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The evaluation of protein supplements for the growing chick Low, Roland Kai-Chong 1986

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THE EVALUATION OF PROTEIN SUPPLEMENTS FOR THE GROWING CHICK By ROLAND KAI-CHONG LOW B . S c , The U n i v e r s i t y of A l b e r t a , 1978 M.Sc, The U n i v e r s i t y of Manitoba, 1980 A THESIS SUBMITTED IN PARTIAL FULFILMENT THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES (Department of Animal Science) We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA October, 1986 © Roland K-C. Low, 1986 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Ro l and Ka i -Chong Low Department of An ima l S c i e n c e The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Oc t obe r 10 , 1986 i ABSTRACT The n u t r i t i v e values of hydrolyzed f e a t h e r meal (HFM) and blood meal (BM), s u p p l i e d by a commercial r e n d e r i n g company, were evaluated using b r o i l e r - t y p e growing c h i c k s . The sample of HFM (89% crude p r o t e i n ) , which was processed at 2.81kg/cm of steam pressure f o r 40 minutes with continuous a g i t a t i o n , c o n t a i n s 1.4, 1.5 and 3.2% a v a i l a b l e h i s t i d i n e , l y s i n e and c y s t i n e , r e s p e c t -i v e l y , and i s w e l l u t i l i z e d by the growing c h i c k . With methionine and l y s i n e supplementation, up to 37% of the d i e t a r y p r o t e i n could be s u p p l i e d by HFM i n c h i c k s t a r t e r r a t i o n s . On the other hand, a crude p r o t e i n content of 95% was obtained from the BM sample processed at 190°C f o r f i v e minutes at a pressure of 1.41kg/cm^. Even though BM c o n t a i n s 8.1 and 10.7% of a v a i l a b l e l y s i n e and l e u c i n e , r e s p e c t i v e l y , i t i s not e f f i c i e n t l y u t i l i z e d by b r o i l e r c h i c k s . With a r g i n i n e , i s o l e u c i n e , methionine and c y s t i n e supplementation, only 17% of the d i e t a r y p r o t e i n could be s u p p l i e d by BM i n c h i c k s t a r t e r r a t i o n s . A combination of 2.5% BM and 7.5% HFM was s a t i s f a c t o r y f o r c h i c k s at the s t a r t i n g phase. The r e s u l t s i n d i c a t e d a p o s s i b l e r e l a t i o n s h i p between p r o t e i n source and d i e t a r y p r o t e i n c o n c e n t r a t i o n on amino a c i d a v a i l -a b i l i t y (AAA). However, no v a l i d c o n c l u s i o n could be drawn from the present i n v e s t i g a t i o n , s i n c e there were no c o n s i s t e n t trends on the AAA with d i f f e r e n t d i e t a r y p r o t e i n c o n c e n t r a t i o n s . i i R egression equations, e s t a b l i s h e d from the r e l a t i o n s h i p s between a l p h a - c e l l u l o s e i n t a k e s and amino a c i d e x c r e t i o n s , were r e l i a b l e f o r the c o r r e c t i o n s of metabolic and endogenous amino a c i d s i n the AAA assay. T h i s should provide a b a s i s f o r f u t u r e s t u d i e s i n v o l v e d i n the development of a u n i v e r s a l method f o r the e s t i m a t i o n of endogenous amino a c i d s i n the growing ch i c k that w i l l save tedious l a b o r , time and expense. F i n a l l y , an eight-hour f e c a l c o l l e c t i o n p e r i o d , with the r e g r e s s i o n endogenous c o r r e c t i o n method, was s u f f i c i e n t f o r the c a l i b r a t i o n of AAA f o r HFM and soybean meal. i i i ACKNOWLEDGEMENTS The author i s indebted to Dr. D.B. Bragg f o r the o p p o r t u n i t y to l e a r n p r a c t i c a l a s p e c t s of a g r i c u l t u r a l r e s e a r c h under h i s s u p e r v i s i o n . D r. B r a g g ' s c o n s i s t e n t a d v i c e , g u i d a n c e , encouragement and c o n s t r u c t i v e c r i t i c i s m d u r i n g the course of t h i s study are g r a t e f u l l y acknowledged. HFM and BM samples were obtained from West Coast Reduction, Vancouver, B r i t i s h Columbia, Canada, through the c o u r t e s y of Mr. B r i a n Osborne. S i n c e r e a p p r e c i a t i o n i s a l s o extended to the members of my t h e s i s committee: P r o f e s s o r B.E. March, Dr. M e l v i n Lee, Dr. R.C. F i t z s i m m o n s , Dr. J.S. Sim, Dr. K.M. Cheng and Dr. J . Vanderstoep f o r t h e i r v a l u a b l e suggestions. The author wishes to thank Mr. R. Soong, Mrs. B. C a r l s o n , Mr. M. Hudson, Miss C. McMillan, Mr. G. Schierman and Mr. B. Chan f o r t h e i r a s s i s t a n c e . Very s p e c i a l thanks must be given to Miss Kamily Cheung f o r her p a t i e n c e , understanding and encouragement d u r i n g the c o u r s e of t h i s s t u d y and the t y p i n g of t h i s m a n u s c r i p t , and to h i s s i s t e r s , V i v i a n and Barbara, f o r t h e i r encouragement and l o v e . F i n a n c i a l s u p p o r t s p r o v i d e d by the Department of P o u l t r y S c i e n c e , C.W. R o b e r t s S c h o l a r s h i p Fund and P a c i f i c Egg and P o u l t r y A s s o c i a t i o n are g r e a t l y a p p r e c i a t e d . i v TABLE OF CONTENTS Page ABSTRACT i ACKNOWLEDGEMENTS i i i TABLE OF CONTENTS i v LIST OF TABLES v i i i LIST OF APPENDIX TABLES x i i LIST OF ABBREVIATIONS x i v INTRODUCTION 1 LITERATURE REVIEW 2 A. U t i l i z a t i o n of hydrolyzed f e a t h e r meal (HFM) i n p o u l t r y f e e d i n g 2 1. I n t r o d u c t i o n 2 2. N u t r i e n t composition of hydrolyzed f e a t h e r meal 3 3. The n u t r i t i v e values of hydrolyzed f e a t h e r meal 10 a) P r a c t i c a l f e e d i n g t r i a l s 10 b) Other animal bioassays 17 c) E n z y m a t i c a l , m i c r o b i o l o g i c a l , and chemical assays .... 20 4. E f f e c t s of p r o c e s s i n g methods on the n u t r i t i v e values of hydrolyzed f e a t h e r meal.. 22 B. U t i l i z a t i o n of blood meal i n p o u l t r y f e e d i n g 25 1. I n t r o d u c t i o n 25 2. N u t r i e n t composition of blood meal 25 3. The n u t r i t i v e values of blood meal 29 C. E s t i m a t i o n by f e c a l analyses of the a v a i l a b i l i t y of amino a c i d s i n f e e d s t u f f s 32 TABLE OF CONTENTS Page 1. I n t r o d u c t i o n 32 2. The c o r r e c t i o n f o r the endogenous amino a c i d s l o s s e s 34 a) E f f e c t of d i e t a r y carbohydrates 36 b) E f f e c t of d i e t a r y p r o t e i n i n t e s t d i e t 39 c) E f f e c t of m i c r o f l o r a i n the g a s t r o -i n t e s t i n a l t r a c t of p o u l t r y 40 d) U r i n a r y c o n t r i b u t i o n 42 3. The q u a n t i t a t i v e c o l l e c t i o n of the t e s t e x c r e t a 44 4. Drying techniques f o r p o u l t r y e x c r e t a 46 MATERIALS AND METHODS 47 A. Feed samples and d i e t s f o r m u l a t i o n 47 B. General b i r d management and experimental d e s i g n . . 48 1. Feeding t r i a l s 48 2. The amino a c i d a v a i l a b i l i t y (AAA) t r i a l s 48 C. Analyses 50 1. General analyses 50 2. Amino a c i d a n a l y s i s 50 D. Chick experiments 54 1. Feeding t r i a l s 54 Experiment I. The e f f e c t s of d i f f e r e n t l e v e l s of hydrolyzed f e a t h e r meal on the growth performance of c h i c k s 54 Experiment I I . The e f f e c t s of d i f f e r e n t l e v e l s of blood meal on the growth performance of c h i c k s 54 v i TABLE OF CONTENTS Page Experiment I I I . The e f f e c t s of blending d i f f e r e n t l e v e l s of blood meal and hydrolyzed f e a t h e r meal on c h i c k performance 57 2. The amino a c i d a v a i l a b i l i t y t r i a l s 57 Experiment IV. The e f f e c t s of p r o t e i n sources (PS) and p r o t e i n l e v e l s (PL) on the amino a c i d a v a i l a b i l i t y i n the growing c h i c k 57 Experiment V. The e f f e c t s of p r o t e i n source (PS) and p r o t e i n l e v e l (PL) and/or endogenous amino a c i d s c o r r e c t i o n method (ECM) on amino a c i d a v a i l -a b i l i t y i n the growing c h i c k 62 Experiment VI. The e f f e c t s of the d u r a t i o n of f e c a l c o l l e c t i o n s on amino a c i d a v a i l a b i l i t y i n the growing c h i c k 65 RESULTS AND DISCUSSION 67 A. The chemical composition of hydrolyzed f e a t h e r meal and blood meal 67 B. Feeding t r i a l s 72 Experiment 1 72 Experiment I I 75 Experiment I I I 79 C. The amino a c i d a v a i l a b i l i t y t r i a l s 82 Experiment IV 82 Experiment V 89 Experiment VI I l l GENERAL DISCUSSION 115 v i i TABLE OF CONTENTS Page SUMMARY AND CONCLUSIONS 123 LITERATURE CITED 125 APPENDIX 137 v i i i LIST OF TABLES Table Page 1 The chemical composition of hydrolyzed f e a t h e r meal 4 2 The amino a c i d (AA) composition of hydrolyzed f e a t h e r meal 8 3 The chemical composition of blood meal 27 4 4 The amino a c i d (AA) composition of blood meal. 28 5 The mean amino a c i d (AA) e x c r e t i o n s of unfed SCWL a d u l t r o o s t e r s 35 6 The mean amino a c i d (AA) e x c r e t i o n s of b i r d s fed N-free d i e t s 37 7 Composition of d i e t s (Experiment I) 55 8 Composition of d i e t s (Experiment I I ) 56 9 Composition of d i e t s (Experiment I I I ) 58 10 Composition of N-free d i e t used i n Experiment IV, V and VI 59 11 D i e t a r y treatments i n Experiment IV 61 12 The o u t l i n e of experimental designs i n Experiment V, Part 2 63 13 D i e t a r y treatments i n Experiment V 64 14 Compositions of hydrolyzed f e a t h e r meal (HFM) and blood meal (BM) 68 15 The mean amino a c i d (AA) composition of hydrolyzed f e a t h e r meal (HFM) and blood meal (BM) 69 16 The performance of b r o i l e r c h i c k s fed d i f f e r e n t l e v e l s of hydrolyzed f e a t h e r meal (HFM) (Experiment I) 73 17 The performance of b r o i l e r c h i c k s fed d i f f e r e n t l e v e l s of blood meal (BM) (Experiment I I ) 76 i x LIST OF TABLES Table Page 18 The performance of b r o i l e r c h i c k s fed d i f f e r e n t 10% blends of blood meal (BM) and hydrolyzed f e a t h e r meal (HFM)(Experiment I I I ) . 81 19 The mean amino a c i d (AA) e x c r e t i o n of b i r d s fed the N-free d i e t (Experiment IV) 83 20 Comparison of amino a c i d (AA) a v a i l a b i l i t y of d i f f e r e n t p r o t e i n source (PS) using 3-week ol d c h i c k s (Experiment IV) 84 21 Comparison of amino a c i d (AA) a v a i l a b i l i t y from d i e t s of d i f f e r e n t p r o t e i n l e v e l (PL) using 3-week o l d c h i c k s (Experiment IV) 85 22 The i n t e r a c t i o n of p r o t e i n source (PS) x p r o t e i n l e v e l (PL) f o r a s p a r t i c a c i d a v a i l a b i l i t y (Experiment IV) 90 23 The i n t e r a c t i o n of p r o t e i n source (PS) x p r o t e i n l e v e l (PL) f o r l y s i n e a v a i l a b i l i t y (Experiment IV) 90 24 The r e l a t i o n s h i p s between amino a c i d (AA) e x c r e t i o n (Ymg) and a l p h a - c e l l u l o s e i n t a k e (Xg) from a N-free d i e t i n Experiment V, Part 1 93 25 Comparison of amino a c i d (AA) a v a i l a b i l i t y from hydrolyzed f e a t h e r meal (HFM) and soybean meal (SBM) i n the growing c h i c k (Experiment V, Part 2a) 95 26 Comparison of amino a c i d (AA) a v a i l a b i l i t y from d i e t s of d i f f e r e n t p r o t e i n l e v e l (PL) i n the growing c h i c k (Experiment V, Part 2a).. 97 27 Comparison of amino a c i d (AA) a v a i l a b i l i t y from three d i f f e r e n t endogenous amino a c i d c o r r e c t i o n method (ECM) i n the growing c h i c k (Experiment V, Part 2a) 98 28 I n t e r a c t i o n s of p r o t e i n source (PS) x p r o t e i n l e v e l (PL) i n amino a c i d a v a i l a b i l i t y (Experiment V, Part 2a) 99 X LIST OF TABLES Table Page 29 Comparison of amino a c i d (AA) a v a i l a b i l i t y from d i e t s of d i f f e r e n t p r o t e i n l e v e l (PL) i n the growing c h i c k (Experiment V, Part 2b).. 101 30 The i n t e r a c t i o n of p r o t e i n source (PS) x p r o t e i n l e v e l (PL) f o r phe n y l a l a n i n e a v a i l a b i l i t y (Experiment V, Part 2b) 102 31 The i n t e r a c t i o n of p r o t e i n source (PS) x p r o t e i n l e v e l (PL) f o r t y r o s i n e a v a i l a b i l i t y (Experiment V, Part 2b) 102 32 Comparison of amino a c i d (AA) a v a i l a b i l i t y from d i e t s of d i f f e r e n t p r o t e i n l e v e l (PL) i n the growing c h i c k (Experiment V, Part 2 c ) . . 103 33 The i n t e r a c t i o n of p r o t e i n source (PS) x p r o t e i n l e v e l (PL) f o r methionine a v a i l a b i l i t y (Experiment V, Part 2c) 104 34 Comparison of amino a c i d (AA) a v a i l a b i l i t y from d i e t s of d i f f e r e n t p r o t e i n l e v e l (PL) i n the growing ch i c k (Experiment V, Part 2d).. 105 35 The i n t e r a c t i o n of p r o t e i n source (PS) x p r o t e i n l e v e l (PL) f o r c y s t i n e a v a i l a b i l i t y (Experiment V, Part 2d) 106 36 The i n t e r a c t i o n of p r o t e i n source (PS) x p r o t e i n l e v e l (PL) f o r phe n y l a l a n i n e a v a i l a b i l i t y (Experiment V, Part 2d) 106 37 The i n t e r a c t i o n of p r o t e i n source (PS) x p r o t e i n l e v e l (PL) f o r t y r o s i n e a v a i l a b i l i t y (Experiment V, Part 2d) 107 38 Comparison of amino a c i d (AA) a v a i l a b i l i t y from d i e t s of d i f f e r e n t p r o t e i n l e v e l (PL) i n the growing c h i c k (Experiment V, Part 2e).. 108 39 Comparison of amino a c i d (AA) a v a i l a b i l i t y from d i e t s of d i f f e r e n t p r o t e i n l e v e l (PL) i n the growing c h i c k (Experiment V, Part 2 f ) . . 109 X I LIST OF TABLES Table Page 40 41 Comparison of amino a c i d (AA) a v a i l a b i l i t y from d i e t s of d i f f e r e n t p r o t e i n l e v e l (PL) i n the growing c h i c k (Experiment V, Part 2g) Comparison of the mean amino a c i d (AA) e x c r e t i o n s of b i r d s fed the N-free d i e t i n Experiment IV, V, and VI 110 112 42 The e f f e c t s of p r o t e i n source (PS) and endogenous amino a c i d (AA) c o r r e c t i o n method on amino a c i d a v a i l a b i l i t y i n the growing c h i c k (Experiment VI) , 114 x i i LIST OF APPENDIX TABLES Table Page 1 A n a l y s i s of v a r i a n c e f o r growth parameters i n Experiment 1 138 2 A n a l y s i s of v a r i a n c e f o r growth parameters i n Experiment I I 138 3 A n a l y s i s of va r i a n c e f o r growth parameters i n Experiment I I I 138 4 A n a l y s i s of v a r i a n c e f o r amino a c i d a v a i l a b i l i t y i n Experiment IV 139 5 A n a l y s i s of va r i a n c e f o r the r e l a t i o n s h i p between dry matter (DM) in t a k e of the N-free d i e t and f e c a l DM output i n Experiment V, Part 1 140 6 A n a l y s i s of va r i a n c e f o r the r e l a t i o n s h i p between dry matter (DM) in t a k e of the N-free d i e t and f e c a l N output i n Experiment V, Part 1 140 7 A n a l y s i s of vari a n c e of the r e l a t i o n s h i p s hetween amino a c i d e x c r e t i o n (Ymg) and a l p h a -c e l l u l o s e i n t a k e (Xg) from a N-free d i e t i n Experiment V, Part 1 141 8 A n a l y s i s of v a r i a n c e f o r amino a c i d (AA) a v a i l a b i l i t y i n Experiment V, Part 2a 142 9 A n a l y s i s of va r i a n c e f o r amino a c i d (AA) a v a i l a b i l i t y i n Experiment V, Part 2b 143 10 A n a l y s i s of va r i a n c e f o r amino a c i d (AA) a v a i l a b i l i t y i n Experiment V, Part 2c 144 11 A n a l y s i s of v a r i a n c e f o r amino a c i d (AA) a v a i l a b i l i t y i n Experiment V, Part 2d 145 12 A n a l y s i s of va r i a n c e f o r amino a c i d (AA) a v a i l a b i l i t y i n Experiment V, Part 2e 146 13 A n a l y s i s of v a r i a n c e f o r amino a c i d (AA) a v a i l a b i l i t y i n Experiment V, Part 2f 147 x i i i LIST OF APPENDIX TABLES Table Page 14 A n a l y s i s of va r i a n c e f o r amino a c i d (AA) a v a i l a b i l i t y i n Experiment V, Part 2g 148 15 A n a l y s i s of va r i a n c e f o r amino a c i d (AA) a v a i l a b i l i t y i n Experiment VI 149 LIST OF ABBREVIATIONS AA(s) - Amino a c i d ( s ) AAA - Amino a c i d a v a i l a b i l i t y ADF - Acid detergent f i b r e AEC - Average endogenous c o r r e c t i o n method BM - Blood meal BV - B i o l o g i c a l value Ca - Calcium CP - Crude p r o t e i n DM - Dry matter ECM - Endogenous amino a c i d c o r r e c t i o n method FCE - Feed conve r s i o n e f f i c i e n c y FDNB - l - f l u o r o - 2 , 4-dinitrobenzene GIT - G a s t r o - i n t e s t i n a l t r a c t HFM - Hydrolyzed f e a t h e r meal IEC - I n d i v i d u a l endogenous c o r r e c t i o n method IVPD - I_n v i t r o pepsin d i g e s t i b i l i t y LEC - Long term endogenous c o r r e c t i o n method ME - M e t a b o l i z a b l e energy MEn - Nitrogen c o r r e c t e d m e t a b o l i z a b l e energy MM - Meat and bone meal NFE - N i t r o g e n - f r e e e x t r a c t NPU - Net p r o t e i n u t i l i z a t i o n X V LIST OF ABBREVIATIONS P - Phosphorus PER - P r o t e i n e f f i c i e n c y r a t i o PL - P r o t e i n l e v e l s PS - P r o t e i n sources REC - Regression endogenous c o r r e c t i o n method SAA(s) - S u l f u r amino a c i d ( s ) SBM - Heat-treated soybean meal SCWL - S i n g l e Comb White Leghorn SEM - Standard e r r o r of the mean TAAE - Metabol i c and endogenous amino a c i d s TAAF - T o t a l f e c a l amino a c i d s excreted from b i r d s fed the feed i n g r e d i e n t s TAAI - T o t a l amino a c i d s i n t a k e from feed i n g r e d i e n t s TD - True d i g e s t i b i l i t y of p r o t e i n TNBS - 2 , 4 , 6 - t r i n i t r o b e n z e n e s u l f o n i c a c i d U-N - U r i n a r y n i t r o g e n UNHg - Ur i n a r y ammonia UUA - U r i n a r y u r i c a c i d 1 INTRODUCTION Hydrolyzed f e a t h e r meal and blood meal are slaughterhouse by-p r o d u c t s f r o m t h e meat p r o c e s s i n g i n d u s t r y . With the v a s t expansion i n the p r o c e s s i n g i n d u s t r y , the a c c u m u l a t i o n of t h e s e p r o d u c t s has imposed a p o l l u t i o n problem to the e n v i r o n m e n t . These by-products, because of h i g h p r o t e i n c o n t e n t s , have a l s o been r e c o g n i z e d as p o t e n t i a l s u b s t i t u t e s or supplements f o r the i n c r e a s i n g l y expensive t r a d i t i o n a l feed p r o t e i n . The l i m i t a t i o n s i n the use of t h e s e p r o d u c t s l i e i n the v a r i a b i l i t y of t h e i r n u t r i t i o n a l q u a l i t y . Samples of hydrolyzed f e a t h e r meal and blood meal were s u p p l i e d by a commercial ren d e r i n g company to e s t a b l i s h the n u t r i t i v e values of these products. The t o t a l f e c a l c o l l e c t i o n method f o r the c a l i b r a t i o n of amino a c i d a v a i l a b i l i t y has been e s t a b l i s h e d f o r q u i t e some time by a g r o u p of s c i e n t i s t s i n A r k a n s a s . The method f o r the c o r r e c t i o n of endogenous amino a c i d e x c r e t i o n s was m o d i f i e d and the e f f e c t of p r o t e i n source and p r o t e i n c o n c e n t r a t i o n on amino a c i d a v a i l a b i l i t y was a l s o examined i n the present i n v e s t i g a t i o n . The o b j e c t i v e of t h i s r e s e a r c h was to p r o v i d e a d d i t i o n a l i n f o r m a t i o n r e g a r d i n g the e v a l u a t i o n of h y d r o l y z e d f e a t h e r meal and b l o o d meal. I t i s an attempt to c o n t r i b u t e d a t a f o r the d e v e l o p m e n t o f a u n i v e r s a l method f o r t h e e s t i m a t i o n o f endogenous amino a c i d e x c r e t i o n s i n the c a l i b r a t i o n of amino a c i d a v a i l a b i l i t y i n the growing c h i c k . 2 LITERATURE REVIEW A. U t i l i z a t i o n of hydrolyzed f e a t h e r meal (HFM) i n p o u l t r y  f e e d i n g 1. I n t r o d u c t i o n P o u l t r y f e a t h e r s , a by-product of the p o u l t r y processing p l a n t , have imposed a p o l l u t i o n problem on the environment. An annual production of 50,000 to 60,000 tons of dry f e a t h e r s was reported i n the 1950's (Wilder et a l . , 1955; L i l l i e et <al. , 1956; S u l l i v a n and Stephenson, 1957). With the vast expansion of the b r o i l e r i n d u s t r y , d i s p o s a l problems a s s o c i a t e d with an accumula-t i o n of t h i s waste m a t e r i a l have l e d many r e s e a r c h e r s to i n q u i r e i n t o the p o s s i b l e uses f o r such waste (Wilder e_t a_l. , 1955; L i l l i e et_ a l . , 1956; Harms and Goff, 1957; Wisman et. a l . , 1958). P o u l t r y f e a t h e r s , c o n s i s t i n g p r i m a r i l y of the s c l e r o -p r o t e i n groups, represent a l a r g e p o t e n t i a l source of p r o t e i n f o r the animal feed i n d u s t r y . Although unprocessed k e r a t i n s are i n d i g e s t i b l e because of the c y s t i n e - d i s u l f i d e bonds, once hydrolyzed, the remaining p r o t e i n f r a c t i o n becomes s u s c e p t i b l e to a t t a c k by p r o t e o l y t i c enzymes (Davis et^ a_l. , 1961). A u t o c l a v i n g seems to be the method mostly used f o r the h y d r o l y s i s of the d i s u l f i d e s i n commercial production of f e a t h e r meal; however, e s c a l a t i n g c o s t s have generated i n t e r e s t i n developing a l t e r n a t i v e methods that w i l l reduce a u t o c l a v i n g requirements. Chicken f e a t h e r s have a l s o been t r e a t e d ^ with v a r i o u s 3 c o n c e n t r a t i o n s of NaOH or HgPO^ and then a u t o c l a v e d . S i g n i f i c a n t improvements i n iii v i t r o pepsin d i g e s t i b i l i t y (IVPD) of fea t h e r p r o t e i n were observed as p r o c e s s i n g time was i n c r e a s e d (P<0.05) and as NaOH (P<0.005) or H 3 P O 4 (P<0.05) c o n c e n t r a t i o n was i n -creased ( S t e i n e r e_t a_l. , 1983). However, the n u t r i t i v e value of fea t h e r meal i s g r e a t l y a f f e c t e d by treatment c o n d i t i o n s such as pressure, temperature, chemicals used and d u r a t i o n of the t r e a t -ment ( S u l l i v a n and Stephenson, 1957; McKerns and R i t t e r s p o r n , 1958; Naber e_t a_l. , 1961; Moran, J r . et a l . , 1966; Eggum, 1970; Mo r r i s and B a l l o u n , 1973a; Latshaw and B i g g e r t , 1983; Papado-poulos ejt a_l. , 1985). The e f f e c t of pr o c e s s i n g on the n u t r i t i v e value of fe a t h e r meal w i l l be d i s c u s s e d i n a l a t e r s e c t i o n . 2. N u t r i e n t composition of hydrolyzed f e a t h e r meal The chemical composition of HFM has been repo r t e d by d i f f e r e n t r e s e a r c h e r s (Gregory e_t a_l. , 1956; Naber and Morgan, 1956; S i b b a l d et^ al_. , 1962; Moran, J r . et al. , 1966; M o r r i s and Ba l l o u n , 1973b; Burgos et a l . , 1974; Matsuda and Shiroma, 1974; MacAlpine and Payne, 1977; NRC, 1977; Johnston and Coon, 1979; B i e l o r a i iet a_l. , 1982; S t e i n e r et^ al.. , 1983; Papadopoulos e_t a l . , 1985). The summarized data (Table 1) r e v e a l the proximate composition to be: moisture, 1.6 to 13.1%; crude p r o t e i n (CP), 74.6 to 90.8%; ether e x t r a c t , 1.2 to 12.7%; ash, 1.0 to 5.0%. Naber and Morgan (1956) repo r t e d that f e a t h e r meal co n t a i n s a t r a c e amount of f i b e r and the n i t r o g e n - f r e e e x t r a c t (NFE) was between 0.3 to 0.4%. B i e l o r a i et al_. (1982) found the calcium 4 Table 1. The chemical composition of hydrolyzed f e a t h e r meal N u t r i e n t (%) Reference number Crude p r o t e i n (Nx6.25) L i p i d (Ether e x t r a c t ) Ash Moisture l a 88.2 2.5 2.1 6.8 2l 85.9 4.0 3.7 6.1 3t 78.5 11.3 4.7 3.5 4 K 74.6 12.7 5.0 5.7 5l 87.4 1.2 2.7 6.7 6 b 80.5 5.1 2.5 7.9 7 C 84.7 2.3 4.0 — 8 C 83.4 1.4 3.6 — 9 c 83.1 2.5 4.2 -10 c 82.9 1.2 3.8 -83.1 2.4 3.6 — 1 2 d 75.8 1.9 1.0 — 13 d 85.4 3.6 1.4 — 14. 78.1 3.0 1.5 — 15 d 84.4 4.5 1.2 — 16 e 80.2 1.7 — 13.1 17 e 90.8 5.2 — 1.6 18 e 86.2 2.2 — 8.2 1 9 e 87.4 3.1 — 6.4 20* 83.9 3.0 — 8.0 21 f 86.4 3.3 - 7.0 Range 74.6-90.8 1 .2-12.7 1.0-5.0 1.6-13.1 Mean+S.D. 83.4+4.2 3 .7+3.0 3.0+1.3 6.8+2.8 Data are rounded o f f to the f i r s t decimal p l a c e . aNaber and Morgan (1956). b B i e l o r a i et a l . (1982). c M o r r i s and B a l l o u n (1973b). aMatsuda and Shiroma (1974). e J o h n s t o n and Coon (1979). fNRC (1977). 