al, 1997). It would be interesting to know whether dietary inclusion o f P K C could reduce the incidence o f bacterial infections in poultry. The use of by-products such as P K C in poultry feeds that are not competing as human food sources w i l l conserve feed resources and should be encouraged. The findings presented in this thesis are valuable to the poultry industry in both developed and developing countries. The new information on the estimated requirements o f poultry for threonine and tryptophan are useful for nutritionists who want to supplement feed-grade threonine and tryptophan in poultry diets. In addition, reduced protein diets are an option for poultry producers who want to reduce the amount of nitrogen in the excreta and minimize nitrogen pollution. The new information on P K C is valuable to nutritionists who want to incorporate P K C into poultry diets. In conclusion, improving utilization o f conventional and non-conventional poultry feedstuffs with supplemental A A and enzymes w i l l help ensure long-term profitability and sustainability of the poultry industry in a very competitive environment. References Abebe, S., and T. R. Morris, 1990. Effects of protein concentration on responses to dietary tryptophan by chicks. Br . Poult. Sci . 31:267-272. Al len , V . M . , F . Fernandez, and M . H . Hinton, 1997. Evaluation of the influence of supplementing the diet with mannose or palm kernel meal on salmonella colonisation in poultry. Br . Poult. Sc i . 38:485-488. Austic, R. E . , 1994. Update on amino acid requirements and ratios for broilers. Pages 114-120 in: Proceedings o f the Maryland Nutrition Conference, College Park, M D . Baker, D . H . , 1997. Pages 1-24 in: Ideal amino acid profiles for swine and poultry and their applications in feed formulation. Biokyowa Publishing Co. , St. Louis, M O . Coon, C , 1998. Amino acid requirements o f commercial laying hens. Pages 70-75 in: Proceedings of 6 t h Asian Pacific Poultry Congress, Nagoya, Japan. Fernandez, S. R., Y . Zhang, and C. M . Parsons, 1995. Dietary formulation with cottonseed meal on a total amino acid versus a digestible amino acid basis. Poultry Sci . 74:1168-1179. 197 Han, Y . , H . Suzuki, and D . H . Baker, 1991. Histidine and tryptophan requirement o f growing chicks. Poultry Sci . 70:2148-2153. Holsheimer, J. P., P. F . G . Vereijken and J. B . Schutte, 1994. Response o f broiler chicks to threonine-supplemented diets to 4 weeks of age. Br . Poult. Sci . 35:551-562. Ishibashi, T., Y . Ogawa, T. Itoh, S. Fujimura, K . Koide, and R. Watanabe, 1998. Threonine requirements of laying hens. Poultry Sci . 77:998-1002. Izat, A . L . , R. E . Hierholzer, J. M . Kopek, M . H . Adams, M . A . Reiber, and J. P. McGinnis , 1990. Research note: Effects o f D-mannose on incidence and levels of salmonellae in ceca and carcass samples o f market age broilers. Poultry Sci . 69:2244-2247. K i d d , M . T., and B . J. Kerr, 1997. Threonine responses in commercial broilers at 30 to 42 days. J. A p p l . Poultry Res. 6:362-367. K i d d , M . T., B . J. Kerr, J. D . Firman, and S. D . Bol ing, 1996. Growth and carcass characteristics o f broilers fed low-protein, threonine-supplemented diets. J. A p p l . Poultry Res. 5:180-190. Leeson, S., and J. D . Summers, 1997. Commercial poultry nutrition. Leeson, S., and J. D . Summers ed. 2 n d edition. University Books, Guelph, Ontario, Canada. Penz, A . M . Jr., G . L . Colnago, and L . S. Jensen. 1997. Threonine supplementation of practical diets for 3- to 6-wk-old broilers. J. App l . Poultry Res. 6:355-361. Rangel-Lugo, M , C. L . Su, and R. E . Austic, 1994. Threonine requirement and threonine imbalance in broiler chickens. Poult. Sci . 73:670-681. Rhodimet Nutrition Guide, 1993. Feed ingredients formulation in digestible amino acids, 2 n d edition 1993 Rhone-Poulenc Animal Nutrition. Rostagno, H . S., J. M . R. Pupa, and M . Pack, 1995. Diet formulation for broilers based on total versus digestible amino acids. J. App l . Poultry Res. 4:293-299. Thomas, O. P., M . Farran, and C . B . Tamplin, 1992. Broiler nutrition update: Threonine requirement for 3-6 week-old broilers. Pages 45-53 in: Proceedings Maryland Nutrition Conference, College Park, M D . Wang, X . amd C . M . Parsons, 1998. Dietary formulation with meat and bone meal on a total versus a digestible or bioavailable amino acid basis. Poultry Sci . 77:1010-1015. Webel, D . M . , S. R. Fernandez, C . M . Parsons, and D . H . Baker, 1996. Digestible threonine requirement o f broiler chickens during the period three to six and six to eight weeks posthatching. Poultry Sci . 75:1253-1257. 198 Yamazaki, M . , Y . Oka, H . Murakami, M . Takemase, M . Ando, and M . Yamazaki, 1997. Available threonine requirement of broiler chickens at two growing stages. Jpn. Poult. Sci. 34:45-51. 

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