5 (Ca) and phosphorus (P) content of four samples of HFM to be 0.2 to 1.3% and 0.1 to 0.6%, r e s p e c t i v e l y . T h i s f i n d i n g was a l s o supported by the r e s u l t s of MacAlpine and Payne (1977), which showed the content of Ca and P i n HFM to be 0.26 and 0.21%, r e s p e c t i v e l y . In a d d i t i o n , S i b b a l d e_t al_. (1962) showed that HFM c o n t a i n s about lOOppm of z i n c and has a gross energy of 5220kcal/kg. The d e r i v e d m e t a b o l i z a b l e energy (ME) of HFM reported by S i b b a l d and S l i n g e r (1962) was lOOOkcal/kg which i s much lower than the value of 3001kcal/kg noted i n Moran, J r . _e_t a l . (1966). On the other hand, the n i t r o g e n - c o r r e c t e d m e t a b o l i z a b l e energy (MEn) values were 3000+411 and 3200+394kcal/kg i n samples of HFM processed at 7.03kg/cm f o r three minutes and 2.81kg/cm f o r 50 minutes, r e s p e c t i v e l y ( B i e l o r a i e_t a_l. , 1982). These MEn values are much higher than the value 2360kcal/kg s t a t e d i n NRC (1977). A n a l y z i n g the vitamin contents of f i v e samples of HFM, Gregory e_t a_l. (1956) i n d i c a t e d that the ranges (means) of r i b o -f l a v i n , n i a c i n , pantothenic a c i d and vitamin B-^ were 1.50 to 2.95 (1.94mg/kg), 13.49 to 22.99 (17.3lmg/kg), 6.11 to 12.61 (8.11mg/kg) and 46.08 to 102.08 (70.99ug/kg), r e s p e c t i v e l y . The v a r i a t i o n s i n chemical composition of HFM might be p a r t i a l l y due to the d i f f e r e n t p r o c e s s i n g methods. Using d i f f e r -ent combinations of steam pressures, a u t o c l a v i n g d u r a t i o n s and a g i t a t i o n methods, M o r r i s and B a l l o u n (1973b) s t a t e d that HFM processed f o r 60 minutes had a marked decrease i n f a t content when compared with those processed f o r 30 minutes. The crude 6 p r o t e i n content was s l i g h t l y decreased by i n c r e a s e d time and pressure of the p r o c e s s i n g . B i e l o r a i jet al_. (1982) a l s o reported that the p r o t e i n contents of two samples of HFM processed at 2.81kg/cm f o r 50 minutes (74.6 and 78.5%) were much lower than that processed f o r 20 minutes (87.4%). However, the f a t content of the l a t t e r sample was much lower (1.2%) than those of the previous two (11.3 and 12.7%). The ash contents were a l s o higher i n those processed f o r 50 minutes (4.7 and 5.0%) than that f o r 20 minutes (2.7) at the same steam pressure s e t t i n g ( B i e l o r a i j3_t a_l. ,1982). The discrepancy might be a t t r i b u t e d to the d i f f e r e n c e i n the f e a t h e r p r o t e i n sources r a t h e r than the p r o c e s s i n g c o n d i t i o n s , s i n c e those samples of HFM with lower amount of p r o t e i n and higher percentages of f a t were processed from slaughterhouse wastes and the HFM with 87.4% CP was obtained from a f e a t h e r meal f a c t o r y . Contamination of f e a t h e r p r o t e i n with p o u l t r y by-products could i n c r e a s e the f a t content of the HFM. Under l a b o r a t o r y c o n d i t i o n s , f e a t h e r p r o t e i n was steam processed at 1.06kg/cm f o r up to 16 hours a f t e r treatment with d i f f e r e n t c o n c e n t r a t i o n s of NaOH and H^PO^. S t e i n e r jet a l . (1983) showed that the p r o t e i n l e v e l s decreased (P<0.01) as the c o n c e n t r a t i o n of NaOH or H3PO4 i n c r e a s e d and the p r o t e i n l e v e l s were p r o p o r t i o n a l to the c o n c e n t r a t i o n s of the chemical added. However, i n c r e a s e d time of p r o c e s s i n g d i d not a f f e c t the p r o t e i n l e v e l . Papadopoulos ejt al_. (1985) a l s o noted an i n c r e a s e i n the ash content i n HFM t r e a t e d with 0.4% NaOH which was due to Na r e t a i n e d i n the samples. The l e v e l s of moisture and p r o t e i n i n 7 HFM (4.63 and 85.21%) prepared under the batch process were higher than those prepared under the continuous process (2.23 and 81.64%) as repo r t e d by Burgos e_t a_l. (1974). The amino a c i d (AA) composition of HFM has been determined by d i f f e r e n t r e s e a r c h e r s (Eggum, 1970; Wessels, 1972; M o r r i s and B a l l o u n , 1973b; Burgos e_t al_. , 1974; MacAlpine and Payne, 1977; NRC, 1977; Kirby et_ a l . , 1978; Wheeler and Latshaw, 1980; Baker e_t al_. , 1981; B i e l o r a i et. al.. , 1982; Papadopoulos et a l . , 1985). The data (Table 2) show that HFM i s r i c h i n c y s t i n e (3.00 to 6.43g/16g N), threonine (2.50 to 5.33g/16g N), and a r g i n i n e (4.33 to 8.08g/16g N). S i m i l a r f i n d i n g s were a l s o r e p o r t e d i n Eggum (1970), who suggested that f e a t h e r meal would be u t i l i z e d i n the f e e d i n g i n d u s t r y i f the p r o t e i n and the AAs contained t h e r e i n could be made b i o l o g i c a l l y a v a i l a b l e . On the other hand, HFM i s low i n l y s i n e (1.67 to 2.33g/16g N), methionine (0.35 to 0.88g/16g N), and tryptophan (0.58 to 0.70g/16g N), and p o s s i b l y h i s t i d i n e (0.39 to 3.89g/l6g N). The wide range i n the AA composition of HFM may be a t t r i b u t e d to the amount of blood contamination and i s v a r i a b l e a c c o r d i n g to the p r o c e s s i n g p l a n t s or where i t i s produced (Wessels, 1972). C y s t i n e content of HFM was decreased with i n c r e a s e d p r o c e s s i n g time and pre s s u r e . However, no in c r e a s e d d e s t r u c t i o n of methionine and l y s i n e occurred i n HFM processed under more strenuous c o n d i t i o n s ( M o r r i s and B a l l o u n , 1973b). The l a t t e r authors a l s o added that HFM hydrolyzed at 3.52kg/cm f o r 60 minutes with i n t e r m i t t e n t a g i t a t i o n , contained the g r e a t e s t 8 Table 2. The amino a c i d (AA) composition of hydrolyzed f e a t h e r meal Reference l a 2 b 3 C 4 d 5 e 6 f 7 8 AA g/16g N — CP 1(Nx6.25), % 83. 13 81, .64 85, .42 97. .50 86. .80 87. .40 86, .40 Al a n i n e 2. 64 5. .82 4, .36 5. .30 4. .97 4. .90 A r g i n i n e 4. 33 8, .08 6. .72 6. .48 6. .89 7. .20 6. .27 A s p a r t i c a c i d 3. 41 7. .04 5. .47 6. ,61 7. .04 6. .40 C y s t i n e 6. 43 3. .67 4. .09 3. .73 4. ,83 3. .00 4. .63 Glutamic a c i d 6. 85 12. .68 11. .54 12. .09 10. .84 10. .20 G l y c i n e 4. 21 8. .39 8. .01 7. .62 8. ,20 7. .90 7, .30 H i s t i d i n e Trace 0. .67 3. .89 0. .68 0. .70 0. .70 0. .39 I s o l e u c i n e 2. 96 5. .21 5. .25 5. .18 5. .00 5. .00 3. .77 Leucine 5. 40 8. ,88 8. ,29 8. ,42 8. ,73 8. . 10 7. .78 L y s i n e 2. 28 2. .33 2. .00 1. ,98 1. .67 1. .90 1. .93 Methionine 0. 35 0. .49 0. .88 0. ,66 0. .52 0. .70 0. .49 Ph e n y l a l a n i n e 3. 15 5. .02 4. .80 4. ,86 4. .90 5. .10 3. .77 P r o l i n e 6. 76 10. .84 10. .75 10. .70 10. .23 10. ,90 Ser i n e 5. 67 11. .33 9. .23 11. .08 12. .33 11. .50 10. .72 Threonine 2. 50 5, .33 4, .62 4. .73 4. .90 4. ,60 3. .97 Tryptophan 0. .70 0. .58 T y r o s i n e 1. 85 2. .94 3. .17 2. ,75 2. .85 2. .70 7. ,30 V a l i n e 4. 01 7, .10 7. .17 7. .52 7. ,93 7. .70 6. .45 CP = crude p r o t e i n . aWessels (1972). DBurgos ej: al_. (1974). cMacAlpine and Payne (1977). d K i r b y et a l . (1978). eBaker et a l . (1981). f B i e l o r a i et a l . (1982). &NRC (1977). 9 amount of l y s i n e , methionine, and h i s t i d i n e ; and had the g r e a t e s t amount of t o t a l f r e e AAs and a v a i l a b l e l y s i n e . Eggum (1970) rep o r t e d that the c y s t i n e content of raw f e a t h e r p r o t e i n (9.02g/16g N) was reduced to l e v e l s between 4.48 to 6.57g/16g N a f t e r d i f f e r e n t p r o c e s s i n g procedures. T h i s r e d u c t i o n was probably a t t r i b u t e d to the c o n v e r s i o n of c y s t i n e to l a n t h i o n i n e . A s i m i l a r f i n d i n g was r e p o r t e d by Papadopoulos et^ a_l. (1985), who showed a decrease i n c y s t i n e and an i n c r e a s e i n l a n t h i o n i n e content i n HFM with i n c r e a s e d p r o c e s s i n g time. Moreover, the l a n t h i o n i n e content of HFM v a r i e d between 1.96 to 3.31% depending on the p r o c e s s i n g c o n d i t i o n s . A study on the u t i l i z a t i o n of l a n t h i o n i n e by c h i c k s with p u r i f i e d c r y s t a l l i n e AA d i e t s (Robbins et a l • , 1980) i n d i c a t e d that the c y s t e i n e moiety of l a n t h i o n i n e i s 32% a v a i l a b l e when fed as the racemic mixture and 52% a v a i l a b l e when fed as the L-DL-isomeric mixture. The s u l f u r amino a c i d (SAA) content of HFM i s u s u a l l y determined a f t e r h y d r o l y s i s at 110°C with 6M HC1 a f t e r performic a c i d o x i d a t i o n . The o x i d i z e d products were c y s t e i c a c i d , methionine s u l f o n e and l a n t h i o n i n e s u l f o n e . Wheeler and Latshaw (1980) found that heating of l a n t h i o n i n e s u l f o n e (SAA standards) i n d i s t i l l e d water at 110°C caused a 95% c o n v e r s i o n of l a n t h i o n i n e s u l f o n e to c y s t e i c a c i d . T h i s might i n t e r f e r e with c a l i b r a t i o n of the a c t u a l amount of the SAA i n HFM. F i n a l l y , pressure cooking a l s o reduced the l e v e l s of threonine and tryptophan (Eggum, 1970). 10 3. The n u t r i t i v e values of hydrolyzed f e a t h e r meal a) P r a c t i c a l f e e d i n g t r i a l s HFM has been shown to be of value as a p r o t e i n source when used to r e p l a c e l i m i t e d amounts of v a r i o u s p r o t e i n f e e d s t u f f s i n p r a c t i c a l b r o i l e r r a t i o n s (Gerry and Smyth, 1954; Wilder et a l . , 1955; Gerry, 1956; L i l l i e et a l . , 1956; Menge ej: a l . , 1956; Naber and Morgan, 1956; Wisman et a l . , 1958; Lewis et_ a l . , 1959; Stephens et a l . , 1959; S i b b a l d e^ a l . , 1962; Tsang et a l . , 1963; Moran, J r . j 3 t aJL. , 1966; M o r r i s and B a l l o u n , 1973a; Matsuda and Shiroma, 1974; Shiroma and Hongo, 1974a and 1974b; MacAlpine and Payne, 1977; Hongo and Shiroma, 1978; Baker e_t a l . , 1981; K o c i et a l . , 1983; S t o j a n o v i c et a l . , 1983). In a l l t e s t s with White Rock b r o i l e r c h i c k s , Gerry and Smyth (1954) repo r t e d that HFM, when used to r e p l a c e up to 10% and 5% of soybean o i l meal and f i s h meal, r e s p e c t i v e l y , had l i t t l e or no d i f f e r e n c e on feed c o n v e r s i o n e f f i c i e n c y (FCE). L i l l i e e_t a_l. (1956) a l s o found no d i f f e r e n c e i n growth response and FCE i n New Hampshire c h i c k s when 5% of f i s h meal was r e p l a c e d by HFM i n d i e t s fed up to ten weeks of age. Moreover, Wisman jet a l . (1958) re p o r t e d that HFM, p o u l t r y by-products meal and p o u l t r y blood meal were s a t i s f a c t o r y sources of animal p r o t e i n i n b r o i l e r r a t i o n s , when used to r e p l a c e up to approximately one-s i x t h of the p r o t e i n content. No a d d i t i o n a l supplementation with AA was employed i n any of the d i e t s . The above f i n d i n g s f a l l i n l i n e with the l i t e r a t u r e of Naber and Morgan (1956), which showed that HFM was capable of r e p l a c i n g one-fourth of the p r o t e i n i n 11 b r o i l e r r a t i o n s c o n t a i n i n g l a r g e amounts of soybean o i l meal and corn f o r t i f i e d with f i s h meal, d r i e d whey product, methionine, m i n e r a l s , v itamins and a n t i b i o t i c s . A l s o , HFM, in c l u d e d to f u r n i s h 5% p r o t e i n i n b r o i l e r r a t i o n s , appears to co n t a i n s u f f i c i e n t v i t a m i n B-^ f o r maximum growth. In c o n t r a s t , Wilder js_t aJL. (1955) r e p o r t e d that supple-mentary l y s i n e might be needed when HFM i s used. E x c e l l e n t chick growth was obtained when HFM was used to supply 2.4% p r o t e i n along with an equal amount of p r o t e i n d e r i v e d from meat and bone scrap or blood meal (BM) and the remaining p r o t e i n from soybean o i l meal, a l f a l f a meal and corn. Supplementation of methionine, l y s i n e and tryptophan i n b r o i l e r r a t i o n when f e a t h e r meal p r o t e i n r e p l a c e d f i s h meal p r o t e i n was reported by Gerry (1956). How-ever, the l e v e l s of replacement and method of p r o c e s s i n g of the f e a t h e r meal p r o t e i n was not reported i n the above study. Sub-s t i t u t i o n of 5% p r o t e i n i n a p r a c t i c a l 20% p r o t e i n corn-soybean r a t i o n with HFM r e s u l t e d i n e q u a l l y good c h i c k performance, whereas, replacement of a l l of the soybean p r o t e i n with corn and HFM s e v e r e l y depressed three weeks chi c k weights (Moran, J r . jet a_l.,1966). However, supplementation with l y s i n e , methionine, and tryptophan completely overcame the d e p r e s s i o n . D i f f e r e n t responses were reported by MacAlpine and Payne (1977), who showed that the maximum r a t e of i n c l u s i o n of HFM i n d i e t s f o r c h i c k s reared i n f l o o r pens and cages, from day 0 to day 35, were 2.5% and 6%, r e s p e c t i v e l y , even with supplementary l y s i n e and methionine. Reduction i n feed consumption was probably 12 the cause, s i n c e there was no s i g n i f i c a n t (P>0.05) d i f f e r e n c e i n FCE. Using 9-day o l d male c h i c k s (New Hampshire x Columbia), Baker e_t a l . (1981) repo r t e d that at l e a s t 10% of the d i e t a r y CP (24%) could be provided as HFM during the 13-day growth t r i a l . With methionine and l y s i n e supplementation i n another t r i a l using 8-day o l d c o c k e r e l s , Baker eJL aJL. (1981) showed that up to 40% of the d i e t a r y p r o t e i n could be s u p p l i e d by HFM with l i t t l e d e p r e ssion i n the c h i c k growth and FCE during the 9-day t r i a l . In another study performed by Lewis et. al_. (1959), methionine supplementation d i d not s i g n i f i c a n t l y i n c r e a s e the growth of males fed the f e a t h e r meal ba s a l d i e t i n one t r i a l , whereas, a d d i t i o n of c y s t i n e to the f e a t h e r meal ba s a l d i e t s i g -n i f i c a n t l y improved the e i g h t weeks weights of males i n another t r i a l . T h i s might be a t t r i b u t e d to a d i f f e r e n c e i n the composition of the f e a t h e r meal basal d i e t s used i n the two d i f f e r e n t t r i a l s . U n f o r t u n a t e l y , the composition of the f e a t h e r meal basal d i e t s was not given i n the r e p o r t . Koci jet j§_l. (1983) repo r t e d that when h a l f or a l l of the animal p r o t e i n i n feed was r e p l a c e d by HFM alone, there was a s i g n i f i c a n t decrease i n r a t e of gain up to 21 days, which p e r s i s t e d f o r up to 53 days. But a 2:1 mixture of HFM and BM, or beef greases, could r e p l a c e h a l f of the animal p r o t e i n i n s t a r t e r d i e t s f o r b r o i l e r s , and a l l of the animal p r o t e i n i n f i n i s h i n g d i e t s , provided that the d i e t a r y requirements of methionine, l y s i n e , Ca, P, and energy are met. S t o j a n o v i c et_ al_. (1983) a l s o pointed out that a 16.75% and 8.75% decrease i n body weight gain 13 and FCE, r e s p e c t i v e l y , when 5% of f i s h meal i n a standard b r o i l e r d i e t was r e p l a c e d by 4% HFM and 1% ground maize. These adverse e f f e c t s were a t t r i b u t e d to a d e f i c i e n c y of l y s i n e and methionine i n the t e s t d i e t s . However, a mixture of 4% HFM and 1% d r i e d blood gave the same FCE as the f i s h meal standard d i e t , but decreased body weight gain by 4.27%. With the supplementation of a low p r o t e i n (15%) c o n t r o l d i e t using d i f f e r e n t l e v e l s of soybean o i l meal, HFM or meat meal, S i b b a l d et^ al_. (1962) repo r t e d that HFM was not capable of r e p l a c i n g e i t h e r soybean o i l meal or meat meal i n low p r o t e i n (15.6%) c h i c k s t a r t e r d i e t s , but was s u p e r i o r to e i t h e r soybean o i l meal or meat meal as supplements to a b a s a l d i e t c o n t a i n i n g 22% p r o t e i n . Consequently, S i b b a l d ejt al_. (1962) came to the c o n c l u s i o n that HFM i s a reasonably good source of " n o n - s p e c i f i c " n i t r o g e n but a poor source of e s s e n t i a l AAs f o r the c h i c k . S i m i l a r trends were repo r t e d i n the study of Tsang e_t a_l. (1963), where HFM was i n c o r p o r a t e d i n a s e r i e s of i s o n i t r o g e n o u s and i s o c a l o r i c b r o i l e r r a t i o n s . The replacement r a t i o was 40 p a r t s of HFM and 35 p a r t s of corn to 70 p a r t s of d e h u l l e d soybean meal and f i v e p a r t s of s t a b i l i z e d animal grease and the C a l o r i e : p r o t e i n r a t i o was kept at 45:1 and 48:1. With the above mani p u l a t i o n s , HFM can be used i n b r o i l e r r a t i o n s c o n t a i n i n g 20% p r o t e i n at l e v e l s up to 4% of the d i e t , and i n r a t i o n s c o n t a i n i n g 22 to 26% p r o t e i n at l e v e l s up to 8% of the d i e t (Tsang ejt al_. , 1963) . 14 A s e r i e s of experiments was c a r r i e d out to evaluate the use of HFM f o r b r o i l e r c h i c k s at the U n i v e r s i t y of Ryukyus, Okinawa. Matsuda and Shiroma (1974) suggested HFM could be used at l e s s than 5% of the d i e t or below 20% of the t o t a l p r o t e i n i n the s t a r t e r d i e t , whereas, i n the f i n i s h e r d i e t , 5% of the d i e t or 25% of the t o t a l p r o t e i n as HFM could be i n c o r p o r a t e d . Moreover, the o l d e r c h i c k s seemed to make b e t t e r use of the f e a t h e r meal p r o t e i n as compared with the c h i c k s of one to four weeks of age. In s p i t e of the r e s u l t s that b i r d s on the r a t i o n with HFM had s i g n i f i c a n t l y lower (P<0.05) d r e s s i n g percentage (69.38%) than those on the r a t i o n with f i s h meal, Shiroma and Hongo (1974a) re p o r t e d that a d d i t i o n of HFM at the r a t e of 2 to 5% i n the b r o i l e r r a t i o n was f e a s i b l e . The examination of body chemical composition by Shiroma and Hongo (1974b) showed no s i g n -i f i c a n t d i f f e r e n c e i n the amount of any proximate n u t r i e n t s i n breast or t h i g h muscles of b i r d s fed f i s h meal or HFM as the p r o t e i n source. Examination of the l i p i d components of the b r o i l e r meat from the same groups of b i r d s (Shiroma and Hongo, 1974b; Hongo and Shiroma, 1978) found that t o t a l l i p i d l e v e l of f r e s h samples and the amount of t r i g l y c e r i d e i n the t o t a l l i p i d were the h i g h e s t i n t h i g h muscle obtained from b r o i l e r chicken fed r a t i o n s high i n energy content and f i s h meal. An u n i d e n t i f i e d growth f a c t o r i n HFM which might be i n -o r g a n i c i n nature, was proposed by L i l l i e et_ al_. (1956). This f i n d i n g was supported i n the l i t e r a t u r e of Menge e_t a_l. (1956), 15 which r e p o r t e d that 4% HFM ashed at 700°C f o r f i v e or more hours was as e f f e c t i v e i n promoting growth of c h i c k s as 4% of the o r i g i n a l m a t e r i a l . In a d d i t i o n , Stephens e_t a_l. (1959) observed that there was s i g n i f i c a n t improvement (P<0.05) i n the growth r a t e of Vantress x N i c h o l s ' 108 c h i c k s i n the four weeks ba t t e r y t r i a l when ash of 14.5% HFM was f u r n i s h e d to a basal d i e t c o n t a i n i n g 6.2% f i s h meal. An attempt was made by S i b b a l d ejt a l • (1962) to confirm that z i n c was the i n o r g a n i c growth f a c t o r i n HFM, s i n c e HFM contained about lOOppm of t h i s m i n e r a l . N e v e r t h e l e s s , there was a s m a l l , but not s i g n i f i c a n t , i n c r e a s e i n c h i c k weight gain a t t r i b u t e d to the z i n c supplementation. The d i f f e r e n c e i n growth response of f e e d i n g HFM to b r o i l e r c h i c k s might be a t t r i b u t e d by the d i f f e r e n c e i n chemical composition of HFM used. A l s o , the e f f e c t s of d i f f e r e n t p r o c e s s -ing methods on the n u t r i t i v e value of HFM should be c o n s i d e r e d . The age of b i r d s could a l s o i n f l u e n c e the u t i l i z a t i o n of HFM. M o r r i s and B a l l o u n (1973a) repo r t e d that b r o i l e r c h i c k s a f t e r four weeks of age made more e f f i c i e n t use of the HFM d i e t . MacAlpine and Payne (1977) a l s o showed that growth depression was apparent when HFM were i n c l u d e d at the r a t e of 2.5% i n the s t a r t e r but no d e p r e s s i o n occurred at 6% i n the f i n i s h e r phase. L i t t l e r e s e a r c h has been repo r t e d on the f e e d i n g value of HFM i n l a y e r r a t i o n s (Gerry and Smyth, 1954; Harms and Goff, 1957; Wisman et al_. , 1958; Moran, J r . ejt aJL. , 1969; Luong and Payne, 1977). A study with White Rock l a y i n g hens (Gerry and 16 Smyth, 1954), repo r t e d that r e p l a c i n g 5 and 3.75% of soybean o i l meal and animal p r o t e i n supplement, r e s p e c t i v e l y , by HFM had no e f f e c t on egg production and feed u t i l i z a t i o n . T h i s f i n d i n g agreed with that of Wisman e_t a_l. (1958), which s t a t e d that i n -c l u s i o n of 3% of HFM i n the t o t a l d i e t at the expense of meat scrap and part of soybean o i l meal had no s i g n i f i c a n t e f f e c t (P>0.05) on r a t e of l a y i n S i n g l e Comb White Leghorn (SCWL) or New Hampshire p u l l e t s . A l s o , Harms and Goff (1957) re p o r t e d that the a d d i t i o n of 5% HFM i n a l a y e r r a t i o n r e s u l t e d i n s i g n i f i c a n t -l y (P<0.05) i n c r e a s e d egg p r o d u c t i o n , when compared to hens r e c e i v i n g the 5% meat scraps, i n a c o r n - s o y b e a n - a l f a l f a meal type d i e t . The l a t t e r workers a l s o r e p o r t e d that the h a t c h a b i l i t y of eggs from hens r e c e i v i n g HFM was s i g n i f i c a n t l y (P<0.01) higher at the end of the seventh month i n p r o d u c t i o n . In c o n t r a s t , Moran, J r . et_ a_l. (1969) showed that supplemental methionine was necessary f o r maximal pr o d u c t i o n and egg weight i n hens fed 5% HFM i n a 10% p r o t e i n b a s a l d i e t . A l s o , using HFM (70g/kg i n a wheat based d i e t ) as an AA source f o r l a y i n g hens, Luong and Payne (1977) noted that supplementation with l y s i n e (0.3%), DL-methionine (0.07%) and tryptophan (0.05%) was needed to maintain d a i l y egg mass output s i m i l a r to that achieved with a c o n v e n t i o n a l l a y e r s ' d i e t . The d i f f e r e n c e i n e f f e c t s of HFM i n l a y e r d i e t s noted i n the above l i t e r a t u r e might be a t t r i b u t e d to the d i f f e r e n c e i n the composition of HFM and/or b a s a l r a t i o n s used. 17 b) Other animal bioassays Other than using growth performances of b i r d s fed p r a c t i c a l r a t i o n s to evaluate the n u t r i t i v e values of HFM, d i f f e r e n t r e s e a r c h e r s have proposed a l t e r n a t e Jji v i v o procedures (McCasland and Richardson, 1966; Moran, J r . ejt aJL. , 1966; Smith, 1968; Eggum, 1970; Wessels, 1972; Burgos et a l . , 1974; Kirby et a l . , 1978; Johnston and Coon, 1979; Kim ejt a l . , 1980; Baker ejt a l . , 1981; B i e l o r a i ejt al_. , 1983; E l Boushy and Roodbeen, 1984; Nordheim and Coon, 1984; Papadopoulos ejt a_l. , 1985). Using HFM as the s o l e source of p r o t e i n i n a semi-p u r i f i e d d i e t , Moran, J r . ejt al_. (1966) found methionine, l y s i n e , h i s t i d i n e , and tryptophan to be the f i r s t through f o u r t h l i m i t i n g AAs. A s i m i l a r procedure was performed by Baker ejt a_l. (1981), who agreed that methionine and l y s i n e were f i r s t - and second-l i m i t i n g AAs, r e s p e c t i v e l y , but h i s t i d i n e and tryptophan were e q u a l l y t h i r d l i m i t i n g . When n i t r o g e n r e t e n t i o n by chickens was used as c r i t e r i o n of the response to evaluate HFM, Wessels (1972) found methionine, l y s i n e , h i s t i d i n e , and t y r o s i n e to be the f i r s t through f o u r t h l i m i t i n g AAs. On the other hand, McCasland and Richardson (1966) claimed that the l i m i t i n g AAs i n HFM were only methionine, l y s i n e , and h i s t i d i n e . Using a c h i c k bioassay with a c r y s t a l l i n e AA standard r e f e r e n c e d i e t , Smith (1968) found the a v a i l a b i l i t y of methionine, l y s i n e , h i s t i d i n e , and tryptophan to be 74.6, 5.3, 0.0, and 45.5%, r e s p e c t i v e l y . The p a r t i c u l a r l y low a v a i l a b i l i t y 18 of h i s t i d i n e and l y s i n e might account f o r the low n u t r i t i v e value of HFM. D i f f e r e n t r e s u l t s were obtained by Nordheim and Coon (1984) using a c h i c k bioassay and d i g e s t i b l e l y s i n e techniques. The authors found that the a v a i l a b i l i t y of l y s i n e from HFM a c c o r d i n g to c h i c k bioassay and d i g e s t i b l e l y s i n e ranged from 64 to 75% and 77 to 89%, r e s p e c t i v e l y . The b i o a v a i l a b l e c y s t i n e c o n c e n t r a t i o n s i n HFM were 2.92 and 2.89% using standard curve method and s l o p e - r a t i o assessment, r e s p e c t i v e l y (Baker ejt a_l. , 1981), and the b i o a v a i l a b i l i t y of the SAA was 52%. Moreover, the a v a i l a b l e SAA content ( p r i m a r i l y c y s t i n e ) of f i v e commercial HFM was s t u d i e d using the growth of b r o i l e r c h i c k s from 11 to 21 days of age as the c r i t e r i o n (Kim e_t a l . , 1980). The authors showed that the a v a i l a b l e SAA content of HFM v a r i e d from 0.92 to 4.01% on a dry matter b a s i s , and the range of a v a i l a b i l i t y of the SAA was from 41 to 82%. Kim ejt a l . (1980) a l s o mentioned that chemical analyses of methionine plus c y s t i n e was a good i n d i c a t o r of the a v a i l a b l e SAA content ( r = 0.996), but crude p r o t e i n or true ME value had no s i g n i f i c a n t c o r r e l a t i o n with the a v a i l a b l e SAA content. Amino a c i d a v a i l a b i l i t y (AAA) s t u d i e s with HFM have been performed by d i f f e r e n t workers (Burgos e_t al_. , 1974; Kirby e_t a l . , 1978; E l Boushy and Roodbeen, 1984; Papadopoulos et a l . , 1985). The mean AAA f o r HFM i n the study of Burgos eit al. (1974) was 97%, and the range f o r i n d i v i d u a l AAs was 95 to 99%. S l i g h t l y lower values have been rep o r t e d by K i r b y et a l . (1978), 19 who found the mean AAA i n HFM to be 94%, ranging between 88 and 97%. In c o n t r a s t , E l Boushy and Roodbeen (1984) showed lower a v a i l a b i l i t y of AAs i n HFM (68%,average) with i n d i v i d u a l AA ranging from 56 to 78%. These f i n d i n g s were supported by Papadopoulos et_ al_. (1985), who reported the AAA i n HFM to range from 47 to 84% f o r i n d i v i d u a l AAs, with a mean value of 69%. The apparent amino a c i d d i g e s t i b i l i t y i n HFM was determined i n the lower ileum of c h i c k s by B i e l o r a i ejt a l . (1983), who found that the average a b s o r p t i o n values f o r HFM was approximately 50%, ranging from 20 to 70%. Higher mean value of 63% with i n d i v i d u a l AA v a r i e d between 41 and 80% were obtained by Papadopoulos e_t a_l. (1985). The p r o t e i n q u a l i t y of s i x samples of HFM has a l s o been evaluated by Johnston and Coon (1979) using c h i c k p r o t e i n e f f i c i e n c y r a t i o (PER) and net p r o t e i n u t i l i z a t i o n (NPU). The c o e f f i c i e n t s of v a r i a t i o n f o r HFM PER's and NPU's were 69 and 22.5%, r e s p e c t i v e l y , which were much l a r g e r than one would expect. The authors found that these v a r i a t i o n s were caused by depressed a p p e t i t e as a r e s u l t of the AA imbalance and suggested that the PER value f o r HFM was not only a r e f l e c t i o n of the r e l a t i v e p r o t e i n q u a l i t y , but a l s o of the gross AA d e f i c i e n c e s . The n u t r i t i v e value of HFM have a l s o been examined by Eggum (1970) using the true d i g e s t i b i l i t y of p r o t e i n (TD), b i o l o g i c a l value (BV), NPU, and AAA of r a t s . The TD, BV, and NPU of HFM ranged from 30.4 to 83.8%, 50 to 83%, and 33.1 to 71.3%, 20 r e s p e c t i v e l y . AAA corresponded mainly to TD, however, the c o r r e l a t i o n s f o r TD versus BV and NPU were poor, which might be due to the lower content of c y s t i n e , methionine and/or tryptophan caused by the more i n t e n s e pressure-cooking and/or the i n c r e a s i n g a c i d i t y and time of h y d r o l y s i s (Eggum, 1970). The d i f f e r e n c e i n f i n d i n g s of d i f f e r e n t r e s e a r c h e r s might be a t t r i b u t e d to the sources of HFM used and/or the methods of p r o c e s s i n g of HFM. c) E n z y m a t i c a l , m i c r o b i o l o g i c a l , and chemical assays The method of i n v i t r o pepsin d i g e s t i b i l i t y (IVPD) have been used by d i f f e r e n t r e s e a r c h e r s to evaluate the n u t r i t i v e values of HFM (Naber jet. al_. , 1961; McCasland and Richardson, 1966; M o r r i s and B a l l o u n , 1973b; Johnston and Coon, 1979; Kim e_t a l . , 1980; B i e l o r a i jejt a l . , 1982; Latshaw and B i g g e r t , 1983; S t e i n e r et a l . , 1983; Nordheim and Coon, 1984). Naber jet al_. (1961) r e p o r t e d that the IVPD of HFM (64 to 83%) d i d not appear to be r e l a t e d to c h i c k growth when fed to f u r n i s h 5% p r o t e i n . M o r r i s and B a l l o u n (1973b) a l s o showed that the range of IVPD of HFM (71.8 to 74.6%), processed under d i f f e r e n t c o n d i t i o n s , d i d not d i f f e r g r e a t l y enough to r e s u l t i n v a l i d c o n c l u s i o n s on t h i s b a s i s . On the other hand, McCasland and Richardson (1966) found good agreement between IVPD (87.1%) and i j i v i v o d i g e s t i o n (82.1%) of HFM by r a t s . The d i f f e r e n c e i n the f i n d i n g s might be a t t r i b u t e d to d i f f e r e n c e s i n the c o n c e n t r a t i o n s of pepsin and procedures used 21 i n the assaying of IVPD. Using four l e v e l s of pepsin (0.2, 0.02, 0.002, a n d 0.0002%), B i e l o r a i et a l . (1982) showed that the d i g e s t i o n c o e f f i c i e n t s of HFM with 0.002% pepsin (44 to 59%) were c l o s e s t to the value f o r i l e a l n i t r o g e n a b s o r p t i o n (55%). Using the same l e v e l of enzyme a c t i v i t y (0.002%), Johnston and Coon (1979) r e p o r t e d that IVPD (12.2 to 34.9%) of HFM c o r r e l a t e d with PER (r = -0.696). S t e i n e r ejt aJL. (1983) a l s o found a p o s i t i v e c o r r e l a t i o n of 0.72 (P<0.05) between IVPD and i n v i t r o rumen dry matter d i g e s t i b i l i t y . Moreover, the a v a i l a b l e SAA content was i n v e r s e l y p r o p o r t i o n a l to IVPD (Kim ejt ajl. , 1980; Latshaw and B i g g e r t , 1983). Other than IVPD, McCasland and Richardson (1966) showed that the ijn v i t r o d i g e s t i o n of HFM with bromelain and "Rhozyme A-4" were 73.3 and 23%, r e s p e c t i v e l y , which were much lower than the value of 87.1% with p e p s i n . M i c r o b i o l o g i c a l e v a l u a t i o n of the p r o t e i n q u a l i t y of HFM using Tetrahymena f u r g a s o n i have been per-formed by Johnston and Coon (1979), who showed that the m i c r o b i o -l o g i c a l assay was not a s a t i s f a c t o r y replacement f o r the c h i c k bioassay due to the t e d i o u s procedure needed f o r c o n s i s t e n t r e s u l t s . Dye b i n d i n g c a p a c i t y of HFM d i d not serve as an adequate i n d i c a t o r of p r o t e i n q u a l i t y and the chemical scores d i d not provide a true e v a l u a t i o n of the p r o t e i n q u a l i t y of HFM due to d i f f e r e n c e s i n amino a c i d a v a i l a b i l i t y . The a v a i l a b l e l y s i n e content of HFM was determined by Nordheim and Coon (1984) with 2 , 4 , 6 - t r i n i t r o b e n z e n e s u l f o n i c a c i d 22 (TNBS) and 1 - f l u o r o - 2 , 4 - d i n i t r o b e n z e n e (FDNB) chemical pro-cedures. The mean a v a i l a b l e l y s i n e content (mean percent l y s i n e a v a i l a b i l i t y ) determined by the TNBS and FDNB procedures were 1.93% (92%) and 2.38% (114%), r e s p e c t i v e l y , f o r HFM. Lower values of 1.28% f o r a v a i l a b l e l y s i n e and 72.5% a v a i l a b i l i t y was r e p o r t e d by M o r r i s and B a l l o u n (1973b) using the TNBS chemical procedure. The v a r i a t i o n s i n a v a i l a b l e l y s i n e might r e f l e c t the p r o c e s s i n g c o n d i t i o n s of the HFM. A l s o , HFM have been repo r t e d by Moran, J r . ejt al_. (1966) as having extremely imbalanced AA contents and i n f e r i o r p r o t e i n d i g e s t i b i l i t y , which suggested that chemical methods might be o v e r r a t i n g the a v a i l a b l e l y s i n e content of HFM. 4. E f f e c t s of p r o c e s s i n g methods on the n u t r i t i v e values of hydrolyzed f e a t h e r meal A s e r i e s of experiments was performed by d i f f e r e n t r e s e a r c h e r s to determine the e f f e c t s of p r o c e s s i n g methods of HFM on c h i c k performance ( S u l l i v a n and Stephenson, 1957; McKerns and R i t t e r s p o r n , 1958; Naber ejt a_l. , 1961; Moran, J r . et a l . , 1966; M o r r i s and B a l l o u n , 1973a, Latshaw and B i g g e r t , 1983). S u l l i v a n and Stephenson (1957) r e p o r t e d that the d i g e s t i b i l i t y of f e a t h e r p r o t e i n was g r e a t l y i n c r e a s e d by steam pressure treatment as mild as 1.06kg/cm f o r 20 minutes, and the optimum c h i c k performance was obtained when f e a t h e r p r o t e i n was autoclaved at 2.46kg/cm f o r 60 minutes. A s i m i l a r f i n d i n g was 23 reported i n the l i t e r a t u r e of McKerns and R i t t e r s p o r n (1958), which s t a t e d that f e a t h e r p r o t e i n autoclaved at 3.52kg/cm f o r 60 minutes could s u b s t i t u t e e f f e c t i v e l y f o r 50% of the soybean o i l meal, or at a l e v e l e q u i v a l e n t to 25% of the t o t a l CP i n a commercial b r o i l e r r a t i o n . M o r r i s and B a l l o u n (1973a) a l s o noted that HFM, processed at 3.52kg/cm steam pressure f o r 60 minutes with i n t e r m i t t e n t a g i t a t i o n , produced the best c h i c k responses. On the other hand, Moran, J r . e_t al_. (1966) repo r t e d that e x t e n s i v e a u t o c l a v i n g treatment of f e a t h e r p r o t e i n (1.06kg/cm^ f o r 18 hours) was needed to support reasonably good c h i c k growth. S i g n i f i c a n t v a r i a t i o n s i n the n u t r i t i v e values of HFM by d i f f e r e n t commercial p r o c e s s i n g methods had a l s o been noted by Naber jet a_l. (1961). F u r t h e r treatments of HFM with v a r i o u s l e v e l s and types of d i s u l f i d e reducing agents (sodium t h i o g l y c o l a t e and sodium s u l f i t e ) did not improve chick performance as compared to the untreated HFM (Moran, J r . e_t a l . , 1966). D i f f e r e n t combinations of pH s e t t i n g s (5, 7, and 9) and cooking pressures (2.11, 2.81, and 3.52kg/cm ) were used by Latshaw and B i g g e r t (1983) to evaluate the u t i l i z a t i o n of SAAs from b r o i l e r f e a t h e r s . Cooking f e a t h e r p r o t e i n at pH 5 or 7 produced b e t t e r c h i c k growth than that at pH 9. The mean AAA were 96.8% and 95.4% f o r batch and continuous p r o c e s s i n g of HFM, r e s p e c t i v e l y , i n d i c a t e d that both methods produced high q u a l i t y HFM (Burgos jet al. , 1974). Combining v a r i o u s p r o c e s s i n g times with/without a d d i t i o n s of 0.4% 24 NaOH or p r o t e o l y t i c enzyme, Papadopoulos jejt a_l. (1985) found that the values i n AAA of HFM ranged from 36.3% f o r a s p a r t i c a c i d to 86.5% f o r i s o l e u c i n e . The l i m i t i n g e s s e n t i a l amino a c i d s , l y s i n e , h i s t i d i n e , and methionine, i n i n c r e a s i n g order, were p a r t i c u l a r l y low i n d i g e s t i b i l i t y . Moreover, the AAA of HFM were a f f e c t e d most s i g n i f i c a n t l y by the p r o c e s s i n g time and i t was concluded that HFM p r o t e i n should be used i n p o u l t r y d i e t s on the b a s i s of the a v a i l a b l e amino a c i d s because of the l a r g e v a r i a t i o n s i n b i o l o g i c a l a v a i l a b i l i t y among the i n d i v i d u a l amino a c i d s (Papadopoulos ejt .al. , 1985) . The e f f e c t s of p r o c e s s i n g on the p r o t e i n values of HFM, expressed as TD, BV, and NPU of r a t s , have been s t u d i e d by Eggum (1970). A u t o c l a v i n g of raw f e a t h e r s at a pressure of 3.10kg/cm^ f o r 75 minutes was s a i d to be i n s u f f i c i e n t as r e f l e c t e d by the low TD and NPU values of 53% and 33.1%, r e s p e c t i v e l y . B o i l i n g of raw f e a t h e r s under r e f l u x f o r 20 hours at pH 4 and 6 were needed to o b t a i n TD values of 73.1% and 71.7%, r e s p e c t i v e l y , however, these treatments were judged to be too severe a c c o r d i n g to BV and NPU. Pressure-cooking of raw f e a t h e r s at 3.31kg/cm during a p e r i o d of 45 minutes and reducing the pressure to normal during a s i m i l a r time was needed to produce the best p r o t e i n q u a l i t y (TD = 83.8%, BV = 77.7%, and NPU = 71.3%) of HFM. Eggum (1970) concluded that p r o c e s s i n g with a c i d alone d i d not have the same s u c e s s f u l e f f e c t on the p r o t e i n of f e a t h e r meal as d i d proper p r e s s u r e - c o o k i n g . 25 HFM processed at 2.81kg/cm f o r 50 minutes r e s u l t e d i n higher value of IVPD (59.2%) than the value of 46.5% i n sample t r e a t e d at 7.03kg/cm f o r three minutes ( B i e l o r a i et a l . , 1982). Regression a n a l y s i s of the NaOH- and HgPO^-treated HFM data i n d i c a t e d that IVPD i n c r e a s e d (P<0.05) as p r o c e s s i n g time i n c r e a s e d and as NaOH (P<0.005) or H 3 P O 4 (P<0.05) c o n c e n t r a t i o n i n c r e a s e d ( S t e i n e r et a l . , 1983). A l s o , low c o n c e n t r a t i o n s of NaOH improved IVPD gre a t e r than d i d HgPO^. S i m i l a r l y , Latshaw and B i g g e r t (1983) showed that b r o i l e r f e a t h e r s processed at pH 9 produced higher values of IVPD than those processed at pH 5 or 7, and the IVPD values were i n v e r s e l y p r o p o r t i o n a l to the a v a i l a b i l i t y of SAA. B. U t i l i z a t i o n of blood meal i n p o u l t r y f e e d i n g 1. I n t r o d u c t i o n Dried blood has been used f o r animal feeds and adhesives f o r many years. Since the replacement of d r i e d blood by m a t e r i a l s that were more water r e p e l l e n t i n the adhesive market, l a r g e r amounts of d r i e d blood have been a v a i l a b l e f o r the feed i n g i n d u s t r y (Doty, 1973). Moreover, blood meal (BM), a s l a u g h t e r -house by-product, could impose a p o l l u t i o n s t r e s s to our environments. 2. N u t r i e n t composition of blood meal The chemical composition of BM has been reported by 26 v a r i o u s workers (Wisman e_t al_. , 1958; Abou-El-Hassan ejt a l . , 1970; Abou-Raya ejt al. , 1971; Hew and Devendra, 1977; NRC, 1977). The data (Table 3) r e v e a l e d the proximate composition to be: moisture 6.7 to 26.3%; crude p r o t e i n , 65.3 to 88.9%; ether e x t r a c t , 0.3 to 11.4%; ash, 3.3 to 10.0%. The crude f i b r e and NFE v a r i e d between 0.2 to 4.56% and 0.15 to 1.39%, r e s p e c t i v e l y (Wisman et a_l. , 1958; Abou-El-Hassan ejt al_. , 1970; Abou-Raya ejt a l . , 1971; Fetuga ejt a_l. , 1973). The Ca and P content ranged from 0.06 to 3.5% and 0.09 to 1.1%, r e s p e c t i v e l y (Wisman e_t a l . , 1958; Abou-Raya et a l . , 1971; Fetuga et a l . , 1973; NRC, 1977). Moreover, Abou-Raya ejt .al. (1971) showed that BM contained 5.01% of s i l i c a . The gross energy of BM was between 4360 and 6200kcal/kg (Fetuga ejt a l . , 1973; King and Campbell, 1978; Nelson et a l . , 1980). Moreover, MEn values of BM were re p o r t e d to l i e between 2830 and 3810kcal/kg ( S i b b a l d and S l i n g e r , 1962; NRC, 1977; Nelson et a l . , 1980). The d i f f e r e n c e s i n chemical composition of BM might be due to the methods of p r o c e s s i n g ( c o n v e n t i o n a l , s p r a y - d r i e d and r i n g - d r i e d ) and/or the d i f f e r e n t combinations of blood from c a t t l e , swine and/or p o u l t r y . The AA composition of BM have been repo r t e d by d i f f e r e n t workers ( F i s h e r , 1968; Fetuga ejt a l . , 1973; NRC, 1977; K i r b y ejt a l . , 1978; King and Campbell, 1978). The summarized data (Table 4) showed t h a t BM i s r i c h i n l y s i n e (8.63 to 9.96g/16g N), l e u c i n e (11.59 to 13.50g/16g N), h i s t i d i n e (5.39 to 6.70g/16g N), Table 3. The chemical composition of blood meal N u t r i e n t m 1 Crude Reference p r o t e i n L i p i d Ash Moisture number (Nx6.25) (Ether e x t r a c t ) l a 68.6 1.0 6.1 26.3 65.3 11.4 8.8 6.7 3 K 85.6 0.4 4.3 9.3 4 b 82.2 0.3 3.3 8.8 5CA 70.0 1.3 10.0 13.1 6 d 68.6 1.1 3.4 -7 e 88.9 1.0 - 7.0 Range 65.3-88.9 0.3-11.4 3.3-10.0 6.7-26.3 Mean+_S. D. 75.6+9.6 2.4+4.0 6.0+2.9 11.9+7.4 ^Data are rounded o f f to the f i r s t decimal place • aWisman et a l . (1958) • Abou-El-Hassan et a l . (1970). cAbou-Raya et a l . (1971). dHew and Devendra (1977). eNRC (1977). 28 Table 4. The amino a c i d (AA) composition of blood meal Reference l a 2 b 3 C 4 d 5 e AA g/16g N c p ^ N x e ^ s ) , % 94, .00 80. .95 98, .80 88, .50 88, .90 Al a n i n e 8. .90 7. ,52 8. .25 A r g i n i n e 4. .70 4. .90 3. .87 4, .05 4. .29 A s p a r t i c a c i d 11. .60 11. .26 10. .24 C y s t i n e 1. .30 0. .58 0. .79 1, .39 0. .97 Glutamic a c i d 11. .10 9. ,55 10. .65 G l y c i n e 4. .50 4. .22 4. .29 4. .50 H i s t i d i n e 6, .70 5. .82 6. .33 5, .39 5. .92 I s o l e u c i n e 1, .20 1. ,17 0. ,76 1. .30 0. ,99 Leucine 13. .50 12. .80 13. .01 11. .59 13. ,30 L y s i n e 9. .90 8. ,78 9. .52 8. .63 9. .96 Methionine 1. .20 0. .38 1. .63 1, .47 0. .84 P h e n y l a l a n i n e 9. .20 7. ,20 7. ,43 6. ,68 7. .37 P r o l i n e 4. .40 3. ,86 3. .70 Ser i n e 5. .50 5. .25 5. .36 4. .34 Threonine 5. .00 5. ,01 4. ,80 5. .16 4. ,43 Tryptophan 0. .72 0. ,65 2. .15 1. ,51 T y r o s i n e 4. .60 3. ,26 3. .00 2. .93 2. ,80 V a l i n e 9. .50 8. ,70 8. .17 8. .75 9. ,67 CP = crude p r o t e i n . a F i s h e r (1968). b F e t u g a et a l . (1973). c K i r b y et. a l . (1978) . a K i n g and Campbell (1978). eNRC (1977). 29 and p h e n y l a l a n i n e (6.68 to 9.20g/16g N). BM a l s o contains r e l a t i v e l y adequate balance of other e s s e n t i a l AAs except methionine (0.38 to 1.63g/16g N), c y s t i n e (0.58 to 1.39g/16g N), and i s o l e u c i n e (0.76 to 1.30g/16g N). However, the o v e r a l l AA composition of d i f f e r e n t samples of BM i s s i m i l a r . T h i s f i n d i n g was supported by the l i t e r a t u r e of Doty (1973), which s t a t e d that the o v e r a l l AA composition of blood d r i e d by d i f f e r e n t methods ( c o n v e n t i o n a l , s p r a y - d r i e d , and r i n g - d r i e d ) was very s i m i l a r . 3. The n u t r i t i v e values of blood meal BM has been r e p o r t e d to be a poor source of p r o t e i n f o r p o u l t r y r a t i o n s ( T i t u s e ± a_l. , 1936) ( c i t e d i n Squibb and Braham). Grau and Almquist (1944) showed that the serum and f i b r i n f r a c t i o n s of beef blood were much b e t t e r q u a l i t y than the blood c e l l f r a c t i o n s i n which i s o l e u c i n e was the p r i n c i p a l l i m i t i n g AA. A l s o , a mixture of blood c e l l p r o t e i n and corn g l u t e n meal p r o t e i n i n a r a t i o of 1:2 produced comparative growth r e s u l t s to s a r d i n e meal i n SCWL c h i c k s . Squibb and Braham (1955) found BM to be most e f f e c t i v e source of l y s i n e when fed at 2 to 4% i n c h i c k r a t i o n s . Wisman ejt al_. (1958) showed that p o u l t r y BM could be i n c o r p o r a t e d to provide 3% of the t o t a l 20% p r o t e i n i n c h i c k s t a r t e r r a t i o n s . Mathur and Ahmed (1971) r e p o r t e d that BM at 2.5% l e v e l was a good supplement of l y s i n e i n a groundnut c a k e - f i s h meal ba s a l r a t i o n f o r Arbor Acre Brown c h i c k s . Moreover, supplementing a maize-wheat b r a n - d e c o r t i c a t e d cotton 30 seed meal ba s a l r a t i o n with 1.3, 2, 2% of BM, meat meal and f i s h meal, r e s p e c t i v e l y , Abou-Raya et a l . (1971) found that the 17-week growth r a t e and FCE of c h i c k s fed BM d i e t s was s u p e r i o r to those on other d i e t s . G a l a l e_t a_l. (1977) showed that BM could be i n c o r p o r t e d at up to 6% i n a t y p i c a l Egyptian b r o i l e r d i e t as a source of l y s i n e . Recent s t u d i e s by Omar e_t a_l. (1985) s t a t e d that the 3-week performance of b r o i l e r - t y p e c h i c k s r e c e i v i n g d i e t s c o n t a i n i n g 4% BM was not d i f f e r e n t from those fed a corn-soy b a s a l d i e t . However, s i g n i f i c a n t growth depression was observed with b i r d s fed 8% BM and no improvement was obtained with e i t h e r a r g i n i n e or potassium supplementations. On the other hand, e v a l u a t i n g BM as the s o l e p r o t e i n source i n d i e t s f o r Columbian x New Hampshire c h i c k s , F i s h e r (1968) found that i s o l e u c i n e , methionine, and a r g i n i n e were the l i m i t i n g amino a c i d s . S t u d i e s with White Leghorn p u l l e t s , Fernandez jajt a l . (1973) showed that hens r e c e i v i n g d i e t s c o n t a i n i n g over 80% t r i t i c a l e and 3.4% BM had pr o d u c t i o n parameters that were f u l l y equal to those hens r e c e i v i n g more c o n v e n t i o n a l d i e t s . Using BM as a source of p r o t e i n i n turkey s t a r t e r d i e t s , Lockhart e_t a l . (1960) showed that n e i t h e r v a t - d r i e d nor f r e e z e - d r i e d BM supported body weight gain equal to that of soybean p r o t e i n , and t h i s d e l e t e r i o u s e f f e c t was not a f f e c t e d by methionine, i s o l e u c i n e , and l y s i n e supplementation, On the other hand, feed i n t a k e was not depressed at 5% l e v e l of e i t h e r v a t - d r i e d or 31 f r e e z e - d r i e d BM. S i m i l a r l y , Novacek and C a r l s o n (1968) found that the growth r a t e of turkeys fed on a d i e t c o n t a i n i n g 13.5% BM was only 10 to 20% of those fed the f i s h meal or soybean meal b a s a l d i e t s . E s t i m a t i o n of NPU i n the mixtures of f r e s h blood and CaO compared with d r i e d skim milk have been performed by Abou-El-Hassan e_t aJL. (1970), who showed that the mixture of blood to CaO at a l e v e l of 8:1 produced the h i g h e s t NPU (84.9%) fo l l o w e d by skim milk (66%). The mean a v a i l a b i l i t y of 17 amino a c i d s i n BM was 89% with ranges from 66% f o r i s o l e u c i n e and 95% f o r h i s t i d i n e . The a v a i l a b i l i t i e s f o r l y s i n e , methionine, and a r g i n i n e were 94, 92, and 92%, r e s p e c t i v e l y ( K i r b y e_t a_l. , 1978). The e f f e c t of p r o c e s s i n g methods on the a v a i l a b l e l y s i n e content of v a t - d r i e d and s p r a y - d r i e d BM have been examined by K r a t z e r and Green (1957). The p r o t e i n content of s p r a y - d r i e d BM was higher than that of v a t - d r i e d BM. L y s i n e a v a i l a b i l i t y f o r s p r a y - d r i e d and v a t - d r i e d samples ranged from 68 to 85% and 49 to 66%, r e s p e c t i v e l y , as determined by both the c h i c k and p o u l t assays. A l s o , s p r a y - d r i e d and v a t - d r i e d BM were found to c o n t a i n approximately 10 to 12% and 6 to 8% l y s i n e , r e s p e c t i v e l y , by c h i c k and p o u l t assay ( K r a t z e r and Green, 1957). Doty (1973) a l s o s t a t e d that the a v a i l a b l e l y s i n e , as determined by turkey bioassay, d i f f e r e d g r e a t l y i n blood that has been s p r a y - d r i e d or r i n g - d r i e d as compared with blood d r i e d by c o n v e n t i o n a l methods of d r y i n g . The e f f e c t of p r o c e s s i n g c o n d i t i o n s on a v a i l a b l e 32 l y s i n e content of BM have a l s o been s t u d i e d by Hamm and Searcy (1976). The c h e m i c a l l y a v a i l a b l e l y s i n e content of p o u l t r y blood was decreased by i n c r e a s i n g temperature and l e n g t h of drying time. A more up-to-date study on the a v a i l a b l e l y s i n e content of d i f f e r e n t l y processed BM has been conducted by N o l l e_t a l . (1984). V a t - d r i e d BM, r e p r e s e n t i n g harsh p r o c e s s i n g c o n d i t i o n s , contained s i g n i f i c a n t l y l e s s t o t a l l y s i n e , FDNB-lysine, and b i o a v a i l a b l e l y s i n e when compared to spray, DeLaval-Anderson, r i n g and f l a s h - d r i e d BM. V a t - d r i e d BM contained 8.74g/16g N and 5.17g/16g N of t o t a l and b i o a v a i l a b l e l y s i n e , r e s p e c t i v e l y . On the other hand, r i n g - d r i e d BM contained 10.13g/16g N and 7.76g/16g N of t o t a l and b i o a v a i l a b l e l y s i n e , r e s p e c t i v e l y . C. E s t i m a t i o n by f e c a l analyses of the a v a i l a b i l i t y of amino  a c i d s i n f e e d s t u f f s 1. I n t r o d u c t i o n The content of AAs i n p r o t e i n s are of fundamental importance i n the e v a l u a t i o n of the n u t r i t i o n a l value of f e e d s t u f f s . On the other hand, the AAs i n c e r t a i n feed i n g r e d i e n t s are poorly u t i l i z e d because of the presence of compounds which reduce a v a i l a b i l i t y ( F l i p o t e_t a_l. , 1971; Nelson et a l . , 1975), the type of p r o t e i n (Naber e_t al_. , 1961; Moran, J r . e_t al_. , 1966; K i r b y ejt al_. , 1978) or damage to p r o t e i n during p r o c e s s i n g (Soares, J r . e_t a_l. , 1971; M o r r i s and B a l l o u n , 1973b). 33 Since not a l l of each AA i n a p r o t e i n becomes a v a i l a b l e to the animal during the course of d i g e s t i o n , a b s o r p t i o n and metabolism, the AA contents of i n d i v i d u a l f e e d s t u f f s determined by physio-chemical methods of analyses are of l i m i t e d v a l u e . Both Iii v i t r o and i j i v i v o methods have been devised by d i f f e r e n t r e s e a r c h e r s to p r e d i c t the a v a i l a b i l i t y of i n d i v i d u a l AAs present i n f e e d s t u f f s . Thorough reviews of these procedures were given i n McNab (1979), and Papadopoulos (1985). In the f o l l o w i n g l i t e r a t u r e review, procedures r e l a t e d to f e c a l analyses w i l l only be d i s c u s s e d . Kuiken and Lyman (1948) ( c i t e d i n Papadopoulous, 1985) f i r s t e s t a b l i s h e d a method to measure the d i g e s t i b i l i t y of i n d i v i d u a l AA based on f e c a l a n a l y s i s , using r a t s as t e s t animals. L a t e r , a t o t a l f e c a l c o l l e c t i o n method based on the measurement of the a c t u a l disappearance of AA from the gut of growing c h i c k s was developed by Bragg e_t aJL. (1969). The l a t t e r method provided an accurate assessment f o r the a v a i l -a b i l i t y of 17 d i e t a r y AAs, as i n d i c a t e d by i t s a p p l i c a t i o n on a v a r i e t y of p l a n t and animal p r o t e i n sources (Ivy ejt aJL. , 1971; Stephenson jajt al_. , 1971; Rostagno et_ aJL. , 1973; Burgos ejt a l . , 1974; Nwokolo ejt a l . , 1976b and 1977; Kirby et_ a l . , 1978; Parsons et a l . , 1981; E l Boushy and Roodbeen, 1984). M o d i f i e d procedures using f o r c e - f e d SCWL r o o s t e r s were a l s o employed ( L i k u s k i and D o r r e l l , 1978; S i b b a l d , 1979b; Engster et a l . , 1985). A l s o , the t o t a l f e c a l c o l l e c t i o n method was modified to estimate the mineral a v a i l a b i l i t y i n f e e d s t u f f s (Nwokolo ejt a_l. , 1976a; Nwokolo and Bragg, 1977; Aw-Yong ejt a l . , 1983). 34 The accuracy of the t o t a l f e c a l c o l l e c t i o n technique f o r AAA of f e e d s t u f f s depends mainly on the c o r r e c t i o n s f o r metabolic plus endogenous amino a c i d s (TAAE) l o s s e s and the q u a n t i t a t i v e c o l l e c t i o n of the t e s t e x c r e t a i n the experimental b i r d s . These parameters w i l l be di s c u s s e d i n the f o l l o w i n g s e c t i o n s . 2. The c o r r e c t i o n f o r the endogenous amino a c i d s l o s s e s The most c r i t i c a l part i n the t o t a l f e c a l c o l l e c t i o n assay i s i n the c o r r e c t i o n f o r AAs of endogenous o r i g i n which appear i n the ex c r e t a with undigested r e s i d u e s . Some r e s e a r c h e r s have u t i l i z e d the ex c r e t a from unfed b i r d s f o r the e s t i m a t i o n of TAAE e x c r e t i o n s ( L i k u s k i and D o r r e l l , 1978; S i b b a l d , 1979b). The mean AA e x c r e t i o n s of f a s t e d SCWL a d u l t r o o s t e r s have been examined by d i f f e r e n t workers ( S i b b a l d , 1979b and 1979d; Muztar and S l i n g e r , 1980; Parsons et_ a_l. , 1983). The summarized data (Table 5) i n d i c a t e that there were v a r i a t i o n s i n the pa t t e r n s of AA e x c r e t i o n s from the f a s t e d r o o s t e r s r e p o r t e d among d i f f e r e n t workers. Moreover, the l e v e l s of AA e x c r e t i o n s of b i r d s i n experiment 2 of Muztar and S l i n g e r (1980) were r e l a t i v e l y higher than those reported i n experiment 1. These o b s e r v a t i o n s suggest that the q u a l i t y and q u a n t i t y of AA excreted by f a s t e d r o o s t e r s are q u i t e v a r i a b l e . The e f f e c t s of d i f f e r e n t d u r a t i o n s of t o t a l f e c a l c o l l e c t i o n p e r i o d and/or age of the b i r d s used should be con s i d e r e d . The c o r r e c t i o n of endogenous amino a c i d s e x c r e t i o n s using f a s t e d b i r d s have a l s o been c i t i c i z e d by Muztar and S l i n g e r 35 Table 5. The mean amino a c i d (AA) e x c r e t i o n s of unfed SCWL ad u l t r o o s t e r s Reference  2 b 3 c 4 d AA mg/bird Alanine 16. 1 17. 3 13. ,7 17. .5 22. 4 A r g i n i n e 17. 1 20. 6 22. ,4 28. ,4 11. 1 A s p a r t i c a c i d 30. 1 30. 2 22, ,8 31. ,4 32. 2 C y s t i n e NM 1 NM 6. .7 6. .8 13. 3 Glutamic a c i d NM 48. 4 41. .7 54. .6 47. 2 G l y c i n e NM NM 140, .8 203. .5 NM H i s t i d i n e 9. 2 12. 0 9. .0 8, .5 NM I s o l e u c i n e 13. 8 13. 0 9. .5 13. .8 14. 0 Leucine 26. 7 25. 5 15. .6 21. .3 22. 7 Ly s i n e 13. 1 21. 4 21. .1 24. .5 25. 0 Methionine NM 5. 0 6, .0 7. .4 6. 0 Phe n y l a l a n i n e 11. 1 12. 2 8. .8 12. .5 11. 8 P r o l i n e 18. 3 24. 4 20. .3 26. ,7 22. 2 Serine 22. 2 26. 6 15. .7 20. ,6 24. 3 Threonine 19. 0 20. 8 13. .6 19. .9 18. 2 T y r o s i n e 12. 2 15. 0 8, .0 9. .2 5. 8 V a l i n e 15. 7 17. 0 13. .3 18. .5 20. 9 iSCWL = S i n g l e Comb White Leghorn. a S i b b a l d (1979b), 24-hour c o l l e c t i o n . b S i b b a l d (1979d), 24-hour c o l l e c t i o n . cMuztar and S l i n g e r (1980), 30-hour c o l l e c t i o n , experiment 1. ^Muztar and S l i n g e r (1980), 30-hour c o l l e c t i o n , experiment 2. e P a r s o n s ejt j§_l. (1983), 48-hour c o l l e c t i o n . NM = not measured. 36 (1980) and Parsons ejt a_l. (1983). Mutzar and S l i n g e r (1980) found that the TAAE of b i r d s fed 20g of c e l l u l o s e was s i g n i f i c a n t l y (P<0.05) higher than those fed a N-free d i e t . Moreover, the TAAE of the l a t t e r groups was higher (<0.01) than those of the unfed groups. S i m i l a r f i n d i n g s was rep o r t e d by Parsons ejt a_l. (1983), who i n d i c a t e d that d i e t a r y carbohydrate s u b s t a n t i a l l y a f f e c t e d the e x c r e t i o n of AA by p o u l t r y . The authors a l s o suggested that f a s t e d b i r d s might not provide an a c c u r a t e estimate of TAAE f o r b i r d s fed high f i b e r f e e d s t u f f s i n AAA t r i a l s . The t o t a l f e c a l c o l l e c t i o n assay of Bragg ejt al_. (1969) i n v o l v e d the use of a N-free d i e t to c o r r e c t f o r the TAAE e x c r e t i o n s . The mean AA e x c r e t i o n s of b i r d s fed d i f f e r e n t compositions of N-free d i e t s have a l s o been examined (Bragg ejt a l . , 1969; Mutzar and S l i n g e r , 1980; Parsons et a l . , 1983). The r e s u l t s (Table 6) show that the AA e x c r e t i o n s of SCWL r o o s t e r s were much higher than those of the 28-day-old b r o i l e r s . In a d d i t i o n , AA contents of the e x c r e t a i n i n t a c t SCWL r o o s t e r s c o l l e c t e d from a 48-hour p e r i o d ( i n Parsons ejt al_. , 1983) were much higher than those c o l l e c t e d from a 30-hour p e r i o d ( i n Muztar and S l i n g e r , 1980). Some f a c t o r s a f f e c t i n g the c o r r e c t i o n of metabolic and endogenous amino a c i d s e x c r e t i o n s i n AAA of f e e d s t u f f s w i l l be presented i n the f o l l o w i n g pages. a) E f f e c t of d i e t a r y carbohydrates The e f f e c t s of d i e t a r y carbohydrates on TAAE e x c r e t i o n s have been examined by d i f f e r e n t workers ( S i b b a l d , 1979b; Muztar 37 Table 6. The mean amino a c i d (AA) e x c r e t i o n s of b i r d s fed N-free d i e t s Reference^ 2 b 3 c 4 d AA mg/bird Al a n i n e 5.0 A.5 25.8 A9.0 20.7 A r g i n i n e 1.8 2.5 13.A 20.2 23.6 A s p a r t i c a c i d A.2 5.8 AO.6 69.5 31.2 C y s t i n e 1.9 1.5 1A.5 < 22.9 6.A Glutamic a c i d 5.5 6.9 51.4 89.2 53.2 G l y c i n e A.5 3.5 NM NM 13A.5 H i s t i d i n e 0.5 1.1 NM NM 8.6 I s o l e u c i n e 1.5 2.2 19.7 32.2 1A.A Leucine 2.A 3.8 3A.6 50.2 22.1 L y s i n e 0.7 2.1 15.A 39.A 22.6 Methionine 0.5 0.9 5.5 11.A 6.8 Phe n y l a l a n i n e 1.9 1.9 19.7 28.A 12.5 P r o l i n e 3.0 3.6 30.2 A3.A 23.5 S e r i n e 3.2 A.O 31.0 A3.8 22.0 Threonine 3.A A . l 31.8 3A.9 19.0 T y r o s i n e l.A 2.2 1A.8 18.0 11.A V a l i n e 1.9 3.7 32.1 A6.7 18.7 *A11 values are rounded o f f to the f i r s t decimal p l a c e . a B r a g g jet aJL. (1969), 28-day-old b r o i l e r s , normal. % d i e t : glucose, 81; c e l l u l o s e , 3; corn o i l , 10; min e r a l and v i t a m i n mix, 6. bBragg ejt a l . (1969), 28-day-old b r o i l e r s , s u r g i c a l l y m o d i fied ( a r t i f i c i a l anus). % d i e t : glucose, 81; c e l l u l o s e , 3; corn o i l , 10; min e r a l and v i t a m i n mix, 6. c P a r s o n s ejt a l . (1983), SCWL r o o s t e r s , A8-hour c o l l e c t i o n , s u r g i c a l l y m o d ified (cecectomy and colostomy). % d i e t : s t a r c h , 50; glucose, A5; c e l l u l o s e , 5. ^Parsons ejt a l . (1983), SCWL r o o s t e r s , A8-hour c o l l e c t i o n , normal. % d i e t : s t a r c h , 50; glucose, A5; c e l l u l o s e , 5. eMuztar and S l i n g e r (1980), SCWL r o o s t e r s , 30-hour c o l l e c t i o n , normal. % d i e t : c e r e l o s e , 90; ground corn cob, 5; animal and vegetable f a t blend, 5. o NM = not measured. 38 and S l i n g e r , 1980; Parsons ejt al_. , 1983; Raharjo and F a r r e l l , 1984). S i b b a l d (1979b), f o r c e - f e e d i n g graded l e v e l s of glucose monohydrate to SCWL r o o s t e r s , found no s i g n i f i c a n t c o r r e l a t i o n s between glucose input and the e x c r e t i o n of 13 AAs. On the other hand, Muztar and S l i n g e r (1980), showed that the e x c r e t i o n of 10 out of the 16 AAs was s i g n i f i c a n t l y (P<0.01) lower f o r the unfed c o n t r o l group compared to those fed the N-free d i e t composed of 90% c e r e l o s e , 5% pure ground corn cob and 5% animal-vegetable f a t blend. S i g n i f i c a n t l y (P<0.05) higher values i n TAAE e x c r e t i o n s were observed i n b i r d s f o r c e - f e d 20g of ground c e l l u l o s e when compared to those fed the N-free d i e t . S i m i l a r f i n d i n g s were repor t e d by Parsons e_t a_l. (1983), who showed that r o o s t e r s fed the h i g h - f i b e r N-free d i e t excreted s u b s t a n t i a l l y more (P<0.05) AAs than did f a s t e d r o o s t e r s or r o o s t e r s fed a l o w - f i b r e N-free d i e t . Moreover, Raharjo and F a r r e l l (1984) found that both N and AA outputs i n c r e a s e d i n i l e a l d i g e s t a and i n e x c r e t a with i n c r e a s e d c o n c e n t r a t i o n s of a c i d d e t e r g e n t - f i b r e (ADF) (15 to 150g/kg) i n the N-free d i e t . The i n c r e a s e i n TAAE e x c r e t i o n s from i n c r e a s e d d i e t a r y f i b r e was probably due to an i n c r e a s e i n the s e c r e t i o n and e x c r e t i o n of endogenous p r o t e i n - r i c h substances. As quoted by McNab and Shannon (1972), f e e d i n g a d i e t composed of 27% c e l l u l o s e contents to b i r d s , B o l t o n (1964) found an i n c r e a s e exceeding 5 0 - f o l d i n the r a t i o of n i t r o g e n to c e l l u l o s e of the nitrogenous m a t e r i a l , 30% was i s o l a t e d mucus. Moreover, Boisen 39 et a l . (1985) re p o r t e d an i n c r e a s e d enzyme s e c r e t i o n , as r e f l e c t e d by an i n c r e a s e i n the weight of pancreas, i n r a t s fed d i e t s c o n t a i n i n g 10% c e l l u l o s e or p e c t i n . The authors proposed that the i n c r e a s e i n the s e c r e t i o n of i n t e s t i n a l enzymes might be a s e c u r i t y mechanism to counteract the reducing e f f e c t s of c e l l u l o s e , and to some degree of p e c t i n , on the enzyme a c t i v i t i e s . On the other hand, Beames and Eggum (1981) repo r t e d that the i n c r e a s e d endogenous n i t r o g e n e x c r e t i o n of r a t s with i n c r e a s i n g d i e t a r y f i b r e was probably due to i n c r e a s e d d i g e s t a passage r a t e and i n c r e a s e d sloughing o f f of c e l l s from the i n t e s t i n a l l i n i n g by the f i b r e . T h i s f i n d i n g was supported by the r e s u l t s of Parsons e_t a_l. (1983), who reported an i n c r e a s e d e x c r e t i o n of glucosamine and hexosamine, the main components of i n t e s t i n a l c e l l mucins and mucoproteins, by r o o s t e r s fed the high f i b r e d i e t s . F i n a l l y , the l a t t e r authors claimed that a s u b s t a n t i a l p r o p o r t i o n of AAs excreted by r o o s t e r s fed the high f i b r e d i e t was a s s o c i a t e d with b a c t e r i a l r e s i d u e s . b) E f f e c t of d i e t a r y p r o t e i n i n t e s t d i e t The t o t a l f e c a l c o l l e c t i o n technique has a l s o been c r i t i c i z e d on the ground that d i e t a r y p r o t e i n i n t e s t i n g r e d i e n t s might i n f l u e n c e the s e c r e t i o n and e x c r e t i o n of endogenous p r o t e i n - r i c h substances, such as d i g e s t i v e j u i c e and enzymes, i n the lumen of the g a s t r o - i n t e s t i n a l t r a c t (GIT) to c a r r y out the d i g e s t i o n process, whereas N-free d i e t f a i l e d to e x h i b i t such an 40 e f f e c t . McNab and Shannon (1972) showed that the n i t r o g e n values of the duodenal contents from 13-week-old p u l l e t s fed the soybean meal and f i s h meal d i e t , i n c r e a s e d 5 and 2 - f o l d , r e s p e c t i v e l y , over t h a t of the food i n g e s t e d . B i r d and Moreau (1 9 7 8 ) , a l s o f o u n d g r e a t e r p r o t e o l y t i c enzyme a c t i v i t i e s ( t r y p s i n and c h y m o t r y p s i n ) i n the p a n c r e a t i c j u i c e of r o o s t e r s f e d a h i g h p r o t e i n (18.5%) c o r n - s o y d i e t than t h o s e f e d the low p r o t e i n (11.5%) r a t i o n s . On t h e o t h e r hand, N a s s e t and J u ( 1 9 6 1 ) reported that a N-free meal produced an AA mixture i n the jejunum which was q u a l i t a t i v e l y i n d i s t i n g u i s h a b l e from t h a t produced by p r o t e i n r i c h f e e d ( c i t e d i n McNab and Shannon, 1972). T h i s o b s e r v a t i o n seems to suggest that the e f f e c t of d i e t a r y p r o t e i n i n t e s t i n g r e d i e n t s c o u l d be s i m u l a t e d by the use of a N-free d i e t . c) E f f e c t of m i c r o f l o r a i n the g a s t r o - i n t e s t i n a l t r a c t of p o u l t r y An average d i r e c t m i c r o s c o p i c count of b a c t e r i a i n dry ex c r e t a from a d u l t SCWL r o o s t e r s of about 8 x 1 0 ^ / g was reported by Parsons ejt aJL. (1982b). T h i s value was s u b s t a n t i a l l y l e s s than 4 x 10^^/g reported f o r dry human feces (Stephen and Cummings, 1980) ( c i t e d i n Parsons ejt a_l. , 1982b). Furthermore, the m i c r o b i a l c o n t r i b u t i o n to AA content of p o u l t r y e x c r e t a was estimated to be 25% (Parsons e_t a_l. , 1982b), which was lower than the value r e p o r t e d f o r m i c r o b i a l c o n t r i b u t i o n to n i t r o g e n content of swine f e c e s . An excess of 50% of the n i t r o g e n i n swine feces was estimated to be of m i c r o b i a l o r i g i n by Mason ejt a_l. (1976) 41 ( c i t e d by Parsons e_t al_. , 1982b). However, the estimate of 25% was suggested to be of s u f f i c i e n t magnitude to r a i s e questions concerning accuracy of the t o t a l f e c a l c o l l e c t i o n method f o r the determination of AAA (Parsons ejt al_. , 1982b). I t has been claimed that the m i c r o f l o r a i n the lower gut might a f f e c t the i n t e r p r e t a t i o n of the data by deaminating undigested AA r e s i d u e s i n t o products having l i t t l e or no n u t r i t i o n a l value, as quoted i n the l i t e r a t u r e of McNab (1979). Parsons e_t a_l. (1982b) i n d i c a t e d that s u b s t a n t i a l metabolism of AAs by the m i c r o f l o r a occurred i n the l a r g e i n t e s t i n e and ceca of p o u l t r y , and that the b a c t e r i a might have s y n t h e s i z e d l a r g e amounts of e x t r a c u l l a r enzymes which were excreted i n f e c e s . Moreover, Mason et a l . (1977) ( c i t e d i n Parsons et a1. , 1982b) rep o r t e d that m i c r o b i a l degradation of the AAs i n the l a r g e i n t e s t i n e of swine r e s u l t e d i n formation of nitrogenous products which were absorbed and excreted i n u r i n e . The above f i n d i n g s seem to suggest that m i c r o b i a l s y n t h e s i s and degradation might change the " t r u e " p a t t e r n of AA excreted by f e e d i n g a t e s t p r o t e i n . T h i s p o s t u l a t i o n however, i s s h a t t e r e d by the f i n d i n g s of S a l t e r and F u l f o r d (1974). The authors, by comparing the AAA i n germ-free and c o n v e n t i o n a l c h i c k s , found that gut m i c r o f l o r a had l i t t l e i n f l u e n c e on the d i g e s t i o n of p r o t e i n but may serve a s i g n i f i c a n t r o l e i n the degradation of endogenous p r o t e i n . Parsons et_ aJL. (1982a) a l s o s t a t e d that the i n t e s t i n a l m i c r o f l o r a appeared to have l e s s e f f e c t on the e x c r e t i o n of AAs by p o u l t r y as compared to r a t s and swine. 42 d) U r i n a r y c o n t r i b u t i o n The u r i n e of the fowl flows from the u r e t e r s i n t o the c l o a c a where i t becomes mixed with f e c e s ; as a r e s u l t , the v a l i d i t y of the e v a l u a t i o n of AAA has been questioned. Techniques f o r the s e p a r a t i o n of u r i n e and f e c e s i n b i r d s has been accomplished by s e v e r a l d i f f e r e n t r e s e a r c h e r s i n hens (Richardson j 3 t a_l. , 1960; Ainsworth, 1965), and growing c h i c k s (Newberne et a l . , 1957; C o l v i n et a l . , 1966; Ivy ejt al. , 1968). Both u r i n e and feces c o n t a i n important amounts of ammonium s a l t s and a small amount of urea. Urine c o n t a i n s , i n a d d i t i o n , small amount of AAs, c r e a t i n i n e , and other N-containing e x c r e t i o n products from ge n e r a l metabolism ( O ' D e l l ejt a_l. , 1960; McNabb and McNabb, 1975; Krogdahl and Dalsgard, 1981). O'Dell et a l . (1960) r e p o r t e d that the u r i n a r y AA n i t r o g e n only made up about 2% of the t o t a l u r i n a r y n i t r o g e n (U-N). E i g h t AAs comprise about three q u a r t e r s of the u r i n a r y AA content. The d i s t r i b u t i o n s of these AAs were g l y c i n e (20.9%), p r o l i n e (11.1%), glutamic a c i d (10.3%), h y d r o x y p r o l i n e (7.9%), a s p a r t i c a c i d (7.4%), l y s i n e (6.5%), o r n i t h i n e (6.1%), and a r g i n i n e ( 2 . 6 % ) . T e r p s t r a (1979) ( c i t e d by Raharjo and F a r r e l l , 1984) a l s o r e p o r t e d a small c o n t r i b u t i o n to e x c r e t a from the AAs i n u r i n e . The author s t a t e d that about 93% of the AAs i n the t o t a l e x c r e t a was c o n t r i b u t e d from f e c e s . D i e t a r y p r o t e i n a l s o i n f l u e n c e d the c o n c e n t r a t i o n s and d i s t r i b u t i o n s of U-N compounds i n the a d u l t r o o s t e r s (Ward ejt 43 a l . , 1975). I n c r e a s i n g the d i e t a r y p r o t e i n l e v e l s from 11 to 33% caused a doubling i n u r i n a r y u r i c a c i d (UUA) c o n c e n t r a t i o n while the c o n c e n t r a t i o n of u r i n a r y ammonia (UNH-j) remained con s t a n t . I f t h i s was the case, the % UUA e x c r e t i o n , r e l a t i v e to the t o t a l U-N output, would i n c r e a s e with i n c r e a s e d d i e t a r y p r o t e i n l e v e l s . T a s a k i and Okumura (1964) a l s o found that the e x c r e t i o n of UUA •4 and UNHg, when expressed as mg N per kg body weight to the power of 0.705, i n c r e a s e d with i n c r e a s e d d i e t a r y c a s e i n from 3 to 12% fed to SCWL r o o s t e r . However, the % UUA and UNHg, r e l a t i v e to the t o t a l U-N, remained at approximately at 77 and 15%, r e s p e c t i v e l y , d i s r e g a r d i n g the p r o t e i n content of the d i e t . The d i f f e r e n c e i n f i n d i n g s might be a t t r i b u t e d to the d i f f e r e n t l e v e l s of d i e t a r y p r o t e i n used and/or to the f a c t that Ward e_t a l . (1975) expressed the U-N compounds i n mg/ml of u r i n e . T a s a k i and Okumura expressed the U-N compounds r e l a t i v e to the metabolic body s i z e of the r o o s t e r s . I t would be of great i n t e r e s t i f the e f f e c t s of d i e t a r y p r o t e i n on u r i n a r y AA e x c r e t i o n were examined. U n f o r t u n a t e l y , the AA contents of the u r i n e samples were not determined i n the previous s t u d i e s ( T a s a k i and Okumura, 1964; Ward e_t al_. , 1975). A study of the d i s t r i b u t i o n of the major nitrogenous compounds and AAs i n chicken u r i n e , O'Dell e_t al.. (1960) showed that the UUA and UNHg made up about 81 and 15%, r e s p e c t i v e l y , of the t o t a l U-N output. T h i s was s i m i l a r to the values r e p o r t e d by Ta s a k i and Okumura (1964). On the other hand, a study with White Leghorn l a y e r s (Krogdahl and Dalsgard, 1981) showed that the U-N 44 d i s t r i b u t i o n was 88, 7, 3, and 2% f o r UUA, UNHg, urea, and u n i d e n t i f i e d m a t e r i a l s , r e s p e c t i v e l y . No s i g n i f i c a n t d i f f e r e n c e s were observed i n the d i s t r i b u t i o n of the AAs found i n h y d r o l y s a t e s of u r i n e from c h i c k s fed a p r a c t i c a l corn-soy d i e t and those fed a p u r i f i e d d i e t c o n t a i n i n g c a s e i n supplemented with methionine and a r g i n i n e ( O ' D e l l et a l . , 1960). I t was demonstrated by Bragg et_ al_. (1969) that the e x c r e t i o n of u r i n e and feces has l i t t l e e f f e c t on the AAA of f e e d s t u f f s . They used both normal and s u r g i c a l l y modified c h i c k s to measure the AAA of g r a i n sorghum ( s e p a r a t i o n of u r i n e and feces by s u r g i c a l m o d i f i c a t i o n to form an a r t i f i c i a l anus). The authors concluded that the method employing the normal c h i c k was simpler and more a c c u r a t e . S i m i l a r c o n c l u s i o n s were repo r t e d by Gruhn (1974) and T e r p s t r a (1977) from s t u d i e s with normal and colostomized hens and cocks, r e s p e c t i v e l y (as quoted by Papadopoulos, 1985). 3. The q u a n t i t a t i v e c o l l e c t i o n of the t e s t e x c r e t a The other c r i t i c a l part i n the t o t a l f e c a l c o l l e c t i o n assay l a y i n the q u a n t i t a t i v e c o l l e c t i o n of the t e s t e x c r e t a from experimental b i r d s . The time r e q u i r e d f o r feed r e s i d u e s to c l e a r the GIT of a d u l t SCWL r o o s t e r s has been i n v e s t i g a t e d e x t e n s i v e l y by S i b b a l d (1979a, 1979c, 1979e, and 1980). F i n e l y ground corn, wheat, oats, and b a r l e y f o r c e - f e d at a l e v e l of 30g c l e a r e d the GIT of ad u l t r o o s t e r s i n 24 hours, whereas, both a l f a l f a meal and HFM r e q u i r e d more than 24 hours to pass through the b i r d s . 45 Parsons ejt al_. (1982a) r e p o r t e d that more than 24 hours were r e q u i r e d f o r the complete i n t e s t i n a l c l e a r a n c e of undigested AA from HFM. Moreover, meat, soybean meal and f i s h meal both r e q u i r e d approximately 30 hours to pass through the GIT of the a d u l t r o o s t e r s ( S i b b a l d , 1979a; K e s s l e r and Thomas, 1981). An extension of the c o l l e c t i o n p e r i o d beyond 30 hours f o r feed which e x h i b i t e d a slower r a t e of passage has a l s o been proposed. The time r e q u i r e d f o r both very f i b r o u s oat and BM to c l e a r the GIT of a d u l t r o o s t e r s was 48 hours ( S i b b a l d , 1980; K e s s l e r and Thomas, 1981). On the other hand, G o l i a n and P o l i n (1984) found that the average t r a n s i t time f o r the f e r r i c oxide marker to pass through the GIT of mixed sex SCWL c h i c k s up to 21 days of age was 167+47 minutes. S i m i l a r l y , Teeter e_t j§_l. (1985) repo r t e d that the i n i t i a l d i g e s t a passage r a t e estimate was 211+6.8 minutes f o r 5-week o l d Arbor Acre x Vantress c h i c k s fed a corn-soybean meal b a s a l d i e t c o n t a i n i n g 1.5% f e r r i c oxide and 1.5% ytterbium-l a b e l e d soybean meal. The p o s s i b i l i t y t h a t f e r r i c oxide may pass through the avian d i g e s t i v e system independent of the p a r t i c u l a t e matter i t i s being used to q u a n t i t a t e was being r a i s e d . Browne (1922) ( c i t e d i n Teeter ej: al_. , 1985) observed that f l u i d s pass more r a p i d l y than s o l i d s through the hen's d i g e s t i v e system. Neverthe-l e s s , the f i n d i n g s of Teeter ejt al_. (1985) showed that f e r r i c oxide flowed with the p a r t i c u l a t e phase, or that the b r o i l e r d i g e s t a l i q u i d and p a r t i c u l a t e passages r a t e s were s i m i l a r . 46 The above f i n d i n g s seem to suggest that there might be an age e f f e c t i n the r a t e of passage of f e e d i n g r e d i e n t s i n b i r d s . The q u a n t i t a t i v e c o l l e c t i o n of the t e s t e x c r e t a might be achieved by the use of f e r r i c oxide as a marker. 4. Drying techniques f o r p o u l t r y e x c r e t a F r e e z e - d r y i n g i s the p r e f e r r e d method f o r the p r e p a r a t i o n of e x c r e t a f o r AA d e t e r m i n a t i o n . However, f r e e z e - d r y i n g equipment i s expensive and not always a v a i l a b l e . A l s o , the time r e q u i r e d to f r e e z e - d r y the same amount of m a t e r i a l i s much longer than oven d r y i n g . T h i s would be a problem w i t h l a r g e e x c r e t a sample s i z e s . The e f f e c t of d r y i n g technique on the s t a b i l i t y of AAs i n e x c r e t a has been examined by W a l l i s and B a l n a v e ( 1 9 8 3 ) . Drying e x c r e t a i n an oven at 60°C f o r 24 hours proved at l e a s t as e f f e c t i v e as f r e e z e d r y i n g f o r the m a j o r i t y of AAs. A s i m i l a r e x p e r i m e n t was c o n d u c t e d by Dale et^ al_. (1985), who showed that the a v a i l a b i l i t y of the t o t a l AAs i n the t e s t i n g r e d i e n t was s i m i l a r l y u n a f f e c t e d by d r y i n g techniques. 47 MATERIALS AND METHODS A. Feed samples and d i e t s f o r m u l a t i o n HFM and BM samples used i n these experiments were s u p p l i e d by Mr. B r i a n Osborne (West Coast Reduction, Vancouver, B.C.). HFM was processed at 2.81kg/cm of steam pressure f o r 40 minutes with continuous a g i t a t i o n . BM was a r i n g - d r i e d sample processed at 190°C f o r f i v e minutes at a steam pressure of 1.41kg/cm . Meat and bone meal (MM) and animal t a l l o w were a l s o obtained from West Coast Reduction. Hea t - t r e a t e d soybean meal (SBM), a l f a l f a meal, wheat, corn, limestone, and calcium phosphate were obtained from Ott e r Farmer Co-op (Langley, B.C.). The composition of calcium phosphate i s 32% and 18% of Ca and P, r e s p e c t i v e l y . A l f a l f a meal was a dehydrated sample with 20% crude p r o t e i n . A l l g r a i n samples were alr e a d y ground to pass through a 2mm scr e e n . D i e t s i n Experiment I, I I , and I I I were formulated to meet the minimum NRC recommendations (1977) f o r b r o i l e r s (0 to 3 weeks). A l l d i e t s were made i s o n i t r o g e n o u s by v a r y i n g the amount of p r o t e i n supplements and g r a i n s . The MEn values used f o r HFM and BM were 2360 and 3420kcal/kg, r e s p e c t i v e l y , based on NRC (1977). The maximum l e v e l of animal t a l l o w i n a l l fee d i n g t r i a l s was maintained at 7%, whereas, a minimum of 6 and 5% was imposed f o r Experiment II and I I I , r e s p e c t i v e l y . A l l d i e t s were fed i n mash form. 4 8 B. General b i r d management and experimental design 1. Feeding t r i a l s Day-old b r o i l e r c h i c k s , purchased from a commercial hatchery, were randomly assigned to the experimental groups ( r e p l i c a t e s ) c o n s i s t i n g of equal number of male and female c h i c k s so as to have the same mean weight. A l l b i r d s were housed i n e l e c t r i c a l l y heated, t h e r m o s t a t i c a l l y c o n t r o l l e d (Petersime) b a t t e r y brooders with r a i s e d wire f l o o r s . Continuous l i g h t i n g was s u p p l i e d by incandescent l i g h t s around the brooders. Feed and water were s u p p l i e d a_d l i b i t u m f o r a l l s t u d i e s . The c h i c k s were weighed weekly throughout each experiment. The d i f f e r e n c e between the i n i t i a l and f i n a l weights of each pen of c h i c k s was taken as the gain i n body weight. The f e e d / g a i n r a t i o was c a l c u l a t e d by d i v i d i n g the feed i n t a k e (g) by the body weight gain ( g ) . A completely randomized design i n v o l v i n g d i f f e r e n t d i e t a r y treatments was employed i n a l l experiments. The d u r a t i o n of the experiment and the average i n i t i a l weight +_ S.D. of the c h i c k s are given i n each experiment. Treatment d i f f e r e n c e s obtained from the a n a l y s i s of v a r i a n c e (Snedecor and Cochran, 1967) were subjected to the Duncan's m u l t i p l e range t e s t (Duncan, 1955). 2. The amino a c i d a v a i l a b i l i t y (AAA) t r i a l s Day-old b r o i l e r c h i c k s purchased from a commercial 49 hatchery were r a i s e d i n e l e c t r i c a l l y heated, t h e r m o s t a t i c a l l y c o n t r o l l e d (Petersime) b a t t e r y brooders with r a i s e d wire f l o o r s . Continuous l i g h t i n g was s u p p l i e d by incandescant l i g h t s around the brooders. The b r o i l e r c h i c k s were fed a commercial ch i c k s t a r t e r (minimum, 20% crude p r o t e i n ) d i e t , purchased from Surrey Co-op. Feed Company i n Abbotsford, B.C., from 1-day o l d to 21 days i n Experiment IV and to 25 days i n Experiment V and VI before commencing the experiments. Feed and water were s u p p l i e d ad l i b i t u m . A randomized complete block design with f a c t o r i a l arrangements was employed i n a l l a v a i l a b i l i t y t r i a l s . Chicks were weighed at 21 days of age (Experiment IV) and 25 days of age (Experiment V, and V I ) , and t r a n s f e r r e d to s t a i n l e s s s t e e l metabolism cages. The same s t a r t e r d i e t was o f f e r e d during the 3-day a c c l i m a t i z a t i o n p e r i o d and water was given ad_ l i b i t u m . The t o t a l f e c a l c o l l e c t i o n method of Bragg ejt al_. , (1969) f o r AAA was performed with minor m o d i f i c a t i o n s . Test d i e t s were o f f e r e d at 25 days of age (Experiment IV) and at 28 days of age (Experiment V, and V I ) . The d e t a i l e d procedure w i l l be d i s c u s s e d i n the a p p r o p r i a t e s e c t i o n l a t e r . A l l d i e t s were fed i n mash form. Test d i e t consumption was recorded f o r each cage and f e c a l c o l l e c t i o n was c a r r i e d out at the end of each t e s t i n g p e r i o d . Treatment d i f f e r e n c e s obtained from the a n a l y s i s of v a r i a n c e (Snedecor and Cochran, 1969) were subjected to the Duncan's m u l t i p l e range t e s t (Duncan, 1955). 50 C. Analyses 1. General analyses Dry matter, crude p r o t e i n (N x 6.25), ether e x t r a c t , and ash content were determined by methods a c c o r d i n g to the A.O.A.C. (1975). 2. Amino a c i d a n a l y s i s The AA compositions of samples were determined a c c o r d i n g to the methods of Bragg e_t a_l. (1966) and Moore (1963) with p a r t i a l m o d i f i c a t i o n s . The procedure was as f o l l o w s : a) Sample p r e p a r a t i o n i ) For feed samples: F i n e l y ground samples c o r r e -sponding to approximately 4mg of N were weighed i n t o 125ml b o i l i n g f l a s k s equipped with connecting tubes. i i ) For f e c a l samples: F i n e l y ground samples c o r r e -sponding to approximately lOmg of N were weighed i n t o 125ml b o i l i n g f l a s k s equipped with connecting tubes. b) H y d r o l y s i s of the samples i ) To each b o i l i n g f l a s k 20ml of 3M HCl was added, i i ) N i trogen gas was bubbled through the mixture f o r one minute to purge the oxygen and connecting tube was attached to the f l a s k . The f i t t i n g s were cleaned and greased with high vacuum s i l i c o n e grease (Dow Corning) f o r good vacuum s e a l under reduced p r e s s u r e . i i i ) The whole apparatus was evacuated v i a the 51 connecting tube to remove the excess n i t r o g e n . i v ) The sample was autoclaved at 121°C at 1.05kg/cm2 f o r 16 hours. c) Ev a p o r a t i o n and f i l t r a t i o n of samples i ) F o l l o w i n g h y d r o l y s i s , the HC1 was removed from the sample h y d r o l y s a t e using a r o t a r y evaporator (Rotavapor-R, Buchi) and a water bath temperature of 60°C. i i ) The d r i e d r e s i d u e was r e d i s s o l v e d i n approximate-l y 15ml of 0.2M sodium c r i t r a t e b u f f e r (pH 2.2). i i i ) The sample was then f i l t e r e d through a Gooch c r u c i b l e (medium p o r o s i t y ) f o llowed by two washings of b u f f e r (10ml) i n t o a f i l t r a t i o n f l a s k . i v ) The f i l t r a t e was q u a n t i t a t i v e l y t r a n s f e r r e d i n t o a 50ml volumeric f l a s k c o n t a i n i n g approximately 3ml of 0.5% NaOH s o l u t i o n , and made to volume. v) The sample was then r e f i l t e r e d using a m i l l i p o r e f i l t e r (Millex-GV, 0.22um f i l t e r u n i t , M i l l i p o r e ) i n t o a screw-capped v i a l , and s t o r e d at 4°C. d) Q u a n t i t a t i o n of amino a c i d s i n h y d r o l y s a t e samples The chromatography was performed with a Beckman System 6300 High Performance Amino Acid Analyzer, on a prepacked Beckman System 6300 High Performance Column. A l l b u f f e r s (Na-A, Na-B, and Na-C) were Beckman System 6300 High Performance Amino Acid A n a l y s i s B u f f e r . i ) H y drolysate sample was f i r s t loaded i n t o the autosampling c o i l which t r a n s f e r r e d an exact amount (50ul) of 52 sample i n t o the column. i i ) The i n i t i a l temperature of the column was 50°C. i i i ) The chromatography e l u t i o n was i n i t i a t e d w i t h a Na-A b u f f e r . i v ) The column t e m p e r a t u r e was i n c r e a s e d at the r a t e of l°C/rainute a f t e r s i x minutes u n t i l i t reached 65°C. v) Na-A b u f f e r was s w i t c h e d to N"a - B b u f f e r a f t e r 20.5 minutes. v i ) The t h i r d b u f f e r , Na-C, came i n t o o p e r a t i o n a f t e r 34.5 minutes. v i i ) The chromatographic run l a s t e d 49 minutes, v i i i ) The AA peaks were i n t e g r a t e d by t h e H e w l e t t -Parkard 3390A I n t e g r a t o r . i x ) F i v e AA s t a n d a r d runs were performed before and a f t e r 20 test-sample runs. The ave r a g e of the ten AA s t a n d a r d runs was used to compute the AA contents of the 20 t e s t samples. The c a l c u l a t i o n of each i n d i v i d u a l AA content was c a r r i e d out acc o r d i n g to the f o l l o w i n g equation: Equation I. , A V a a x M W a a x u M s t d x 1 0 " 6 x D F Amino a c i d (%) = X 100 A V s t d x W s Where A V a a = area value of the AA i n h y d r o l y s a t e . MWga = molecular weight of the AA. uM g t (j = umole of the AA i n the standard s o l u t i o n . DF = d i l u t i o n f a c t o r of the h y d r o l y s a t e (1000). A V g t ( j = average area value of the AA i n standard s o l u t i o n . W = sample weight i n grams. 53 e) C y s t i n e d e t e r m i n a t i o n C y s t i n e was determined as c y s t e i c a c i d a f t e r performic a c i d o x i d a t i o n . Methionine s u l f o n e was not c a l i b r a t e d due to the p a r t i a l o v e r l a p p i n g of peaks between a s p a r t i c a c i d and methionine s u l f o n e . Sample p r e p a r a t i o n was the same as s t a t e d i n s u b s e c t i o n 2, part a. i ) To each b o i l i n g f l a s k 2ml of o x i d a t i o n reagent was added and mixed by s w i r l i n g of the f l a s k (the o x i d a t i o n reagent was composed of one part 30% hydrogen peroxide ( P e r h y d r o l , Merck) mixed with nine p a r t s of 90% formic a c i d ( P u r i f i e d , F i s h e r S c i e n t i f i c ) mixed at room temperature f o r one hour, and then cooled i n an i c e water bath f o r ten minutes before adding to the sample). i i ) Let the r e a c t i o n mixture to o x i d i z e f o r four hours i n an i c e water bath. i i i ) The o x i d a t i o n procedure was terminated by the a d d i t i o n of 0.3ml of 48% hydrobromic a c i d (Reagent grade, A l l i e d Chemical). i v ) The o x i d i z e d sample was then d r i e d by using a r o t a r y evaporator. The d r y i n g took approximately three minutes at a water bath temperature of 60°C. v) The same h y d r o l y z i n g procedure mentioned i n s u b s e c t i o n 2, part b was repeated. 54 D. Chick experiments 1. Feeding t r i a l s The f o l l o w i n g experiments i n v o l v e d a d m i n i s t e r i n g d i f f e r e n t d i e t a r y treatments to growing b r o i l e r c h i c k s from day 1 to day 28. * Experiment I. The e f f e c t s of d i f f e r e n t l e v e l s of hydrolyzed f e a t h e r meal on the growth performance of c h i c k s . The experimental design i n v o l v e d e i g h t treatments c o n s i s t i n g of i n c r e a s i n g l e v e l s of HFM (0, 1, 2, 4, 6, 8, 10, and 15%) r e -p l a c i n g SBM (Table 7) i n four r e p l i c a t e s ; each r e p l i c a t e contained ten b i r d s . Feed i n t a k e and weight gain were determined and the corresponding feed/gain r a t i o c a l c u l a t e d f o r the 28-day p e r i o d . Experiment I I . The e f f e c t s of d i f f e r e n t l e v e l s of blood meal on the growth performance of c h i c k s . The experimental design i n v o l v e d e i g h t treatments c o n s i s t i n g of d i f f e r e n t l e v e l s of BM (0, 1, 2, 4, 6, 8, 10, and 15%) r e -p l a c i n g SBM (Table 8) i n four r e p l i c a t e s ; each r e p l i c a t e contained ten b i r d s . The minimum l e v e l of i n c l u s i o n of animal t a l l o w was set at 6%. As a r e s u l t , the c a l c u l a t e d MEn values were about 3.5 to 5% higher i n D i e t s D to G, and 10% higher i n D i e t H when compared to that of the c o n t r o l r a t i o n ( D i e t A), a f t e r a l l d i e t s were balanced i s o n i t r o g e n o u s l y . Feed i n t a k e and weight gain were determined and the corresponding feed/gain r a t i o c a l c u l a t e d f o r the 28-day p e r i o d . 55 Table 7. Composition of d i e t s (Experiment I) I n g r e d i e n t s D i e t (%) B D E H Soybean meal 38 .0 36 .0 34. 0 30.0 26.0 22 .0 18 .1 8.0 Corn HFM1 25 .1 26 .1 27. 6 29.5 32.4 35 .0 36 .9 43.5 1 .0 2. 0 4.0 6.0 8 .0 10 .0 15.0 Wheat 25 .0 25 .0 25. 0 25.0 25.0 25 .0 25 .0 25.0 Animal t a l l o w 7 .0 7 .0 6. 5 6.5 5.6- 5 .0 5 .0 3.5 Calcium phosphate 1 .1 1 .2 1. 2 1.3 1.3 1 .3 1 .4 .1.5 Limestone Premix 1 .3 1 .2 1. 2 1.2 1.2 1 .2 1 .1 1.0 2 .5 2 .5 2. 5 2.5 2.5 2 .5 2 .5 2.5 C a l c u l a t e d composition MEn, k c a l / k g 3062 3071 3062 3077 3062 3059 3076 3068 Crude p r o t e i n , % 23.2 23.3 23.2 23.3 23.3 23.3 23.3 23.3 Methionine, % 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 L y s i n e , % 1.35 1.31 1.26 1.20 1.20 1.20 1.20 1.20 C y s t i n e , % 0.43 0.43 0.43 0.48 0.54 0.59 0.65 0.79 HFM = hydrolyzed f e a t h e r meal. Premix was composed of 1.5% of AA mix , 0.5% of vit a m i n mix and 0.5% of mineral mix . Composition of DL-methionine, 0. 0.15; L - c y s t i n e , methionine, 0.16 methionine, 0.18 methionine, 0.20 methionine, 0.22 methionine, 0.25; to 1.5% of d i e t wi the AA mix (% of d i e t ) was as f o l l o w s : d i e t A, 14; L - c y s t i n e , 0.05; d i e t B, DL-methionine, 0.03; d i e t C, DL-methionine, 0.15; d i e t D, DL-L - l y s i n e h y d r o c h l o r i d e , 0.03; d i e t E, DL-L - l y s i n e h y d r o c h l o r i d e , 0.11; d i e t F, DL-L - l y s i n e h y d r o c h l o r i d e , 0.21: d i e t G, DL-L - l y s i n e h y d r o c h l o r i d e , 0.28; d i e t H, DL-0.52; and L - l y s i n e h y d r o c h l o r i d e th ground wheat. was made up Vitamin mix per kg of d i e t s u p p l i e d : r e t i n y l p a l m i t a t e , 4.500IU; c h o l e c a l c i f e r o l , 1.000ICU; a l p h a - t o c o p h e r o l , 25IU; menadione, l.Omg; r i b o f l a v i n , 2.4mg; pantothenic a c i d , 9.0mg; n i a c i n , 25.0mg; c h o l i n e c h l o r i d e , 500mg; vi t a m i n B ] ^ , 0.0132mg; ethoxyquin, 500mg; and the vit a m i n mix was made up to 0.5% of d i e t by ground wheat. ^M i n e r a l mix (mg/kg of d i e t ) was composed o f : manganese, 247 as MnS0 4.5H 20 ; z i n c , 38 as ZnO; copper, 12 as CuS0^.5H 20; and i o d i z e d NaCl, 4,703. 56 Table 8. Composition of d i e t s (Experiment I I ) Die t (%) In g r e d i e n t s A B C D E F G H Soybean meal 36.5 34.5 32.0 27.0 22.0 16.5 10.4 Corn 26.6 27.6 29.5 33.0 33.0 31.0 23.0 37.0 Blood meal — 1.0 2.0 4.0 6.0 8.0 10.0 15.0 Wheat 24.0 24.0 24.0 24.0 27.0 32.5 44.5 35.8 A l f a l f a meal 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 Animal t a l l o w 7.0 7.0 6.6 6.0 6.0 6.0 6.0 6.0 Calcium phosphate 1.2 1.2 1.3 1.4 1.4 1.6 1.6 2.0 Limestone 1.2 1.2 1.1 1.1 1.1 0.9 1.0 0.7 Premix 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 C a l c u l a t e d composition MEn, kc a l / k g 3013 3033 3043 3119 3149 3169 3150 3304 Crude p r o t e i n , % 23.0 23.1 23.1 23.0 23.0 23.0 23.0 23.0 Methionine, % 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 L y s i n e , % 1.33 1.37 1.38 1.40 1.43 1.47 1.47 1.59 I s o l e u c i n e , % 1.20 1.16 1.11 1.01 0.91 0.80 0.80 0.80 Leucine, % 1.93 1.99 2.03 2.11 2.18 2.23 2.27 2.43 V a l i n e , % 1.30 1.34 1.36 1.41 1.48 1.52 1.56 1.72 Premix was composed of 1.5% of AA mix , 0.5% of vit a m i n mix and 0.5% of mineral mix . Composition of the AA mix (% of d i e t ) was as f o l l o w s : d i e t A, DL-methionine, 0.14; L - c y s t i n e , 0.05; d i e t B, DL-methionine, 0.13; L - c y s t i n e , 0.06; d i e t C, DL-methionine, 0.14; L - c y s t i n e , 0.07; d i e t D, DL-methionine, 0.16; L - c y s t i n e , 0.08; d i e t E, DL-methionine, 0.17, L - c y s t i n e , 0.10; L - a r g i n i n e , 0.06; d i e t F, DL-methionine, 0.18; L - c y s t i n e , 0.10; L - a r g i n i n e , 0.18; d i e t G, DL-methionine, 0.20; L - c y s t i n e , 0.11; L - a r g i n i n e , 0.29; L-i s o l e u c i n e , 0.08; d i e t H, DL-methionine, 0.26; L - c y s t i n e , 0.18; L - a r g i n i n e , 0.46; L - i s o l e u c i n e , 0.31; and was made up to 1.5% of d i e t with ground wheat. Same as those i n Experiment I. 57 Experiment I I I . The e f f e c t s of blending d i f f e r e n t l e v e l s of blood meal and hydrolyzed f e a t h e r meal on c h i c k performance. The experimental design i n v o l v e d s i x treatments c o n s i s t i n g of d i f f e r e n t blends of BM and HFM at the 10% l e v e l r e p l a c i n g SBM i n the d i e t s (Table 9) and fed to four r e p l i c a t e s of ten b i r d s each. The minimum l e v e l of animal t a l l o w was set at 5%. As a r e s u l t , the MEn of D i e t s B to F was about 3.5% higher than that of the c o n t r o l r a t i o n ( D i e t A), a f t e r a l l d i e t s were balanced i s o n i t r o -genously. Feed i n t a k e and weight gain were determined and the corresponding feed/gain r a t i o c a l c u l a t e d f o r the 28-day p e r i o d . 2. The amino a c i d a v a i l a b i l i t y t r i a l s Experiment IV. The e f f e c t s of p r o t e i n sources (PS) and p r o t e i n l e v e l s (PL) on the amino a c i d a v a i l a b i l i t y i n the growing c h i c k . A 4 x 3 f a c t o r i a l experiment was conducted to estimate the AAA of d i f f e r e n t PS (BM, HFM, MM, and SBM) at d i f f e r e n t PL (10, 20, and 30% CP). Each treatment c o n s i s t e d of two r e p l i c a t e s of four b i r d s each. N i n e t y - s i x uniform s i z e d 21-day o l d c h i c k s were employed. B i r d s were t r a n s f e r r e d randomly to s t a i n l e s s s t e e l metabolism cages with four b i r d s per cage. The t e s t b i r d s remained on the c h i c k s t a r t e r d i e t u n t i l 24-days of age to a c c l i m a t i z e to the new environment. S t a r t i n g i n the evening of the 24th day, a l l b i r d s were f a s t e d f o r approximately 16 hours. In the morning of the 25th day f o l l o w i n g the f a s t i n g p e r i o d , b i r d s were fed a N-free d i e t (Table 10) f o r four hours. B i r d s were then f a s t e d f o r another hour before f e e d i n g the c h i c k s t a r t e r d i e t c o n t a i n i n g 0.3% f e r r i c oxide marker f o r three hours. 58 Table 9. Composition of d i e t s (Experiment I I I ) In g r e d i e n t s D i e t (%) A B C D E F Soybean meal 36.5 10.4 12.0 13.5 14.5 15.5 Corn 26.6 24.0 38.5 43.0 48.5 52.0 HFM1 0.0 0.0 2.5 5'.0 7.5 10.0 Blood meal 0.0 10.0 7.5 5.0 2.5 0.0 Wheat 24.5 45.0 29.0 23.0 16.5 12.0 A l f a l f a meal 1.0 1.0 1.0 1.0 1.0 1.0 Animal t a l l o w 7.0 5.0 5.0 5.0 5.0 5.0 Calcium phosphate 1.2 1.6 1.6 1.5 1.5 1.3 Limestone 1.2 1.0 0.9 1.0 1.0 1.2 Premix 2 2.0 2.0 2.0 2.0 2.0 2.0 C a l c u l a t e d composition MEn, kc a l / k g 3013 3114 3124 3120 3125 3117 Crude p r o t e i n , % 23.0 23.1 23.0 23.0 23.1 23.0 Methionine, % 0.50 0.50 0.50 0.50 0.50 0.50 L y s i n e , % 1.33 1.47 1.30 1.20 1.20 1.20 C y s t i n e , % 0.43 0.43 0.43 0.46 0.54 0.62 I s o l e u c i n e , % 1.19 0.80 0.80 0.85 0.93 1.00 Leucine, % 1.92 2.27 2.22 2.15 2.05 1.95 V a l i n e , % 1.29 1.55 1.51 1.46 1.38 1.34 1HFM = Hydrolyzed f e a t h e r meal. 9 " ^ 4 Premix was composed of 1.0% of AA mix , 0.5% of vit a m i n mix and 0.5% of mineral mix . •^Composition of the AA mix (% of d i e t ) was as f o l l o w s : d i e t A, DL-methionine, 0.14; L - c y s t i n e , 0.05; d i e t B, DL-methionine, 0.20; L - c y s t i n e , 0.10; L - i s o l e u c i n e , 0.08; L - a r g i n i n e , 0.29; d i e t C, DL-methionine, 0.20; L - c y s t i n e , 0.04; L - i s o l e u c i n e , 0.02; L-a r g i n i n e , 0.21; d i e t D, DL-methionine, 0.20; L - l y s i n e h y d r o c h l o r i d e , 0.05; L - a r g i n i n e , 0.12; d i e t E, DL-methionine, 0.22; L - l y s i n e h y d r o c h l o r i d e , 0.19; L - a r g i n i n e , 0.05; d i e t F, DL-methionine, 0.23; L - l y s i n e h y d r o c h l o r i d e , 0.35; was made up to 1.0% of the d i e t with ground wheat. ^Same as those i n Experiment I . 59 Table 10. Composition of N-free d i e t used i n Experiment IV, V and VI I n g r e d i e n t s % D i e t S t a r c h 40.6 Sucrose 40.6 A l p h a - c e l l u l o s e 13.8 Corn o i l 5.0 T o t a l 100.0 60 Unmarked feces were c o l l e c t e d beginning two hours a f t e r the feedi n g of the N-free d i e t and ending two hours a f t e r the appearence of the marked e x c r e t a . The c o l l e c t i o n p e r i o d l a s t e d approximately e i g h t hours. In the evening of the 25th day, the same procedure was repeated except the t e s t d i e t s (Table 11) re p l a c e d the N-free d i e t . Feed i n c l u d i n g the N-free, t e s t , and marked d i e t s and water were s u p p l i e d ad l i b i t u m during the t e s t i n g p e r i o d . A measured volume of water was o f f e r e d during the 16 hours f a s t i n g p e r i o d . Feed consumption of both the N-free d i e t and t e s t d i e t s were recorded at the end of each p e r i o d . Feces c o l l e c t e d from each cage, were f r o z e n u n t i l l y o p h i l i z e d . The f r e e z e - d r i e d feces were weighed and ground i n t o f i n e powder. A l l samples were f r o z e n before AA a n a l y s i s and e q u i l i b r a t e d at room temperature f o r one hour before being analyzed f o r AA contents. The formula used to c a l c u l a t e AAA a f t e r a n a l y s i s of i n g r e d i e n t s and ex c r e t a was f i r s t developed by Bragg ejt a_l. (1969) as f o l l o w s : Equation I I . TAAI - (TAAF - TAAE) % Amino Acid A v a i l a b i l i t y = X 100 TAAI Where TAAI = T o t a l AA in t a k e from feed i n g r e d i e n t s . TAAF = T o t a l f e c a l AA excreted from b i r d s fed the feed i n g r e d i e n t s . TAAE = T o t a l metabolic and endogenous AA. The TAAE i n t h i s t r i a l was obtained by p o o l i n g the f e c a l samples of the 24 groups of b i r d s fed the N-free d i e t . 61 Table 11. D i e t a r y treatments i n Experiment IV In g r e d i e n t s (%) Diet N-free d i e t * BM 2 HFM 2 MM3 SBM 3 BIO 89.3 10.7 B20 78.6 21.4 - — — B30 67.9 32.1 - - -H10 88.8 _ 11.2 H20 77.6 — 22.4 — — H30 66.4 - 33.6 - -M10 79.7 20.3 M20 59.4 - — 40.6 — M30 39.1 - - 60.9 -S10 78.2 _ 21.8 S20 56.4 — _ — 43.6 S30 34.6 — — — 65.4 h- f r e e d i e t as i n d i c a t e d i n Table 10. 2BM = blood meal; HFM = hydrolyzed f e a t h e r meal. 3MM = meat and bone meal; SBM = soybean meal. 62 Experiment V. The e f f e c t s of p r o t e i n source (PS) and p r o t e i n l e v e l (PL) and/or endogenous amino a c i d s c o r r e c t i o n method (ECM) on amino a c i d a v a i l a b i l i t y i n the growing c h i c k . The o b j e c t i v e s of t h i s e x p e r i m e n t were as f o l l o w s : (1) to measure the e f f e c t of a l p h a - c e l l u l o s e intake on AA e x c r e t i o n and to use t h i s r e l a t i o n s h i p to e s t a b l i s h a new r e g r e s s i o n endogenous AA c o r r e c t i o n method (REC) to c o r r e c t f o r the endogenous AA e x c r e t i o n s i n AAA s t u d i e s ( P a r t 1); (2) to re-examine the e f f e c t of d i e t a r y PS and PL on AAA i n the growing c h i c k s as used i n Experiment IV, using d i f f e r e n t combinations of the REC p r o c e d u r e and/or two o t h e r methods f o r the c o r r e c t i o n s of endogenous AA e x c r e t i o n s ( P a r t 2). The e x p e r i m e n t a l d e s i g n s (see T a b l e 12) a r e o u t l i n e d as f o l l o w s : A 2 (PS) x 3 (ECM) x 4 (PL) f a c t o r i a l e x p e r i m e n t was f i r s t employed ( P a r t 2a) and s u b d i v i d e d i n t o t h r e e 2 (PS) x 2 (ECM) x 4 (PL) ( P a r t 2b, 2c, and 2d), and t h r e e 2 (PS) x 4 (PL) ( P a r t 2e, 2f, and 2g) f a c t o r i a l arrangements. One hundred and t w e n t y - e i g h t u n i f o r m l y s i z e d 25-day o l d b r o i l e r c h i c k s were e m p l o y e d . T h e r e were e i g h t d i e t a r y treatments (Table 13) c o n s i s t i n g of two PS (HFM, SBM) and four PL (10, 15, 20, and 25% CP). Each treatment u t i l i z e d four r e p l i c a t e s of four b i r d s each. The AAA procedure was b a s i c a l l y the same as o u t l i n e d i n E x p e r i m e n t IV e x c e p t t h a t the c h i c k s were f i r s t f a s t e d i n the evening of the 28th day of t h i s experiment. A l s o , the TAAE value i n Equation II was dependent on the ECM. The r e l a t i o n s h i p s b e t w een a 1 p h a - c e 1 1 u 1 o s e i n t a k e and 63 Table 12. The o u t l i n e of exper Experiment V, Part 2 1 imental designs i n P r o t e i n Endogenous amino source (PS) c o r r e c t i o n method a c i d (ECM) J P r o t e i n l e v e l ( P L ) 2 A E C a b c e 1 0 a b c e ^abce 2Qabce 2 5 a b c e H F M a b c d e f g I E C a b d f ^Qabdf I c^abdf 2 0 a b d f 2 ^ a b d f R E C a c d g 1 0 a c d g 1 5 a c d g 2 0 a c d g 2 5 a c d g A E C a b c e jQabce 1 cjabce 2Qabce 2^abce ggj^abcdefg j E ^ a b d f j^Qabdf j^abdf 2Qabd f 25abdf R E C a c d S 1 0 a c d g 1 5 a c d g 2 0 a c d g 2 5 a c d g ^Seven randomized complete block designs com-posed of one 2 (PS) x 3 (ECM) x 4 (PL), three 2 (PS) x 2 (ECM) x 4 (PL), and three 2 (PS) x 4 (PL) f a c t o r i a l arrangements were employed i n t h i s experiment. Each separate design (P a r t 2a to 2g) was denoted by s h a r i n g the same s u p e r s c r i p t . PS composed of hydrolyzed f e a t h e r meal (HFM) and soybean meal (SBM); PL composed of 10, 15, 20 and 25% crude p r o t e i n . JECM composed of average endogenous c o r r e c t i o n s (AEC), i n d i v i d u a l endogenous c o r r e c t i o n s (IEC), and endogenous c o r r e c t i o n s by the r e g r e s s i o n method (REC). Table 13. D i e t a r y treatments i n Experiment V Diet I n g r e d i e n t s (%) Alpha-c e l l u l o s e (%) N-free d i e t * HFM 2 SBM 2 H10 89.0 11.0 12.28 H15 83.5 16.5 — 11.52 H20 78.0 22.0 — 10.76 H25 72.5 27.5 - 10.01 SIO 78.0 _ 22.0 10.76 S15 67.0 — 33.0 9.25 S20 56.0 - 44.0 7.73 S25 45.0 — 55.0 6.21 N-free d i e t as i n d i c a t e d i n Table 10. HFM = hydrolyzed f e a t h e r meal; SBM = soybean meal. •^Calculated by m u l t i p l y i n g 0.138 by the % of N-free d i e t i n the same row (used f o r the c a l i b r a t i o n of endogenous AA e x c r e t i o n s by the r e g r e s s i o n method). 65 i n d i v i d u a l AA e x c r e t i o n s were s t u d i e d by monitoring the amount of vo l u n t a r y i n t a k e of N-free d i e t (13.8% a l p h a - c e l l u l o s e ) and the AA e x c r e t i o n s . . The REC procedure i n v o l v e d the use of the r e g r e s s i o n equations f o r i n d i v i d u a l AAs, e s t a b l i s h e d from these r e l a t i o n s h i p s , to c a l i b r a t e the TAAE value f o r AAA. The TAAE f o r a s p e c i f i c group ( r e p l i c a t e ) was obtained by s u b s t i t u t i n g the t o t a l a l p h a - c e l l u l o s e intake from feed i n g r e d i e n t of that group i n t o the r e g r e s s i o n equations. The t o t a l a l p h a - c e l l u l o s e i n t a k e was c a l i b r a t e d by m u l t i p l y i n g the amount of feed i n t a k e to the percentage a l p h a - c e l l u l o s e i n the d i e t as l i s t e d i n Table 13. The mean i n d i v i d u a l AA e x c r e t i o n s from the 32 cages were used as the TAAE i n the average endogenous c o r r e c t i o n s (AEC) method. Whereas, the i n d i v i d u a l endogenous c o r r e c t i o n s (IEC) method u t i l i z e d the AA e x c r e t i o n s from the i n d i v i d u a l cages. Experiment VI. The e f f e c t s of the d u r a t i o n of f e c a l c o l l e c t i o n s on amino a c i d a v a i l a b i l i t y i n the growing c h i c k . A 2 x 2 f a c t o r i a l experiment was conducted to examine the e f f e c t s of f e c a l c o l l e c t i o n times on AAA i n the growing c h i c k . Two P S ( H F M and S B M ) and two methods of endogenous AA c o r r e c t i o n were s t u d i e d . Each treatment c o n s i s t e d of four r e p l i c a t e s of four b i r d s each. The d i e t s used were the same as H ^ Q and S-^Q f o r H F M and S B M , r e s p e c t i v e l y , i n Table 13. The short term (8-hour) REC method as de s c r i b e d f o r Experiment V and a long term (30-hour) f e c a l c o l l e c t i o n (LEC) procedure were employed. The AAA procedure f o r the REC method i n v o l v e d the use of 66 r e g r e s s i o n equations f o r the c o r r e c t i o n s of endogenous AA e x c r e t i o n s , and was the same as that used i n Experiment V. On the other hand, no marked d i e t was employed i n the LEC method. Four separate groups of four b i r d s each were used to c o r r e c t e x c r e t a f o r the endogenous AA. F e c a l c o l l e c t i o n s f o r the LEC groups s t a r t e d two hours a f t e r the f e e d i n g of the N-free or t e s t d i e t s and ended 30 hours l a t e r . A l l b i r d s i n t h i s experiment were 28 days of age when the i n i t i a l 16-hour f a s t i n g commenced. 67 RESULTS AND DISCUSSION A. The chemical composition of hydrolyzed f e a t h e r meal and blood  meal The o b j e c t i v e of t h i s study was to e s t a b l i s h the chemical composition of HFM and BM used i n the present study, s i n c e l a r g e v a r i a t i o n s have been rep o r t e d by previous authors. R e s u l t s from the chemical composition of HFM (Table 14) f a l l w i t h i n the range rep o r t e d by other r e s e a r c h e r s (Naber and Morgan, 1956; M o r r i s and B a l l o u n , 1973b; Matsuda and Shiroma, 1974; NRC, 1977; Johnston and Coon, 1979; B i e l o r a i et a_l. , 1982). The AA composition of HFM (Table 15) a l s o agrees with that r e p o r t e d by a number of other workers ( V e s s e l s , 1972; Burgos ejt al_. , 1974; MacAlpine and Payne, 1977; NRC, 1977; Kirby et al., 1978; Baker et a l . , 1981; B i e l o r a i ejt a_l. , 1982) with two e x c e p t i o n s . The c o n c e n t r a t i o n of h i s t i d i n e i n HFM that was analyzed i n the c u r r e n t study was much higher than those reported p r e v i o u s l y except f o r the value given i n MacAlpine and Payne (1977). On the other hand, the c o n c e n t r a t i o n of t y r o s i n e i n HFM was i n c l o s e agreement with those of the previous authors except the value re p o r t e d by NRC (1977) which was more than two-fold higher. The higher l e v e l s of h i s t i d i n e found i n the present study and that of MacAlpine and Payne (1977) were probably the r e s u l t of contamination with a small amount of blood, which would i n c r e a s e the h i s t i d i n e content. The high l e v e l of t y r o s i n e i n the NRC r e p o r t (1977) appears to be out of order. Table 14. Compositions of blood meal (BM) ^hydrolyzed f e a t h e r meal (HFM) and N u t r i e n t (%, as fed) HFM BM Nitrogen (N) 14.28+0. 13 15.16+0. 02 Crude p r o t e i n (Nx6.25) 89.30+0. 80 94.80+0. 10 Dry matter 94.30+0. 06 94.80+0. 05 L i p i d ( e t h e r e x t r a c t ) 2.72+0. 05 T r a c e 2 Ash 1.97+0. 05 1.34+0. 03 Average values +_ S.D. were from t r i p l i c a t e d e t e r m i n a t i o n s . Less than 0.005%. 69 Table 15. The mean amino a c i d (AA) composition of hydrolyzed f e a t h e r meal (HFM) and blood meal (BM) 1 HFM BM AA as- fed g/16g N as- fed g/16g N Alanine 3. 85+0 .26 4 .31+0. 29 7. 04+0 .06 7 .42+0 .07 A r g i n i n e 6. 19+0 .04 6 .93+0. 05 4. 18+0 .02 4 .41 + 0 .02 A s p a r t i c a c i d 6. 00+0 .08 6 .72+0. 08 9. 03+0 .10 9 .53+0 .11 C y s t i n e 3. 64+0 .17 4 .08+0. 19 0. 39+0 .01 0 .41+0 .01 Glutamic a c i d 11. 08+0 .73 12 .40+0. 82 8. 17+0 .03 8 .61+0 .03 G l y c i n e 6. 59+0 .06 7 .37+0. 06 2. 19+0 .03 2 .31+0 .03 H i s t i d i n e 1. 84+0 .17 2 .07+0. 19 7. 78+0 .04 8 .21+0 .04 I s o l e u c i n e 4. 05+0 .22 4 .54+0. 25 0. 87+0 .03 0 .92+0 .03 Leucine 7. 29+0 .37 8 .16+0. 42 11. 48+0 .06 12 .10+0 .06 L y s i n e 1. 86+0 .25 2 .08+0. 28 8. 68+0 .55 9 .16+0 .58 Methionine 0. 53+0 .05 0 .60+0. 06 0. 76+0 .01 0 .80+0 .01 P h e n y l a l a n i n e 4. 15+0 .04 4 .65+0. 05 6. 14+0 .06 6 .48+0 .06 P r o l i n e 9. 17+0 .21 10 .27+0. 24 3. 54+0 .03 3 .73+0 .04 Seri n e 9. 18+0 .28 10 .28+0. 31 5. 15+0 .06 5 .43+0 .06 Threonine 4. 64+0 .11 5 .19+0. 12 4. 30+0 .02 4 .54+0 .02 T y r o s i n e 2. 89+0 .33 3 .24+0. 37 2. 51+0 .02 2 .64+0 .02 V a l i n e 6. 09+0 .09 6 .82+0. 10 6. 93+0 .06 7 .31+0 .06 Average values +_ S.D. were from q u a d r u p l i c a t e h y d r o l y s e s . The crude p r o t e i n content i s given i n Table 14. 70 Higher percentages of crude p r o t e i n and dry matter, but much lower l e v e l s of ether e x t r a c t and ash were obtained i n the BM sample used i n the present study as compared to those r e p o r t e d by other workers (Wisman ejt al_. , 1958; Abou-El-Hassan ejt al.. , 1970; Abou-Raya et a l . , 1971; Hew and Devendra, 1977; NRC, 1977). T h i s might be a r e f l e c t i o n of the p r o c e s s i n g method f o r BM. The AA analyses of BM (Table 15) re v e a l e d s l i g h t l y lower c o n c e n t r a t i o n s of a l a n i n e , a s p a r t i c a c i d , glutamic a c i d , p h e n y l a l a n i n e , t y r o s i n e and v a l i n e , and very low l e v e l s of c y s t i n e and g l y c i n e as compared to r e s u l t s of other r e s e a r c h e r s ( F i s h e r , 1968; Fetuga eji a_l., 1973; NRC, 1977; King and Campbell, 1978; Ki r b y et a l . , 1978). In c o n t r a s t , a much higher c o n c e n t r a t i o n of h i s t i d i n e was obtained i n the c u r r e n t study. The remaining AAs were in t e r m e d i a t e to the l e v e l s r e p o r t e d p r e v i o u s l y . The low c o n c e n t r a t i o n of c y s t i n e might a l s o r e f l e c t the over-h e a t i n g of the BM sample. H u r r e l l e_t al_. (1976) repo r t e d that o v e r h e a t i n g could cause f i s s i o n of the d i s u l p h i d e bond i n c y s t i n e , y i e l d i n g dehydroalanine which may condense with c y s t e i n e to form l a n t h i o n i n e . Furthermore, heat damage would lead to the formation of new enzyme-resistant i s o p e p t i d e l i n k a g e s by the r e a c t i o n of the epsilon-amino group of l y s i n e with the c a r b o x y l group of e i t h e r a s p a r t i c or glutamic a c i d s ( H u r r e l l ejt al_. , 1976). The lower l e v e l s of a s p a r t i c and glutamic a c i d s but i n t e r -mediate c o n c e n t r a t i o n of l y s i n e i n the present sample might imply a moderate degree of heat damage. 71 Other than the p o s s i b i l i t y of o v e r p r o c e s s i n g , the d i s t r i b u t i o n of the v a r i o u s f r a c t i o n s of blood used i n the p r o d u c t i o n of the meal could i n f l u e n c e the percent d i s t r i b u t i o n of AA. Grau and Almquist (1944) r e p o r t e d that the p r o t e i n q u a l i t y of serum and f i b r i n f r a c t i o n s of beef blood were s u p e r i o r to the blood c e l l f r a c t i o n i n which i s o l e u c i n e was the p r i n c i p a l l i m i t i n g AA. Since the c o n c e n t r a t i o n of i s o l e u c i n e i n the sample of BM used i n the present i n v e s t i g a t i o n was second lowest to that reported by the previous workers, i t may suggest that a higher p o r t i o n of blood c e l l than serum or f i b r i n f r a c t i o n i s present i n the BM sample. The v a r i a t i o n i n f i n d i n g s of the present study and those i n the previous r e p o r t s might be a t t r i b u t e d to the c o n d i t i o n s of p r o c e s s i n g , methods of c o l l e c t i o n of blood samples at the s l a u g h t e r house and/or p o s s i b l y the use of blood from d i f f e r e n t kinds or combinations of animals. In c o n c l u s i o n , the chemical composition of HFM i n the present experiment r e v e a l e d a sample that i s r i c h i n c y s t i n e , t h r e o n i n e , a r g i n i n e and i s o l e u c i n e , and c o n t a i n s adequate l e v e l s of other amino a c i d s except l y s i n e and methionine, when s u p p l i e d as the s o l e source of p r o t e i n f o r b r o i l e r s (0 to 3 weeks). On the other hand, the BM sample may have been overprocessed and c o n t a i n s a r e l a t i v e l y high percentage of the blood c e l l s as r e f l e c t e d by the low c o n c e n t r a t i o n of i s o l e u c i n e . N e v e r t h e l e s s , the sample i s r i c h i n l y s i n e , h i s t i d i n e and l e u c i n e , and c o n t a i n s an adequate balance of the remaining AAs except methionine, c y s t i n e , 72 a r g i n i n e , and i s o l e u c i n e . The r a t i o of i s o l e u c i n e : l e u c i n e (1:13) was imbalanced when compared to those i n HFM (1:1.8), SBM (1:1.5), and MM (1:2). T h i s wide r a t i o could impose a problem i n feed f o r m u l a t i o n . B. Feeding t r i a l s The performance data i n Experiment I, I I and I I I are presented on an c h i c k average b a s i s . The a n a l y s i s of va r i a n c e f o r growth parameters i n Experiment I, II and I I I are given i n Table 1, 2 and 3 (Appendix). Experiment I. The o b j e c t i v e of t h i s study was to examine the feed i n t a k e , body weight gain and feed/gain r a t i o of b r o i l e r c h i c k s fed d i e t s c o n t a i n i n g v a r y i n g l e v e l s of HFM from day 1 to 28 days post-h a t c h i n g . The r e s u l t s (Table 16) show that c h i c k s can t o l e r a t e up to 10% HFM i n d i e t s with performance s i m i l a r to those fed the soy-wheat-corn b a s a l d i e t . T h i s i n c l u s i o n r a t e of 10% HFM i n d i e t s i s e q u i v a l e n t to 37% of the d i e t a r y p r o t e i n (23%). A s i m i l a r f i n d i n g was repor t e d by Baker e_t al_. (1981), using male c h i c k s from 9 to 22 days pos t h a t c h i n g , i n which up to 40% of the d i e t a r y p r o t e i n could be s u p p l i e d by HFM. On the other hand, t h i s l e v e l (10%) of i n c l u s i o n with methionine and l y s i n e supplementation was much higher than those reported by other workers ( S i b b a l d et a l . , 1962; Tsang et a l . , 1963; Moran, J r . et a l . , 1966; Matsuda and Shiroma, 1974; MacAlpine and Payne, 1977). Table 16. The performance of b r o i l e r c h i c k s fed d i f f e r e n t l e v e l s of hydrolyzed f e a t h e r meal (HFM) (Experiment I) Response c r i t e r i o n L e v e l s of Feed HFM consumption Weight gain Feed/gain (%) (g) (g) (g/g) 0 (A) 1477 A 955 A 1.55 A 1 (B) 1482 A 1435 A 972 A 1.53 A 2 (C) 922 A 1.56 A 4 (D) 1432 A 932 A 1.54 A 6 (E) 1453 A 940 A 1.55 A 8 (F) 1412 A 9 0 7 » 1.56 A 10 (G) 1455 A 1290 B 924 A 1.58 A 15 (H) 788 B 1.64 B SEM 3 29 21 0.014 L e t t e r s i n brackets r e f e r to d i e t d e s i g n a t i o n s i n Table 7. o The performance data i n t h i s experiment i s presented on an average c h i c k b a s i s . The i n i t i a l average weight of the day-old c h i c k s was 36.0+0.9g. The d u r a t i o n of the experiment was 28 days. Means w i t h i n each column not s h a r i n g a common s u p e r s c r i p t are s i g n i f i c a n t l y d i f f e r e n t at P<0.01. SEM = standard e r r o r of the treatment means. 74 The d i f f e r e n c e i n these f i n d i n g s might be due to the n u t r i t i v e q u a l i t y of HFM processed under d i f f e r e n t c o n d i t i o n s . The HFM used i n the present experiment was processed at 2.81kg/cm of steam pressure f o r 40 minutes with continuous a g i t a t i o n . These p r o c e s s i n g c o n d i t i o n s seem to be s u f f i c i e n t f o r reasonably good c h i c k performance. Feed consumption, weight gain and FCE were s i g n i f i c a n t l y . (P<0.01) depressed i n c h i c k s fed d i e t s c o n t a i n i n g 15% HFM during the 28-day t r i a l . The depressions i n feed consumption, weight gain and FCE as compared to those fed the soy-wheat-corn b a s a l d i e t (or 0% HFM) were 13, 18 and 6%, r e s p e c t i v e l y . The d e l e t e r i o u s e f f e c t may be a t t r i b u t e d to a minor degree of AA imbalance. The l e v e l of c y s t i n e i n the 15% HFM d i e t (as s t a t e d i n Table 7) was c a l c u l a t e d to be 0.79% of the d i e t which i s 84% higher than the suggested l e v e l of 0.43 (as recommended by NRC, 1977). Fe a t h e r s t o n and Rogler (1978) repo r t e d the e x i s t e n c e of an antagonism of c y s t i n e on methionine u t i l i z a t i o n only when the d i e t a r y l e v e l of methionine was suboptimal. T h i s seems not be p o s s i b l e , s i n c e an adequate l e v e l of d i e t a r y methionine (0.5%) was being maintained i n the present study. Another probable reason f o r the poor performance might be the high c o n c e n t r a t i o n s of the t o t a l SAAs i n the 15% HFM d i e t (1.29%) as compared to the 0.93% recommended by NRC (1977). T h i s 39% i n c r e a s e i n t o t a l SAA might impose a problem to the u t i l i z a t i o n of the other l i m i t i n g AA. However, no i n f o r m a t i o n on the maximum 75 t o l e r a b l e l e v e l f o r t o t a l SAA and i t s e f f e c t on AA imbalance i s c u r r e n t l y a v a i l a b l e . On the other hand, the AA imbalance might a l s o be imposed from low AAA i n HFM. The mean AAA of HFM was rep o r t e d to be as high as 97% (Burgos ejt al_. , 1974) to a l e v e l as low as 68% ( E l Boushy and Roodbeen, 1984). F u r t h e r experiments are needed to e s t a b l i s h the AAA of the HFM used i n the present study. Experiment I I . The o b j e c t i v e of t h i s experiment was to examine the performance of c h i c k s fed d i e t s c o n t a i n i n g i n c r e a s i n g l e v e l s of BM. The r e s u l t s (Table 17) show that feed consumption and body weight gain were s i g n i f i c a n t l y depressed when 15% BM was i n c o r p o r a t e d i n the c h i c k s t a r t e r d i e t s . The depressions (P<0.01) of feed i n t a k e and weight gain were 58 and 62% i n groups fed the 15% BM as compared to those fed the soy-wheat-corn b a s a l d i e t (or 0% BM). On the other hand, there was no s i g n i f i c a n t d i f f e r e n c e i n FCE between the two groups. F i s h e r (1968) a l s o found severe growth de p r e s s i o n i n c h i c k s fed a s e m i - p u r i f i e d d i e t c o n t a i n i n g 14% BM. These authors concluded that t h i s d e p ression could be c o r r e c t e d by the a d d i t i o n of i s o l e u c i n e , methionine and a r g i n i n e to the d i e t . The d e l e t e r i o u s e f f e c t i n t h i s study was probably caused by an AA imbalance r e s u l t i n g from a high c o n c e n t r a t i o n of l e u c i n e i n the 15% BM d i e t . The l e v e l of l e u c i n e was 2.43% i n the 15% BM d i e t (as l i s t e d i n Table 8) which i s 80% higher than the value of Table 17. The performance of b r o i l e r c h i c k s fed d i f f e r e n t l e v e l s of blood meal (BM) (Experiment I I ) • 2 Response c r i t e r i o n L e v e l s of Feed BM consumption Weight gain Feed/gain T%) (?) Tgl WT) 0 (A) 1 2 5 6 » 7 2 8 A 1 . 7 3 a b c 1 (B) 1260 A 718 A 1 . 7 6 a b c 2 (C) 1 1 4 5 A 6 7 5 A 1.70 a 4 (D) 1201 A 730 A 1 ' 6 5 B u 6 (E) 1 1 1 6 A 6 4 8 A 1.72 a b 8 (F) 1079 A 645 A l - 6 7 a 10 (G) H 5 1 A 610 A 1.89 b c 15 (H) 526 B 275 B 1.91 c SEM 46 40 0.058 L e t t e r s i n brackets r e f e r to d i e t d e s i g n a t i o n s i n Table 8. 2The performance data i n t h i s experiment i s presented on an average c h i c k b a s i s . The i n i t i a l average weight of the day-old c h i c k s was 40.0+1.7g. The d u r a t i o n of the experiment was 28 days. Means w i t h i n each column not s h a r i n g a common c a p i t a l i z e d or n o n - c a p i t a l i z e d s u p e r s c r i p t are s i g n i f i c a n t l y d i f f e r e n t at P<0.01 or P<0.05, r e s p e c t i v e l y . 77 1.35 as recommended by NRC (1977). T h i s excess of l e u c i n e could r e s u l t i n a severe d i s p r o p o r t i o n of AA and lead to the adverse e f f e c t s on c h i c k growth but not the FCE which i s supported by the f i n d i n g s of Ueda ejt a l . (1981). Those authors r e p o r t e d that i s o -nitrogenous supplement of e x c e s s i v e amount of l e u c i n e i n c h i c k d i e t s r e s u l t e d i n depressed growth r a t e but not i n the f e e d / g a i n r a t i o . The d e p r e s s i o n was, however, overcome by f o r c e -f e e d i n g the l e u c i n e - e x c e s s d i e t . R e s u l t s (Table 17) show that no s i g n i f i c a n t (P>0.01) d i f f e r e n c e i n c h i c k performances was observed with d i e t a r y l e v e l s from 1 to 10% BM. The AA imbalance caused a high degree of v a r i a t i o n i n r e s u l t s between 0 to 10% d i e t a r y BM and became s i g n i f i c a n t at the 15% l e v e l . T h i s i s r e f l e c t e d by the l a r g e value f o r the standard e r r o r of the treatment means. From the c l o s e examination of the feed i n t a k e data, a d e p r e s s i o n (P>0.05) was observed from i n c r e a s i n g l e v e l s of BM beyond the 1% l e v e l . Depressions i n feed i n t a k e f o r c h i c k s fed d i e t s c o n t a i n i n g 2, 4, 6, 8 and 10% BM as compared to those fed the soy-wheat-corn c o n t r o l d i e t (or 0% HFM) were 9, 4, 11, 14 and 8%, r e s p e c t i v e l y . The c a l c u l a t e d MEn of d i e t s (as s t a t e d i n Table 8) c o n t a i n i n g the 4, 6, 8 and 10% BM were about 3.5 to 5% higher than that of the c o n t r o l as mentioned i n the previous s e c t i o n . Assuming the c h i c k s consumed a c c o r d i n g to t h e i r energy requirements, the depressions i n feed consumptions a f t e r c o r r e c t i o n f o r the d i f f e r e n c e i n energy i n t a k e were 8, 1, 7, 9 and 4% i n groups fed 78 the 2, 4, 6, 8 and 10% BM as compared to those fed the c o n t r o l d i e t . With t h i s c o r r e c t i o n , the group fed the 4% BM d i e t seem to have the best c h i c k performance as r e f l e c t e d by s u p e r i o r weight gain and FCE. S i m i l a r f i n d i n g s were repo r t e d by Wisman et a l . (1958). Those authors showed that BM could be i n c o r p o r a t e d to provide 3% of the t o t a l 20% CP i n ch i c k s t a r t e r r a t i o n s . Moreover, Omar e_t a l . (1985) r e p o r t e d that the 3-week performance of b r o i l e r - t y p e c h i c k s r e c e i v i n g d i e t s c o n t a i n i n g 4% BM was not d i f f e r e n t from those fed the corn-soy b a s a l d i e t . The authors a l s o added that s i g n i f i c a n t growth de p r e s s i o n was observed with b i r d s fed 8% BM with or without the supplementations of a r g i n i n e or potassium. The d e l e t e r i o u s e f f e c t of the higher l e v e l of BM i n the study of Omar e_t a_l. (1985) would a l s o be due to an imbalance of the branched-chain AA as s t a t e d p r e v i o u s l y i n the present study. On the other hand, G a l a l ejt al_. (1977) showed that BM could be in c o r p o r a t e d at up to 6% i n t y p i c a l Egyptian b r o i l e r d i e t as a source of l y s i n e . The reason that G a l a l ejt al_. (1977) could use a higher l e v e l of BM (6%) than the present i n v e s t i g a t i o n and that of Omar e_t al_. (1985) may be due to the use of d i f f e r e n t b a s a l d i e t s . The composition of the Egyptian b r o i l e r d i e t s used by G a l a l e_t al_. (1977) was very d i f f e r e n t from the soy-wheat-corn based d i e t s used i n the present i n v e s t i g a t i o n or the corn-soy b a s a l d i e t i n the study of Omar ejt a_l. (1985). The r a t i o of i s o l e u c i n e : l e u c i n e ( r e f e r to Table 8) i n d i e t A, 79 B, C, D, E, F, G and H were 1:1.6, 1:1.7, 1:1.8, 1:2.1, 1:2.4, 1:2.8, 1:2.8 and 1:3, r e s p e c t i v e l y . The i n c r e a s e i n d i s p r o p o r -t i o n a l i t y between i s o l e u c i n e and l e u c i n e could a l s o i n f l u e n c e the v a r i a t i o n s i n performance of treatment groups. The improvement i n c h i c k s fed the 4% BM d i e t was probably due to a number of f a c t o r s i n c l u d i n g a r e d u c t i o n i n feed consumption which p a r t i a l l y a l l e v i a t e d the de p r e s s i v e e f f e c t of AA imbalance. Even though AA supplementation was provided (Table 8, footnote 2) to r a i s e the l e v e l s of a r g i n i n e , i s o l e u c i n e , methionine and c y s t i n e i n d i e t s c o n t a i n i n g more than 4% BM to AA l e v e l s recommended by NRC (1977), the performance of c h i c k s was depressed by the imbalance of the branched-chain AA. The wide range of AAA from BM (as repo r t e d by K i r b y e_t al_. , 1978) could accentuate the AA imbalance of BM. I t may be concluded t h a t , with methionine supplementation, BM can be i n c o r p o r a t e d i n a soy-wheat-corn based d i e t at a maximum l e v e l of 4% provided that the energy l e v e l i s kept i n balance. F u r t h e r s t u d i e s are needed to e s t a b l i s h the AAA of BM used i n the present experiment. Experiment I I I . The o b j e c t i v e of t h i s experiment was to examine the e f f e c t s of i n c o r p o r a t i n g d i f f e r e n t blends of BM:HFM (10:0, 7.5:2.5, 5:5, 2.5:7.5 and 0:10) i n c h i c k s t a r t e r d i e t s at the 10% l e v e l . In the previous s e c t i o n i t was shown that HFM i s r i c h i n c y s t i n e , t h r e o n i n e , i s o l e u c i n e and a r g i n i n e but low i n l y s i n e and methionine. BM i s r i c h i n l y s i n e , h i s t i d i n e and l e u c i n e but i s 80 d e f i c i e n t i n c y s t i n e , i s o l e u c i n e and methionine. T h e r e f o r e , the p o t e n t i a l b e n e f i t of combining HFM and BM was i n v e s t i g a t e d i n t h i s study. The r e s u l t s (Table 18) show no s i g n i f i c a n t (P>0.05) d i f f e r e n c e i n feed i n t a k e and weight gain of b i r d s fed the d i f f e r e n t blends of BM and HFM. However, the FCE i n groups fed the 10:0 and 7.5:2.5 (BM:HFM) blends were s i g n i f i c a n t l y (P<0.05) lower than those of the other groups. The b e n e f i c i a l e f f e c t of blending HFM and BM was a l s o observed by Koci et. a l . (1983). The authors r e p o r t e d that 2:1 mixture of HFM and BM could r e p l a c e 50 and 100% of the animal p r o t e i n i n b r o i l e r s t a r t e r and f i n i s h e r d i e t s , r e s p e c t i v e l y . Upon examination of the r a t i o of i s o l e u c i n e : l e u c i n e i n the d i e t s (Table 9), the values of 1:1.6, 1:2.8, 1:2.8, 1:2.5, 1:2.2 and 1:2 were obtained f o r d i e t s A, B, C, D, E and F, r e s p e c t i v e l y , i n the present study. The s e v e r e l y imbalanced r a t i o of i s o l e u c i n e : l e u c i n e i n d i e t B and C may be the c a u s t i v e f a c t o r f o r the decreases i n FCE i n the two groups. Even though there was no s i g n i f i c a n t d i f f e r e n c e i n feed i n t a k e among the groups fed the 5:5, 2.5:7.5 and 0:10 (BM:HFM) blends, the data showed that the group fed the 10% HFM had the high e s t feed consumption and weight gain of the three groups. The decreases i n weight gain of groups fed the 2.5:7.5 and 5:5 (BM:HFM) were 2 and 5%, r e s p e c t i v e l y , i n feed consumption as compared to the group fed 10% HFM. From t h i s study, i t appears f e a s i b l e to i n c o r p o r a t e 2.5% BM and 7.5% HFM i n ch i c k s t a r t e r 81 Table 18. The performance of b r o i l e r c h i c k s fed d i f f e r e n t 10% blends of blood meal (BM) and hydrolyzed f e a t h e r meal (HFM) (Experiment I I I ) 1 • 2 Response c r i t e r i o n L e v e l s of Feed BM HFM consumption Weight gain Feed/gain — % — Til (I) (g/g) 0.0 0.0 (A) 1343 842 1*60t 10.0 0.0 (B) 1319 771 1'7K 7.5 2.5 (C) 1303 771 l - 6 ^ \ 5.0 5.0 (D) 1295 786 i - 6 5 * ? 2.5 7.5 (E) 1334 812 i - 6 * 3 ? 0.0 10.0 (F) 1360 831 1.64 a b SEM 31 19 0.022 •"•Letters i n brackets r e f e r to d i e t d e s i g n a t i o n s i n Table 9. 2 The performance data i n t h i s experiment i s presented on an average c h i c k b a s i s . The i n i t i a l average weight of the day-old c h i c k s was 34.2+0.9g. The d u r a t i o n of the experiment was 28 days. Means w i t h i n each column not sha r i n g a common s u p e r s c r i p t are s i g n i f i c a n t l y d i f f e r e n t at P<0.05. 82 d i e t s p r o v i d i n g the market and the p r i c e of BM and HFM are comparable. C. The amino a c i d a v a i l a b i l i t y t r i a l s  Experiment IV. T h i s experiment was conducted to estimate the AAA of HFM and BM samples used i n the previous experiments and to compare them to those i n two other commonly used p r o t e i n supplements namely, SBM and MM. Since l i t t l e r e s e a r c h was conducted to evaluate the e f f e c t s of d i f f e r e n t l e v e l s of p r o t e i n - r i c h supplements on AAA i n the growing c h i c k s , three l e v e l s of p r o t e i n i n c l u s i o n s were employed si m u l t a n e o u s l y with the d i f f e r e n t p r o t e i n c o n c e ntrates so as to provide a d d i t i o n a l i n f o r m a t i o n about the AAA assay. Data d e c r i b i n g the AA e x c r e t i o n s of the groups fed the N-free d i e t s are i l l u s t r a t e d i n Table 19. T h i s was used to c o r r e c t f o r the TAAE i n the c a l c u l a t i o n of AAA. The AAA values f o r the d i f f e r e n t PS (HFM, BM, SBM and MM) and PL (10, 20 and 30% CP) are presented i n Table 20 and Table 21, r e s p e c t i v e l y . From the a n a l y s i s of va r i a n c e i n Appendix Table 4 , i t i s c l e a r that both PS and PL were s i g n i f i c a n t v a r i a b l e s . Moreover, a s p a r t i c a c i d and l y s i n e were a f f e c t e d by PS x PL i n t e r a c t i o n s at P<0.05 and P<0.01, r e s p e c t i v e l y . The mean AAA i n HFM was 86.9%. The a v a i l a b i l i t y of the i n d i v i d u a l AA ranged from 75.9% f o r h i s t i d i n e to 92.3% f o r i s o l e u c i n e . The mean AAA i n HFM was lower than the values of 97 Table 19. The mean amino a c i d (AA) e x c r e t i o n of b i r d s fed the N-free d i e t (Experiment I V ) 1 Mean AA e x c r e t i o n AA mg per group mg per b i r d A l a n i n e 7.8 2.0 A r g i n i n e 7.2 1.8 A s p a r t i c a c i d 13.8 3.5 Glutamic a c i d 18.0 4.5 H i s t i d i n e 24.6 6.2 I s o l e u c i n e 13.8 3.5 Leucine 13.2 3.3 L y s i n e 13.8 3.5 P h e n y l a l a n i n e 6.6 1.7 P r o l i n e 12.6 3.2 Ser i n e 11.4 2.9 Threonine 11.4 2.9 T y r o s i n e 9.6 2.4 V a l i n e 6.6 1.7 •"•There were four b i r d s per group. The average i n t a k e of the N-free d i e t from the 24 groups was 61.5g, ranging from 43 to 83g. The average amount of endogenous f e c a l e x c r e t i o n s was 6g/group. 84 Table 20. Comparison of amino a c i d (AA) a v a i l a b i l i t y of d i f f e r e n t p r o t e i n source (PS) using 3-week o l d c h i c k s (Experiment I V ) 1 PS AA HFM"1 BM' SBM' MM' SEM Alanine A r g i n i n e A s p a r t i c a c i d Glutamic a c i d H i s t i d i n e I s o l e u c i n e Leucine L y s i n e P h e n y l a l a n i n e P r o l i n e S e r i n e Threonine T y r o s i n e V a l i n e Average 89 89 80 86 75 92 89 82 89 87 87 86 89 89 86.9 l 3C,3 h 8 c 9 B 3c 9 c 0 C 93 91 91 91 91 86 93 92 92 90 92 91 90 92 >B 91.5 B 96. 5 A 95. oAB » 97. 4A 95. 5 A 95. 9 A 93. oAB 97. 5 t 95. 92. 8 t 92. 97. 7 A 96. 6 A 96. 8 A 94. 6 A B 96. 6 A 94. 6 A B 92. 2. 91. 8. 96. l A 95. 0 A 96. 6 A 94. 2 A B 95. 8 A 94. 2A® 96. 8 A 92. 7 A B 96. 7 A 94. 4 A B 96. 1 A 94. 3 A 0.56 0.71 0.67 0.82 0.74 0.89 0.60 0.70 0.74 0.84 0.67 0.80 1.03 0.60 0.58 An eight-hour f e c a l c o l l e c t i o n p e r i o d was used. 2HFM = hydrolyzed f e a t h e r meal; BM = blood meal; SBM = soybean meal; MM = meat and bone meal. Means with d i f f e r e n t s u p e r s c r i p t s i g n i f i c a n t l y d i f f e r e n t (P<0.01). w i t h i n row are 85 Table 21. Comparison of amino a c i d (AA) a v a i l a b i l i t y from d i e t s of d i f f e r e n t p r o t e i n l e v e l (PL) using 3-week o l d c h i c k s (Experiment IV) PL (%)  AA 10 20 . 30 SEM Alanine 92. 4 A r g i n i n e 92. 7 A s p a r t i c a c i d 88. 2 Glutamic a c i d 90. 6 H i s t i d i n e 86. 7 I s o l e u c i n e 91. 2 Leucine 92. 1 Ly s i n e 88. 3 Phe n y l a l a n i n e 88. 8 P r o l i n e 90. 7 Seri n e 91. 0 Threonine 89. 9 Ty r o s i n e 88. 6 V a l i n e 92. 1 Average 90. 2 B 94.6 a 93.9 a 0.48 94.5 93.0 A 0.61 91.6 A 91.5 A 0.58 94.0 a 9 3 . l a 0.71 89.9 a 8 8 . 3 a b 0.64 94.5 a 93.7 a 0.77 94.5 a 9 4 . l a 0.52 93.2 A 92.9 A 0.61 92.6 A 92.8 A 0.65 93.5 a 9 2 . 6 a b 0.73 94.0 A 9 3 . 2 A B 0.58 93.7 A 9 2 . 4 A B 0.69 94.0 A 93.8 A 0.89 94.2 a 9 3 . 4 a b 0.52 93.5 A 92.8 A 0.05 An eight-hour f e c a l c o l l e c t i o n p e r i o d was used. o Means w i t h i n each row not sh a r i n g a common n o n - c a p i t a l i z e d or c a p i t a l i z e d s u p e r s c r i p t are s i g n i f i c a n t l y d i f f e r e n t at P<0.05 or P<0.01, r e s p e c t i v e l y . 86 and 94% r e p o r t e d by Burgos ejt al_. (1974) and K i r b y elt a_l. (1978), r e s p e c t i v e l y . On the other hand, i t was s i g n i f i c a n t l y higher than the values of 68 to 69% r e p o r t e d i n E l Boushy and Roodbeen (1984) and Papadopoulos e_t a_l. (1985). Furthermore, a much wider range of values f o r the a v a i l a b i l i t y of i n d i v i d u a l AAs was r e p o r t e d by the two l a t t e r groups. The d i f f e r e n c e between the present f i n d i n g s and those reported by the previous authors (Burgos j3_t al_. , 1974; K i r b y ejt al_. , 1978; E l Boushy and Roodbeen, 1984; Papadopoulos e_t al_. , 1985) may be due to the use of HFM samples processed under d i f f e r e n t c o n d i t i o n s . S i g n i f i c a n t v a r i a t i o n s i n AAA of HFM from d i f f e r e n t p r o c e s s i n g methods have been r e p o r t e d by d i f f e r e n t workers (Eggum, 1970; Papadopoulos, 1985). Eggum (1970) showed that the AAA of HFM i n r a t s v a r i e d between 30 and 84% f o r d i f f e r e n t p r o c e s s i n g methods. S t u d i e s by Papadopoulos (1985) on the e f f e c t s of p r o c e s s i n g time, with/with-out a d d i t i o n s of NaOH or p r o t e o l y t i c enzyme i n d i c a t e d that the AAA of HFM i n growing c h i c k s were most s i g n i f i c a n t l y a f f e c t e d by the p r o c e s s i n g time. Since l y s i n e i s o f t e n the f i r s t or second l i m i t i n g AA i n p o u l t r y feeds, the amount of d i e t a r y l y s i n e which i s b i o l o g i c a l l y a v a i l a b l e i s of extreme importance. The a v a i l a b i l i t y of l y s i n e i n HFM was 82% which i s i n t e r m e d i a t e to the values r e p o r t e d by p r e v i o u s r e s e a r c h e r s . With the t o t a l l y s i n e c o n c e n t r a t i o n of 1.86% (Table 15), the a v a i l a b l e l y s i n e i n HFM was c a l c u l a t e d to be 1.53% i n the present i n v e s t i g a t i o n . T h i s f a l l s w i t h i n the 87 range of values r e p o r t e d by Nordheim and Coon (1984). The mean a v a i l a b l e l y s i n e values determined by the TNBS, FDNB, CBA and DL procedures were 1.93, 2.38, 1.41 and 1.73%, r e s p e c t i v e l y , from four samples of HFM (Nordheim and Coon, 1984). The AAA of BM i n the present study were s i g n i f i c a n t l y (P<0.01) higher than those i n HFM. The mean AAA i n BM was 91.5% with i n d i v i d u a l AA ranging between 86% f o r i s o l e u c i n e and 94% f o r a l a n i n e . The AAA i n BM were s i m i l a r to those observed by Kirby et a l . (1978), with the exception that the a v a i l a b i l i t y of i s o l e u c i n e (86%) was much higher than the value of 66% reported by these workers. The major disadvantage of BM has been the poor d i g e s t i b i l i t y of p r o t e i n and, e s p e c i a l l y the poor a v a i l a b i l i t y of l y s i n e ( K r a t z e r and Green, 1957). The percent a v a i l a b i l i t y of l y s i n e i n BM and i t s corresponding a v a i l a b l e l y s i n e value were 93 and 8.1%, r e s p e c t i v e l y , i n the present study. The a v a i l a b l e l y s i n e content was lower than those (10 to 12%) reported by K r a t z e r and Green (1957) using both the chi c k and poult assays f o r s p r a y - d r i e d BM. On the other hand, i t was higher than those r e p o r t e d by Hamm and Searcy (1976) and N o l l £t al. (1984). The lower content of a v a i l a b l e l y s i n e from BM i n the present study as compared to those r e p o r t e d by K r a t z e r and Green was probably due to a r e l a t i v e l y low content of t o t a l l y s i n e content (8.68%) i n s t e a d of poor a v a i l a b i l i t y , s i n c e the l y s i n e a v a i l a b i l i t y f o r HFM i n t h i s study was estimated to be 93%. 88 The AAA values i n SBM were s i g n i f i c a n t l y (P<0.01) s u p e r i o r to those i n BM and HFM. The mean AAA was 96.1% with ranges from 92.2% f o r phen y l a l a n i n e to 97.7% f o r i s o l e u c i n e . The mean AAA values f o r SBM were s l i g h t l y b e t t e r than the values reported by L i k u s k i and D o r r e l l (1978) and Engster jet a_l. (1985) using the r o o s t e r assay. On the other hand, they were s i m i l a r to those s t a t e d by Ivy ejt al_. (1971) and Nwokolo jet a_l. (1976b) using a s i m i l a r procedure. The AAA values f o r MM were s i m i l a r to those f o r SBM i n t h i s experiment. The mean AAA was 94.3% ranging between 91.8 and 96.6% f o r phen y l a l a n i n e and i s o l e u c i n e , r e s p e c t i v e l y . The AAA values i n MM were much b e t t e r than those re p o r t e d i n Engster jet j a l . (1985) using the r o o s t e r assay. The r e s u l t s (Table 21) show that there were s i g n i f i c a n t (P<0.01) improvements i n mean AAA when the p r o t e i n - r i c h supplements (HFM, BM, SBM and MM) were i n c o r p o r a t e d with N-free d i e t s to provide more than 10% p r o t e i n . In other words, the AA i n the p r o t e i n supplements might be more a v a i l a b l e to the c h i c k s when fed at l e v e l s that supply more than 10% of the p r o t e i n i n the d i e t s . Nearly a l l AA followed the same trends as the average values except a r g i n i n e . The data seem to imply that the AAA values i n 20% CP l e v e l was s l i g h t l y b e t t e r , even though not s i g n i f i c a n t , than those i n 30% CP l e v e l . S i b b a l d (1979d) a l s o found d i f f e r e n c e s i n AAA between three l e v e l s of input with f i v e g r a i n s v a r i e t i e s . The author reported that the d i f f e r e n c e s were r e l a t i v e l y small and i n c o n s i s t e n t , and suggested that the 89 d i f f e r e n c e s might be a t t r i b u t e d to experimental e r r o r r a t h e r than to some b i o l o g i c a l phenomenon. The d i f f e r e n c e i n AAA with v a r y i n g l e v e l s of p r o t e i n i n the present study seem to be i n f l u e n c e d by p r o t e i n source. The a n a l y s i s of v a r i a n c e (Table 4, Appendix) show that the mean squares f o r PL were much s m a l l e r than f o r PS, t h e r e f o r e , the t o t a l experimental v a r i a t i o n i s i n f l u e n c e d to a g r e a t e r extent by PS. On the other hand, s i g n i f i c a n t PS x PL i n t e r a c t i o n s (Table 4, Appendix) were obtained f o r a s p a r t i c a c i d (P<0.05) and l y s i n e (P<0.01). The a v a i l a b i l i t y of a s p a r t i c a c i d and l y s i n e was s i g n -i f i c a n t l y improved when HFM was i n c o r p o r a t e d at l e v e l s g r e a t e r than 10% CP (Table 22 and Table 23). Fu r t h e r r e s e a r c h i s needed to c l a r i f y the e f f e c t s of PL on AAA and the i n t e r a c t i o n s between PL and PS on AAA f o r p r o t e i n - r i c h feed i n g r e d i e n t s f o r the growing c h i c k s . In c o n c l u s i o n , the samples of HFM and BM used i n the present study were q u i t e d i g e s t i b l e by the growing c h i c k s . However, AAA f o r HFM and BM were s t i l l i n f e r i o r to those f o r SBM and MM, except f o r p h e n y l a l a n i n e . The a v a i l a b i l i t y of a s p a r t i c a c i d and l y s i n e f o r HFM improved when the l e v e l s of i n c l u s i o n i n c r e a s e d . Moreover, there might be a r e l a t i o n s h i p between PS versus the l e v e l of i n c l u s i o n s , and some i n g r e d i e n t s might be a f f e c t e d more than the o t h e r s . Experiment V. The o b j e c t i v e s of t h i s experiment were as f o l l o w s : (1) to 90 Table 22. The i n t e r a c t i o n of p r o t e i n source (PS) x p r o t e i n l e v e l (PL) f o r a s p a r t i c a c i d a v a i l a b i l i t y (Experiment IV) PL (%) PS 10 20 30 HFM 2 75.6 d 82.5 C • 84.4 C Blood meal 90.0 b 9 2 . 7 a b 9 2 . 7 a b Soybean meal 96.0 a 96.2 a 9 5 . 5 a b MM2 9 1 . 3 a b 9 5 . 0 a b 9 3 . 5 a b *Means with d i f f e r e n t s i g n i f i c a n t l y d i f f e r e n t at P<0 s u p e r s c r i p t s .05. The SEM i s are ; 1.15. 2 HFM = hydrolyzed f e a t h e r meal; MM = meat meal. and bone Table 23. The i n t e r a c t i o n of p r o t e i n source p r o t e i n l e v e l (PL) f o r a v a i l a b i l i t y (Experiment IV) (PS) x l y s i n e PL (%) PS 10 20 30 HFM 2 72.3 D 86.2 C 8 7 . 6 B C Blood meal 9 1 . 5 A B C 93.6 A 9 2 . 8 A B Soybean meal 96.8 A 96.6 A 96.3 A MM2 9 2 . 5 A B 96.3 A 94.9 A Means with d i f f e r e n t s u p e r s c r i p t s are s i g n i f i c a n t l y d i f f e r e n t at P<0.01. The SEM i s 1.22. HFM = hydrolyzed f e a t h e r meal; MM = meat and bone meal. 91 examine the r e l a t i o n s h i p between vo l u n t a r y i n t a k e of a l p h a -c e l l u l o s e from a N-free d i e t and AA e x c r e t i o n s i n the growing c h i c k s , and to use the r e g r e s s i o n equations e s t a b l i s h e d from these r e l a t i o n s h i p s to c o r r e c t f o r the TAAE value used f o r the c a l i b r a t i o n s of AAA (Equation I I ) i n f e e d s t u f f s . (2) to r e -examine the e f f e c t s of PL and PS on AAA i n the growing c h i c k s using three d i f f e r e n t ECM s i n g l y or i n combination i n d i f f e r e n t f a c t o r i a l arrangements. P a r t 1 of t h i s experiment i n v o l v e s the c a l i b r a t i o n of AA e x c r e t i o n s from b i r d s with d i f f e r e n t amount of v o l u n t a r y a l p h a -c e l l u l o s e i n t a k e . There were s i g n i f i c a n t (P<0.01) e f f e c t s of dry matter (DM) i n t a k e on f e c a l DM output i n b i r d s fed the N-free d i e t s ( r e f e r to Table 5, Appendix). However, no s i g n i f i c a n t (P>0.05) c o r r e l a t i o n was observed between DM i n t a k e and f e c a l N output ( r e f e r to Table 6, Appendix). D i f f e r e n t r e s u l t s were obtained by Raharjo and F a r r e l l (1984) who r e p o r t e d that there were s i g n i f i c a n t (P<0.01) e f f e c t s of ADF content of the d i e t on N i n i l e a l d i g e s t a and e x c r e t a of b i r d s fed on N-free d i e t s . The d i f f e r e n c e between the present f i n d i n g s and those of Raharjo and F a r r e l l may be a t t r i b u t a b l e to methods of f e e d i n g of the N-free d i e t s . Voluntary N-free d i e t i n t a k e was employed i n t h i s study where there was no c o n t r o l over the l e v e l s of feed i n p u t . On the other hand, the b i r d s used by the other workers were t r a i n e d to consume lOOg of f eed. A l s o , the ages of the b i r d s were d i f f e r e n t i n the two t r i a l s . 92 Despite the f a c t that there were no s i g n i f i c a n t c o r r e l a t i o n s between DM i n t a k e and N output, there were s i g n i f i c a n t c o r r e l a t i o n s between a l p h a - c e l l u l o s e i n t a k e s and AA e x c r e t i o n s f o r h i s t i d i n e , l e u c i n e , l y s i n e , p h e n y l a l a n i n e , threonine and t y r o s i n e (Table 24). Less s i g n i f i c a n t (P<0.10) data were obtained f o r a l a n i n e , a s p a r t i c a c i d , i s o l e u c i n e , p r o l i n e and s e r i n e (Appendix Table 7 ). The i n t e r c e p t s of the r e g r e s s i o n s ranged from 4.8 f o r methionine to 39.2 f o r glutamic a c i d . S i m i l a r f i n d i n g s were obtained by Raharjo and F a r r e l l (1984). These authors r e p o r t e d s i g n i f i c a n t (P<0.05) i n c r e a s e s i n l y s i n e , t h reonine and i s o l e u c i n e output with i n c r e a s i n g r i c e h u l l s i n the d i e t . D i f f e r e n t f i n d i n g s were obtained by S i b b a l d (1979b). They administered graded l e v e l s of glucose monohydrate i n the a d u l t SCWL r o o s t e r and found no c o r r e l a t i o n between glucose input (Xg) and AA e x c r e t i o n (Ymg). The d i f f e r e n c e between the present f i n d i n g s and those of S i b b a l d (1979b) might be due to the carbohydrate sources (glucose verus a l p h a - c e l l u l o s e ) . Part 2 of t h i s experiment was subdivided i n t o seven d i f f e r e n t s u b s e c t i o n s ( r e f e r to Table 12). Part 2a was designed to i n v e s t i g a t e the e f f e c t s of two PS (HFM and SBM), three methods of ECM (AEC, IEC and REC) and four PL (10, 15, 20 and 25% CP). A n a l y s i s of v a r i a n c e i n Appendix Table 8 shows that there were s i g n i f i c a n t (P<0.01) d i f f e r e n c e s i n AAA r e s u l t i n g from PS i n a l l 16 AAs s t u d i e d . On the other hand, 11 out of the 16 AAs were s i g n i f i c a n t l y a f f e c t e d by PL. A l s o , no s i g n i f i c a n t d i f f e r e n c e 93 Table 24. The r e l a t i o n s h i p s between amino a c i d (AA) e x c r e t i o n (Ymg) and a l p h a - c e l l u l o s e i n t a k e (Xg) from a N-free d i e t i n Experiment V, Part 1 C o r r e l a t i o n Regression c o e f f i c i e n t I n t e r c e p t c o e f f i c i e n t Mean AA ( r ) (a) . (b) of Y Ala n i n e 0.328 14.6 0.906+ 23.3 A r g i n i n e 0.281 15.7 0.750 23.0 A s p a r t i c a c i d 0.331 28.4 1.784+ 45.6 C y s t i n e 0.244 13.9 0.404 17.8 Glutamic a c i d 0.261 39.2 1.472** 56.7 H i s t i d i n e 0.513 24.7 39.0 I s o l e u c i n e 0.310 9.3 0.581+ 14.9 Leucine 0.372 20.9 34.9 L y s i n e 0.347 27.8 1.048 38.0 Methionine 0.207 4.8 0.199. 6.8 Phe n y l a l a n i n e 0.443 13.3 0.999 23.0 P r o l i n e 0.297 26.7 1.077+ 37.1 S e r i n e 0.297 20.8 0.936+ 29.9 Threonine 0.381 19.5 29.9 T y r o s i n e 0.375 10.4 0.709 17.3 V a l i n e 0.265 14.4 0.658 20.8 There were 32 p a i r s of o b s e r v a t i o n s f o r each AA. The average in t a k e of the N-free d i e t from the 32 groups of four b i r d s each was 74g, ranging from 45 to l O l g . The average amount of endogenous f e c a l e x c r e t i o n s was 13.4g/group. The r e g r e s s i o n c o e f f i c i e n t s are s i g n i f i c a n t at P<0.10. The r e g r e s s i o n c o e f f i c i e n t s are s i g n i f i c a n t at P<0.05. The r e g r e s s i o n c o e f f i c i e n t i s s i g n i f i c a n t at P<0.01. 94 was observed i n AAA with the three ECM. Moreover, a s p a r t i c a c i d , c y s t i n e , p h e n y l a l a n i n e and t y r o s i n e were a f f e c t e d by PS x PL i n t e r a c t i o n s at d i f f e r e n t l e v e l s of p r o b a b i l i t y . The r e s u l t s (Table 25) show a s i g n i f i c a n t d i f f e r e n c e i n AAA f o r HFM and SBM. These f i n d i n g s , s i m i l a r to those i n Experiment IV, were d i s c u s s e d i n the previous s e c t i o n . However, i n t h i s experiment, two a d d i t i o n a l AAs were a l s o s t u d i e d . The c y s t i n e and methionine a v a i l a b i l i t i e s f o r HFM were 78.6 and 83.9%, r e s p e c t i v e l y . These values were s i g n i f i c a n t l y lower than those reported by Burgos jet al_. (1974) and K i r b y ejt a l . (1978). The values r e p o r t e d by these authors were a l l g r e a t e r than 93%. On the other hand, much lower values than those obtained i n the present i n v e s t i g a t i o n were r e p o r t e d by E l Boushy and Roodbeen (1984) and Papadopoulos ejt al. (1985). The a v a i l a b i l i t y of c y s t i n e and methionine was each reported to be 65% i n the study of E l Boushy and Roodbeen (1984) and much lower values of 30 to 60% were obtained by Papadopoulos je_t .al. (1985). The a v a i l a b l e c y s t i n e i n HFM used i n the present study was c a l c u l a t e d to be 3.2%, which i s higher than the values of 2.92 and 2.89% obtained by the standard curve method and s l o p e - r a t i o assessment, r e s p e c t i v e l y (Baker je_t a l . , 1981). The a v a i l a b i l i t e s of c y s t i n e and methionine f o r SBM were 90.5 and 93.2%, r e s p e c t i v e l y , which were lower than those reported by Ivy e_t a_l. (1971) and Nwokolo et. a l . (1976b). S i m i l a r values were repo r t e d by L i k u s k i and D o r r e l l (1978). 95 Table 25. Comparison of amino a c i d (AA) a v a i l a b i l i t y from hydrolyzed f e a t h e r meal (HFM) and soybean meal (SBM) i n the growing c h i c k (Experiment V, Part 2 a ) 1 ' 2 P r o t e i n source AA HFM SBM SEM Alanine A r g i n i n e A s p a r t i c a c i d C y s t i n e Glutamic a c i d H i s t i d i n e I s o l e u c i n e Leucine L y s i n e Methionine P h e n y l a l a n i n e P r o l i n e S e r i n e Threonine T y r o s i n e V a l i n e Average 90 92 82 78 86 78 93 93 86 83 90 88 88 86 90 91 0 B 0 B iB 0 B B ,B 87.4 B 95 97 95 90 96 91 96 96 97 93 93 95 95 94 94 96 95.1 J 0.22 0.16 0.32 0.34 0.26 0.38 0.19 0.20 0.42 0.39 0.25 0.24 0.24 0.28 0.32 0.22 0.20 An eight-hour f e c a l c o l l e c t i o n p e r i o d was used. Means w i t h i n each row not s h a r i n g a common s u p e r s c r i p t are s i g n i f i c a n t l y d i f f e r e n t at P<0.01. 96 The r e s u l t s (Table 26) show that the mean AAA values were s i g n i f i c a n t l y lower at PL of 25%. The a v a i l a b i l i t i e s of a l a n i n e , a r g i n i n e , i s o l e u c i n e , l e u c i n e , l y s i n e , p r o l i n e , s e r i n e , and v a l i n e f o l l o w the same p a t t e r n as the mean AAA. On the other hand, the AAA values f o r methionine, p h e n y l a l a n i n e and t y r o s i n e were the hi g h e s t at the 20% PL. The r e s u l t s of t h i s experiment * were d i f f e r e n t than those obtained i n Experiment IV where the AAA were s i g n i f i c a n t l y lower at the 10% PL. Comparing of the AAA using the three ECM, the r e s u l t s (Table 27) show no s i g n i f i c a n t d i f f e r e n c e i n AAA among the three methods. T h i s may suggest that the use of the r e g r e s s i o n equations, e s t a b l i s h e d from a l p h a - c e l l u l o s e i n t a k e s and AA e x c r e t i o n s , i s r e l i a b l e f o r the AAA assay u t i l i z e d . The i n t e r a c t i o n s between PS and PL (Table 28) show no c o n s i s t e n t trends with the four AAs. The d i f f e r e n c e i n AAA with graded l e v e l s of p r o t e i n seem to be caused by the complexity of the experiment which r e s u l t e d i n a small e r r o r mean square. Attempts were made to re-a n a l y s e the data by reducing the l e v e l of complexity from the o r g i n a l design as d e s c r i b e d p r e v i o u s l y i n Table 12. Part 2b, 2c and 2d of t h i s study i n v o l v e d the r e c a l c u l a t i o n s of the data i n part 2a a f t e r o m i t t i n g only one ECM at a time. The three r e s u l t a n t experimental designs were 2 PS x 2 ECM x A PL f a c t o r i a l arrangements. From the analyses of v a r i a n c e (Appendix Table 9 to Table 11) , i t i s c l e a r that PS was the main s i g n i f i c a n t v a r i a b l e . 97 Table 26. Comparison of amino a c i d (AA) a v a i l a b i l i t y from d i e t s of d i f f e r e n t p r o t e i n l e v e l .(EL) i n the growing c h i c k (Experiment V, Part 2a) ' PL (%) AA 10 15 20 . 25 SEM Alanine 9 3 - 7 a 9 3 - 4 3 9 3 - 4 A D 92.3 b 0.31 Ar g i n i n e 95.5 A 95.2 A 9 4 . 9 A B 94.3 B 0.23 A s p a r t i c a c i d 88.8 88.6 89.1 88.2 0.46 Cy s t i n e 83.7 85.0 84.2 85.3 0.48 Glutamic a c i d 91.8 91.9 91.7 90.9 0.37 H i s t i d i n e 8 4 - 3 » 8 5 - 1 A 8 5 - 1 A 8 4 - ° N ° - 5 3 I s o l e u c i n e 9 5 - 6 A R 95.7 A 9 ^ . 2 A 94.1 B 0.27 Leucine 9 4 . 6 A B 95.2 A 9 5 . 1 A , 93.8 B 0.29 Ly s i n e 93.2^ 92.3 a 9 1 . 7 a b 90.5 b 0.59 Methionine 8 8 . 8 A B 8 7 . 2 B 90.4 A 8 7 . 8 B 0.56 Pheny l a l a n i n e 90.5 B 91.3 B 93.8 A 90.9 B 0.53 P r o l i n e 9 2 - 5 a i < 9 2 - 4 A 92.3 a 91.2J 0.34 Serine 9 1 . 7 A B 92.5 A 92.2 A 90.6 B 0.34 Threonine 9 1 - 2 n 9 0 - 0 A N 9 1 - ° A 9 0 - 0 D ° - 4 0 T y r o s i n e 9 1 - 3 A 9 2 . 6 A B 9 ^ . 2 A I J 9 l - 0 ? 0.46 V a l i n e 93.9 A 94.3 A 9 3 . 5 A B 92.6 B 0.31 Average 91.3 a 91.5 a 91.7 a 90.5 b 0.28 *An eight-hour f e c a l c o l l e c t i o n p e r i o d was used. 2 Means w i t h i n each row not sha r i n g a common n o n - c a p i t a l i z e d or c a p i t a l i z e d s u p e r s c r i p t are s i g n i f i c a n t l y d i f f e r e n t at P<0.05 or P<0.01, r e s p e c t i v e l y . 98 Table 27. Comparison of amino a c i d (AA) a v a i l a b i l i t y from three d i f f e r e n t endogenous amino a c i d c o r r e c t i o n method (ECM) i n the growing c h i c k (Experiment V, Part 2 a ) 1 ' 2 ECM AA I E C 3 AEC 3 * REC 3 SEM Ala n i n e 93. .5 93. .2 92, .8 0. ,27 A r g i n i n e 95. .1 95. .1 94. .8 0. .20 A s p a r t i c a c i d 89, .0 88. .2 88. .8 0, .40 C y s t i n e 84. .2 84. .8 84, .7 0. .42 Glutamic a c i d 91, .7 91. .5 91. .5 0. .32 H i s t i d i n e 84, .8 84. .5 84, .5 0. .46 I s o l e u c i n e 95. .3 95. .1 95. .0 0. .23 Leucine 95. .0 94. .5 94. .5 0. .25 Ly s i n e 92, .3 92. .1 91. .3 0. .52 Methionine 88. .9 88. ,2 88. .5 0, .48 Ph e n y l a l a n i n e 91. .7 91. .6 91. .5 0. ,31 P r o l i n e 92. .2 92. .1 92, .0 0. .30 Serine 92, .0 91. .5 91, .8 0. .30 Threonine 90. .9 90. .8 90. .6 0. ,35 T y r o s i n e 92. .5 92. .1 92. .2 0. ,39 V a l i n e 93, .9 93. .4 93. .5 0. ,27 Average 91, .5 91. .2 91. .1 0. ,24 An eight-hour f e c a l c o l l e c t i o n p e r i o d was used. None of the ECM are s i g n i f i c a n t l y d i f f e r e n t at the 5% l e v e l of p r o b a b i l i t y . o IEC = i n d i v i d u a l endogenous c o r r e c t i o n method; AEC = average endogenous c o r r e c t i o n method; REC = r e g r e s s i o n endogenous c o r r e c t i o n method. 99 Table 28. I n t e r a c t i o n s of p r o t e i n source (PS) x p r o t e i n l e v e l (PL) i n amino a c i d a v a i l a b i l i t y (Experiment V, Part 2 a ) 1 ' 2 PS HFM" SBM" PL (%) 10 15 20 25 10 15 20 25 SEM Ala Arg Asp Cys Glu His H e Leu Lys Met Phe Pro Ser Thr Tyr V a l 7 5 7^ 9 6 6 C 90. 92. 81. 78. 86.4 77.8 93.5 94.3 90 92 81 77.4' 86.1 77.1 BC 92.8 87.1 84 90 88 87 86 90 91.0 2 C D 6 6 ?DE D 93 86 81 89 88 89.1 8 6 . 8 _ 9 0 . 4 D E 91.6 91.3 92.2 83.4^ 77.4° 86.6 78.5 93.8 93.2 86.3 8 5 . 3 ^ 9 2 . 0 B C 88.8 88.9 87.5 91.2 D 91.4 89.9 92.0 80.8 B 85.6 78.6 92.2 92.2 84.6 83.8 89.0° 87.2 86 86, 88.1 J 90.1 96.6 98.4 95. 9 a 90.0 A 97.4 91.5 97.6 96.3 99.2 9 2' 7CD 9 0 . 7 L U96.4 95.8 9 2 . 2 L U 96.9 95, 97, 95, 91, 97, 92, 97, 9 9 1a 4 3 1 ab 96.6 98.2 92.9 93.0 B 96.2 95.9 94.9 94.8 B 97.1 95.5 97.6 94.9 A 9 1 . 0 A 96.8 91.6 96.6 97.0 97.0 95.4, 95.7 A 95.9 95.5 94.6 A 97.3 A 95.6 ab 94.6 96.7 93.8° 89.8 A 96.2 89.4 95.9 95.5 96.3 9 1 . 9 92.8 B 95.2 95 93 93.9 95.1 B 0.44 0.33 0.65 0.68 0.52 0.76 0.38 0.41 0.84 0.79 0.50 0.48 0.48 0.57 0.64 0.44 Ave 87.4 87.5 88.0 86.8 95.2 95.4 95.5 94.1 0.39 •*An eight-hour f e c a l c o l l e c t i o n p e r i o d was used. 2 Means w i t h i n each row not sh a r i n g a common n o n - c a p i t a l i z e d or c a p i t a l i z e d s u p e r s c r i p t are s i g n i f i c a n t l y d i f f e r e n t at P<jD.05 or P<0.01, r e s p e c t i v e l y . o JHFM = hydrolyzed f e a t h e r meal; SBM = soybean meal. 100 S i g n i f i c a n t d i f f e r e n c e s i n AAA r e s u l t e d from PL f o r 7, 10 and 8 out of the t o t a l 16 AAs i n pa r t s 2b, 2c and 2d, r e s p e c t i v e l y . The s i g n i f i c a n t PS x PL i n t e r a c t i o n s were 2, 1 and 3 i n pa r t s 2b, 2c and 2d, r e s p e c t i v e l y . No s i g n i f i c a n t d i f f e r e n c e was obtained from the ECM. The r e s u l t s (Table 29 to Table 37) show no c o n s i s t e n t trends i n AAA from d i f f e r e n t PL or PS x PL i n t e r a c t i o n s . P a r t 2e, 2f and 2g i n t h i s t r i a l i n v o l v e d the se g r e g a t i o n s of the data i n part 2a i n t o three 2 PS x 4 PL f a c t o r i a l arrangements. The r e s u l t s show that only the a v a i l a b i l i t y of p h e n y l a l a n i n e was s i g n i f i c a n t l y a f f e c t e d by PL i n each of p a r t s 2e, 2f and 2g (Tables 38, 39 and 40) . T h i s i m p l i e s that the a v a i l a b i l i t y of phe n y l a l a n i n e was optimumized at 20% CP l e v e l . T y r o s i n e was a l s o s i g n i f i c a n t l y a f f e c t e d i n p a r t s 2e and 2f with a maximum value at the 20% CP l e v e l . A n a l y s i s of va r i a n c e (Appendix Tables 12, 13 and 14) shows that there were s t i l l s i g n i f i c a n t (P<0.01) d i f f e r e n c e s i n AAA r e s u l t i n g from PS. However, only 5, 3 and 4 out of the 16 AAs i n p a r t s 2e, 2f and 2g, r e s p e c t i v e l y , were s i g n i f i c a n t l y a f f e c t e d by the v a r y i n g PL. No s i g n i f i c a n t d i f f e r e n c e was obtained f o r the PS x PL i n t e r a c t i o n s . In c o n c l u s i o n , there were s i g n i f i c a n t c o r r e l a t i o n s between a l p h a - c e l l u l o s e i n t a k e and AA e x c r e t i o n s . Regression equations e s t a b l i s h e d from t h i s r e l a t i o n s h i p could be used to c o r r e c t f o r the TAAE i n the c a l c u l a t i o n of AAA. The s i g n i f i c a n t d i f f e r e n c e s 101 Table 29. Comparison of amino a c i d (AA) a v a i l a b i l i t y from d i e t s of d i f f e r e n t p r o t e i n l e v e l (PL) i n the growing c h i c k (Experiment V, Part 2b) • AA 10 PL (%) 15 20 25 SEM Ala n i n e A r g i n i n e A s p a r t i c a c i d C y s t i n e Glutamic a c i d H i s t i d i n e I s o l e u c i n e Leucine L y s i n e Methionine Phe n y l a l a n i n e P r o l i n e S e r i n e Threonine T y r o s i n e V a l i n e Average .7 ,4 ,5. 93 95 88 83.2 91.8 84.0 95 AB 9 4 . 7 a b 93.3 88, 90. 92, 9 1 . 9 1 . 91. ab 4 B 4 6AB ?B 93.8 93.5 95.3 88.6 8 5 . l a 92.0 85. 2 A 95.8 A 95.3 a 9 2 . 5 . 87.0 b 91.3 B 92.5 A 92.6 A 91.0._ 9 2 . 8 A B 94.4 93.5 95.0 89.2 84.3 91.8 85.2 95 95 92 90 93 92 92 91 ab AB 91.2 ab 91.5 ab 3 2 a 4 9 A 4 2 94.3 A 93.6 91.9 a AB 92.7 94.6 88.2 85.3 a 90.9 84.3 94.2 B 93.8 b 90.7 V 88.2 91.1 91.3 90.6 90.0 91. 0 B 92.8 90.6 b B B 0.35 0.27 0.57 0.56 0.45 0.60 0.33 0.34 0.74 0.74 0.42 0.42 0.41 0.46 0.57 0.39 0.32 1 An eight-hour f e c a l c o l l e c t i o n p e r i o d was used Means w i t h i n each row not s h a r i n g a common n o n - c a p i t a l i z e d or c a p i t a l i z e d s u p e r s c r i p t are s i g n i f i c a n t l y d i f f e r e n t at P<0.05 or P<0.01, r e s p e c t i v e l y . 102 Table 30. The i n t e r a c t i o n of p r o t e i n source (PS) x p r o t e i n l e v e l (PL) f o r phe n y l a l a n i n e a v a i l a b i l i t y (Experiment V, P a r t 2b) PL (%) PS 10 15 20 25 HFM 2 90.3 d 89.7 d 92. l*bc 8 9 . l d Soybean meal 9 0 . 5 c d 92.9 b 95. 8 a 9 3 . l b Means with d i f f e r e n t s u p e r s c r i p t are s i g n i f i c a n t l y d i f f e r e n t at P<0.05. The SEM i s 0.6. HFM = hydrolyzed f e a t h e r meal. Table 31. The i n t e r a c t i o n of p r o t e i n source (PS) x p r o t e i n l e v e l (PL) f o r t y r o s i n e a v a i l a b i l i t y (Experiment V, Part 2b) PL (%) PS 10 15 20 25 HFM 2 9 0 . 3 d e 90.7 d 91.3 d 88. 0 e Soybean meal 9 2 . 0 c d 94.8 b 97.4 a 9 4 . 1 b c Means with d i f f e r e n t s u p e r s c r i p t are s i g n i f i c a n t l y d i f f e r e n t at P<0.05. The SEM i s 0.8. HFM = hydrolyzed f e a t h e r meal. 103 Table 32. Comparison of amino a c i d (AA) a v a i l a b i l i t y from d i e t s of d i f f e r e n t p r o t e i n l e v e l .(EL) i n the growing c h i c k (Experiment V, Part 2c) • PL (%) AA 10 15 20 25 SEM Ala n i n e A r g i n i n e A s p a r t i c a c i d C y s t i n e Glutamic a c i d H i s t i d i n e I s o l e u c i n e Leucine L y s i n e Methionine Ph e n y l a l a n i n e P r o l i n e S e r i n e Threonine T y r o s i n e V a l i n e Average 93.5 a 95.4 a 88.5 84.6 91.8 84.4 A 95.4 A 9 4 . 2 a b 92.7 A_ 8 8 . 8 A B 90.4 B 92.5 a 91.6 91.0 AB 91 93 ,2 B 6 a 93.4 a 95.3 a 88.5 85.1 91.9 85.0. 95 95 92 86 91 92 92 90 ,7* 2 a ,6 A ,9 91.2 ab 9 2 . 6 A B 94.4 a 91.5 a 93.3 a 9 4 . 9 a b 89.1 84.0 91.7 9 5 . 1 A B 95.0 a 91.3 A 90.4 A 93.8 A 92.3 a 92.1 A 91.0 A 94.2 A 9 3 . 4 a b 91.7 a 91.8 b 94.2 b 87.8 85, 90, 83, 93. 93, 90, 87, B ;B B 90.8 91.0 b 90.4 B 89.7 90.6 92.3 1 90.2' B 0.41 0.30 0.58 0.63 0.48 0.69 0.34 0.40 0.73 0.69 0.46 0.43 0.42 0.49 0.59 0.42 0.36 An eight-hour f e c a l c o l l e c t i o n p e r i o d was used. "Means w i t h i n each row not s h a r i n g a common n o n - c a p i t a l i z e d or c a p i t a l i z e d s u p e r s c r i p t are P<0.05 or P<0.01, r e s p e c t i v e l y . s i g n i f i c a n t l y d i f f e r e n t at 104 Table 33. The i n t e r a c t i o n of p r o t e i n source (PS) x p r o t e i n l e v e l (PL) f o r methionine a v a i l a b i l i t y (Experiment V, Part 2c) PL (%) PS 10 15 20 25 HFM 2 85.2 C 80. 5 d 85.6 C 83.8 C Soybean meal 9 2 . 4 a b 9 2 . 9 a b 95.2 a 91.3 b Means with d i f f e r e n t s u p e r s c r i p t are s i g n i f i c a n t l y d i f f e r e n t at P<0.05. The SEM i s 0.98. 2HFM = hydrolyzed f e a t h e r meal. 105 Table 34. Comparison of amino a c i d (AA) a v a i l a b i l i t y from d i e t s of d i f f e r e n t p r o t e i n l e v e l (PL) growing c h i c k (Experiment V, Part 2d) * i n the PL (%) AA 10 15 20 25 SEM Ala n i n e A r g i n i n e A s p a r t i c a c i d C y s t i n e Glutamic a c i d H i s t i d i n e I s o l e u c i n e Leucine L y s i n e Methionine Ph e n y l a l a n i n e P r o l i n e S e r i n e Threonine T y r o s i n e V a l i n e 93.7 95. 5 a 89.4 83.3 91.8 84.5 A 95.8 A 94.8 93. 5 a ab 88 90 92 92 91 91 94 7 B 6 , 0 a b 93.3 95.2 a 88.6 84.9 91.8 85.0, 95.6 A 95.0 v 9 1 . 9 a b 87 91 92 92 90 92 94 2B 3 4 a .7 cAB 93, 94, 89, 3 , o,ab 1 84.1 91.7 85.0 95, 95, 91, 90, 93, 92, 92, AB 1 3 3 a 91.0 94, 93, 92 94, 88 85 91 6 4 2 84.0 94.1 B 94.2 90.5 b 87.8 b 90.9 B 91.3, 90.8 b 90.2„ ;AB 91.4 92.7 0.38 0.28 0.53 0.57 0.43 0.66 0.32 0.31 0.72 0.61 0.42 0.41 0.43 0.52 0.52 0.35 Average 91.5 91.4 91.7 90.6 0.33 An eight-hour f e c a l c o l l e c t i o n p e r i o d was used. Means w i t h i n each row not s h a r i n g a common n o n - c a p i t a l i z e d or c a p i t a l i z e d s u p e r s c r i p t are s i g n i f i c a n t l y d i f f e r e n t at P<0.05 or P<0.01, r e s p e c t i v e l y . 106 Table 35. The i n t e r a c t i o n of p r o t e i n source (PS) x p r o t e i n l e v e l (PL) f o r c y s t i n e a v a i l a b i l i t y (Experiment V, Part 2d) PL (%) PS 10 15 20 25 HFM 2 77.3 C 78.9 C 77.3 C 81.2 b Soybean meal 89.4 a 90.9 a 91.0 a 89.7 a Means with d i f f e r e n t s u p e r s c r i p t are s i g n i f i c a n t l y d i f f e r e n t at P<0.05. The SEM i s 0.8. HFM = hydrolyzed f e a t h e r meal. Table 36. The i n t e r a c t i o n of p r o t e i n source (PS) x p r o t e i n l e v e l (PL) f o r phe n y l a l a n i n e a v a i l a b i l i t y (Experiment V, Part 2d) PL (%) PS 10 15 20 25 HFM 2 Soybean meal 9 0 . 4 c d e 9 0 . 9 c d 8 9 . 3 d e 9 3 . l b 9 1 . 9 b c 95.6 a 88. 9 e 92. 8 b Means with d i f f e r e n t s u p e r s c r i p t are s i g n i f i c a n t l y d i f f e r e n t at P_<0.05. The SEM i s 0.59. HFM = hydrolyzed f e a t h e r meal. 107 Table 37. The i n t e r a c t i o n of p r o t e i n source (PS) x p r o t e i n l e v e l (PL) f o r t y r o s i n e a v a i l a b i l i t y (Experiment V, Part 2d) PL (%) PS 10 15 20 25 HFM 2 9 0 . 6 c d 9 0 . 3 c d 9 1 . 0 c d 88.9 d Soybean meal 9 2 . 5 b c 94.6 b 97.3 a 93.9 b Means with d i f f e r e n t s u p e r s c r i p t are s i g n i f i c a n t l y d i f f e r e n t at P<0.05. The SEM i s 0.73. 2HFM = hydrolyzed f e a t h e r meal. 108 Table 38. Comparison of amino a c i d (AA) a v a i l a b i l i t y from d i e t s of d i f f e r e n t p r o t e i n l e v e l .(£L) i n the growing c h i c k (Experiment V, Part 2e) ' PL (%) AA 10 15 20 . 25 SEM Ala n i n e 93.5 93.7 93.5 92.3 0.53 A r g i n i n e 95.4 95.4 95.0 94.6 0.41 A s p a r t i c a c i d 87.6 88.5 89.3 87.5 0.87 C y s t i n e 84.5 85.4 84.3 84.9 0.89 Glutamic a c i d 91.8 92.0 91.8 90.3 0.70 H i s t i d i n e 83.8 85.3 85.2 83.9 0.89 I s o l e u c i n e 95.1 95.9 95.4 94.1 0.49 Leucine 9 4 . 1 a b 95.4 a 95.2 a 9 3 . l b 0.61 Ly s i n e 92.5 93.0 92.4 90.4 1.06 Methionine 8 8 . 4 a b 85.7 b 90.6 a 8 8 . 0 a b 1.14 Ph e n y l a l a n i n e 90.1 B 9 1 . 4 A B 94.0 A 91.0 B 0.66 P r o l i n e 92.4 92.7 92.4 91.0 0.63 Seri n e 9 1 . 2 a b 92.8 a 92.0 a 90.2 b 0.57 Threonine 9 1 - 1 v 9 1 - 3 v 9 1 - 2 8 9 ' 5 u ° ' 6 1 T y r o s i n e 90.8 b 9 2 . 9 a b 94.4 a 90.3 b 0.89 V a l i n e 93.2 94.4 93.5 92.4 0.63 Average 91.0 91.6 91.9 90.2 0.51 ^An eight-hour f e c a l c o l l e c t i o n p e r i o d was used. Means w i t h i n each row not sh a r i n g a common n o n - c a p i t a l i z e d or c a p i t a l i z e d s u p e r s c r i p t are s i g n i f i c a n t l y d i f f e r e n t at P<0.05 or P<0.01, r e s p e c t i v e l y . 109 Table 39. Comparison of amino a c i d (AA) a v a i l a b i l i t y from d i e t s of d i f f e r e n t p r o t e i n l e v e l (PL) i n the growing c h i c k (Experiment V, Part 2f) ' PL (%) AA 10 15 20 . 25 SEM Alan i n e 93.9 93.4 93.5 93.2 0.47 A r g i n i n e 95.5 95.2 95.0 94.7 0.36 A s p a r t i c a c i d 8 9 « 4 B 8 8 > 7 A 89.2 8 9 - ° » 0.72 C y s t i n e 81.9 B 84.9 A 8 4 . 4 A B 85.6 A 0.69 Glutamic a c i d 91.8 91.9 91.8 91.5 0.57 H i s t i d i n e 84.2 85.1 85.2 84.6 0.80 I s o l e u c i n e 95.9 95.6 95.3 94.3 0.44 Leucine 95.3 95.1 95.2 94.5 0.31 Lys i n e 94.1 91.9 92.4 91.0 1.02 Methionine 8 8 * 7 R 8 8 ' 2 n 9 0 * 3 A 8 8 ' 4 i 3 ° ' 3 5 P h e n y l a l a n i n e 90.7 B 91.2 B 93.9 A 91.1 B 0.53 P r o l i n e 92.5 92.3 92.4 91.6 0.54 Seri n e 92.0 92.4 92.5 91.0 0.58 Threonine 9 1 - 4 L 9 0 - 8 ^ 91.1 90.5 0.69 Ty r o s i n e 91.5 b 9 2 . 6 a b 94.3 a 91.8 b 0.69 V a l i n e 94.5 94.3 93.6 93.2 0.44 Average 91.4 91.5 91.9 91.0 0.40 *An eight-hour f e c a l c o l l e c t i o n p e r i o d was used. 9 Means w i t h i n each row not sh a r i n g a common n o n - c a p i t a l i z e d or c a p i t a l i z e d s u p e r s c r i p t are s i g n i f i c a n t l y d i f f e r e n t at P<0.05 or P<0.01, r e s p e c t i v e l y . 110 Table 40. Comparison of amino a c i d (AA) a v a i l a b i l i t y from d i e t s of d i f f e r e n t p r o t e i n l e v e l (EL) i n the growing c h i c k (Experiment V, Part 2g) ' PL (%)  AA 10 15 20 . 25 SEM Ala n i n e 93, .6 93, .2 93. .2 91. 0, .61 A r g i n i n e 95, .5 a 95, , l a 94. . 8 a b 93, .8 b 0, .42 A s p a r t i c a c i d 89, .4 88, .5 88. .9 88, . 1 0, .78 C y s t i n e 84. .8 84. .9 83. .8 85. .2 0. .91 Glutamic a c i d 91. .8 91. .7 91. ,6 90. .9 0. .64 H i s t i d i n e 84. .9 84. .8 84. 83. .4, 1. .06 I s o l e u c i n e 95. .7 a 95. .5 a 94. 9 a b 93. .8 b 0. .46 Leucine 94. .3 94. .9 94. .9 93. .9 0. .53 L y s i n e 92. .9 92. .0 90. ,2 90. .0 1. .01 Methionine 89, 87. • 7AT>. 90. .1. 87. 0. .79 P h e n y l a l a n i n e 90. .6 B 91. . 2 93. ,6 A 90. .6 B 0. .64 P r o l i n e 92, .6 92. .3 92. ,1 90. .9 0. .60 Seri n e 92, . 1 92, .4 92. ,2 90. .5 0, .62 Threonine 91, .0 90. .6 90. .8 89. .9 0, .77 Ty r o s i n e 91. .6 92. .3 94. •° i. 91. • ° K 0. .77 V a l i n e 94, . l a 94. .3 a 93. , 3 a b 92. .2 b 0. .54 Average 91, .5 91. .3 91. ,5 90. .2 0. .52 An eight-hour f e c a l c o l l e c t i o n p e r i o d was used. Means w i t h i n each row not s h a r i n g a common n o n - c a p i t a l i z e d or c a p i t a l i z e d s u p e r s c r i p t are s i g n i f i c a n t l y d i f f e r e n t at P<0.05 or P<0.01, r e s p e c t i v e l y . I l l i n AAA r e s u l t e d from v a r y i n g l e v e l s of p r o t e i n i n c l u s i o n s i n the present and previous experiments was probably the r e s u l t of s e v e r a l f a c t o r s i n c l u d i n g experimental design. F u r t h e r s t u d i e s r e l a t e d to the establishment of a set of u n i v e r s a l r e g r e s s i o n equations f o r the c a l i b r a t i o n s of TAAE, s i m i l a r to the REC, used f o r AAA assay w i l l save time and tedious l a b o u r . Experiment VI. The o b j e c t i v e of t h i s experiment was to examine the e f f e c t s of the d u r a t i o n of f e c a l c o l l e c t i o n s on AAA i n the growing c h i c k s . The technique of the AAA assay used i n the present i n v e s t i g a t i o n has been c i t i c i z e d on the grounds that the short d u r a t i o n of f e c a l c o l l e c t i o n p e r i o d might not be s u f f i c i e n t f o r t e s t r e s i d u e s to c l e a r the GIT of the growing b i r d s . As a r e s u l t , higher values i n AAA f o r f e e d s t u f f s were obtained by t h i s procedure. Comparison of the mean AA e x c r e t i o n s i n groups fed the N-free d i e t i n t h i s and the previous two experiments are presented i n Table 41. There were d i f f e r e n c e s i n f e c a l outputs f o r the three t r i a l s , which r e s u l t e d from a d i f f e r e n c e i n N-free d i e t i n t a k e s . However, the mean AA e x c r e t i o n s were not p r o p o r t i o n a l to the amount of f e c a l DM outputs. The f e c a l DM output obtained from four b i r d s of the same age, i n a 30-hour f e c a l c o l l e c t i o n period was only 1.26 times more than that from an 8-hour p e r i o d . Whereas the mean AA e x c r e t i o n s were between 1.96 ( p r o l i n e ) and 2.47 (methionine) times higher from the 30-hour c o l l e c t i o n period 112 Table 41. Comparison of the mean amino a c i d (AA) e x c r e t i o n s of b i r d s fed the N-free d i e t i n Experiment IV, V, and VI Mean AA e x c r e t i o n ^ 2 3 mg/group mg/bird AA E I V 4 EV 4 E V I 4 E I V 4 EV 4 E V I 4 A l a n i n e 7. 8 23. .3 A r g i n i n e 7. 2 23. ,0 A s p a r t i c a c i d 13. 45. ,6 Cy s t i n e NMJ 17. ,8 Glutamic a c i d 18. 0 56. ,7 H i s t i d i n e 24. 6 39. ,0 I s o l e u c i n e 13. 8 14. .9 Leucine 13. 2 34. .9 Ly s i n e 13. 8 38. .0 Methionine NM 6. .8 Phe n y l a l a n i n e 6. 6 23. .0 P r o l i n e 12. 6 37. .1 Ser i n e 11. 4 29. .9 Threonine 11. 4 29. .9 T y r o s i n e 9. 6 17. .3 V a l i n e 6. 6 20. .8 54.0 2.0 5.8 13.5 55.2 1.8 5.8 13.8 96.8 3.5 11.4 24.2 40.0 NM 4.5 10.0 118.0 4.5 14.2 29.5 78.8 6.2 9.8 19.7 36.4 3.5 3.7 9.1 78.4 3.3 8.7 19.6 88.8 3.5 9.5 22.2 16.8 NM 1.7 4.2 48.0 1.7 5.8 12.0 72.8 3.2 9.3 18.2 61.6 2.9 7.5 15.4 62.4 2.9 7.5 15.6 40.0 2.4 4.3 10.0 48.0 1.7 5.2 12.0 The mean endogenous f e c a l output f o r Experiment IV, V and VI i s 6.0, 13.4 and 16.9g, r e s p e c t i v e l y . A 30-hour f e c a l c o l l e c t i o n p e r i o d was used i n Experiment VI, and an 8-hour f e c a l c o l l e c t i o n p e r i o d was employed i n both Experiment IV and V. 2Mean AA e x c r e t i o n s were c a l i b r a t e d from 24, 32 and 4 groups of b i r d s i n Experiment IV, V and VI, r e s p e c t i v e l y . There are four b i r d s per group i n a l l three experiments. 3The b i r d s i n Experiment IV, V and VI were 24, 28 and 28-day o l d r e s p e c t i v e l y . 4 E I V = Experiment IV, EV = Experiment V, EVI = Experiment VI. -*NM = not measured. 113 used i n t h i s experiment as compared to those from the 8-hour p e r i o d . In s p i t e of t h i s unusual d i f f e r e n c e , the mean AAA were very much the same f o r the LEC and REC groups (Table 42). There were s i g n i f i c a n t (P<0.05) d i f f e r e n c e s i n the a v a i l a b i l i t y of c y s t i n e , i s o l e u c i n e and t y r o s i n e between the two methods. A n a l y s i s of va r i a n c e (Appendix Table 15) shows that these d i f f e r e n c e s may be caused by the low e r r o r mean square i n the f a c t o r i a l arrangement. The AAA values f o r both HFM and SBM i n t h i s study were comparable to those i n the previous experiments. T h i s suggests that the REC method may apply to b i r d s of the same age and type. Moreover, the TAAE e x c r e t i o n s (mg/bird) obtained i n 28-day o l d b i r d s from the 30-hour c o l l e c t i o n p e r i o d i n the present study were 2 to 3 times l e s s than those obtained from a 36-hour c o l l e c t i o n p e r i o d (Papadopoulos ejt a_l. , 1985). As a consequence, the AAA values f o r HFM i n the r e p o r t of Papadopoulos ejc a l . (1985) should be much higher than the values reported i n the present study. The AAA values f o r HFM i n t h i s experiment were much higher than the other workers. T h i s might be a t t r i b u t a b l e to d i f f e r e n t p r o c e s s i n g methods used f o r the p r o t e i n source. Upon examination of the equation used by Papadopoulos et a l . (1985) f o r the c a l i b r a t i o n of AAA, the low AAA values may be r e s u l t e d from the i n c o r r e c t c a l c u l a t i o n of the endogenous l o s s e s . \ 114 Table 42. The e f f e c t s of p r o t e i n source (PS) and endogenous amino a c i d (AA) c o r r e c t i o n method (ECM) on amino a c i d a v a i l a b i l i t y i n the growing c h i c k (Experiment VI) Treatment^ 2 3 PS ECM AA HFM 4 SBM 4 SEM L E C 4 REC 4 SEM Alan i n e A r g i n i n e A s p a r t i c a c i d C y s t i n e Glutamic a c i d H i s t i d i n e I s o l e u c i n e Leucine L y s i n e Methionine Ph e n y l a l a n i n e P r o l i n e S e r i n e Threonine T y r o s i n e V a l i n e Average 90 93 83 77 86 77 93 92 89 85 88 87 87 87 91 91 ,B,5 .B :5 'B *B ;B 96 98 94 89 95 93 96 96 98 94 90 95 95 94 93 96 9 A 3 A X A 7 A 4 t 1 A 87.8 B 94.9 A 0.52 93.4 93.6 0.52 0.44 96.1 95.5 0.44 0.86 88.7 89.4 0.86 0.83 82.0 b 84.8 a 0.83 0.72 90.4 91.8 0.72 1.25 85. 8 u 84.9 1.25 0.46 94.2 b 95.7 a 0.46 0.66 94.7 94.3 0.66 1.29 95.0 92.9 1.29 0.83 90.6 89.2 0.83 0.75 88.8 90.6 0.75 0.73 90.6 92.6 0.73 0.64 90.8 92.1 0.64 0.78 91.0 91.0 V 0.78 0.49 9 3 . l a 91.6 b 0.49 0.49 93.3 94.1 0.49 0.58 91.2 91.5 0.58 There were four treatments c o n s i s t e d of four r e p l i c a t e s of four b i r d s each. o A l l d i e t s were composed of 10% CP. o A 30- and 8-hour f e c a l c o l l e c t i o n p e r i o d was employed i n the LEC and REC method, r e s p e c t i v e l y . 4HFM = hydrolyzed f e a t h e r meal; SBM = soybean meal; LEC = long term endogenous c o r r e c t i o n method; REC = r e g r e s s i o n endogenous c o r r e c t i o n method. ^Means w i t h i n each row of each treatment not sh a r i n g a non-c a p i t a l i z e d or c a p i t a l i z e d s u p e r s c r i p t are s i g n i f i c a n t l y d i f f e r e n t at P^0.05 and P<0.01, r e s p e c t i v e l y . 115 GENERAL DISCUSSION The p o t e n t i a l of HFM and BM as p r o t e i n supplements f o r growing c h i c k s has been examined. The chemical and AA composition of HFM (Table 14 and 15) r e v e a l a sample that i s high i n crude p r o t e i n and i s r i c h i n c y s t i n e , t h r e o n i n e , a r g i n i n e and i s o l e u c i n e , but i s d e f i c i e n t i n l y s i n e and methionine. A s i m i l a r f i n d i n g was reported by Eggum (1970), who suggested that HFM would be u t i l i z e d i n the fe e d i n g i n d u s t r y i f the p r o t e i n and AAs contained t h e r e i n could be made b i o l o g i c a l l y a v a i l a b l e . Although the chemical analyses would suggest that HFM should be a good p r o t e i n supplement f o r p o u l t r y , f e e d i n g t r i a l s i n d i c a t e that the maximum amount of HFM that can be i n c o r p o r a t e d i n a b r o i l e r s t a r t e r d i e t i s 10% even with supplemental l y s i n e and methionine. T h i s could be p a r t i a l l y e x p l a i n e d by the high l e v e l of c y s t i n e which might r e s u l t i n a s l i g h t degree of AA imbalance when 15% HFM i s i n c o r p o r a t e d i n t o the c h i c k s t a r t e r d i e t . Moreover, the AAA i n HFM (Table 20) i n d i c a t e d t h a t , on average, 87% of the AAs i n HFM was a v a i l a b l e to the growing c h i c k . The a v a i l a b i l i t y of the i n d i v i d u a l AA ranged from 76% f o r h i s t i d i n e to 92% f o r i s o l e u c i n e i n HFM. With t h i s i n mind, the i n c o r p o r a t i o n of 15% HFM i n a d i e t could c r e a t e a s l i g h t degree of AA d e f i c i e n c y i f the AA content of the d i e t i s balanced a c c o r d i n g to the chemical analyses of HFM and not to the a v a i l a b l e AA content. T h i s i s supported by the r e s u l t s of 116 Papadopoulos ejt al_. (1985), who suggested that HFM should be used i n p o u l t r y d i e t s on the b a s i s of the a v a i l a b l e AA because of the l a r g e v a r i a t i o n s i n d i g e s t i b i l i t y among the i n d i v i d u a l AA. The i n c l u s i o n r a t e of 10% HFM i n d i e t s i s e q u i v a l e n t to 37% of the d i e t a r y p r o t e i n ( 2 3 % ) . Baker et. al_. (1981) a l s o r e p o r t e d that up to 40% of the d i e t a r y p r o t e i n could be s u p p l i e d by HFM to male c h i c k s from 9 to 22 days of age. On the other hand, t h i s l e v e l (10%) of i n c o r p o r a t i o n with methionine and l y s i n e supplementation was much higher than those r e p o r t e d p r e v i o u s l y . The d i f f e r e n c e i n f i n d i n g s might be a t t r i b u t e d to the n u t r i t i v e value of HFM processed under d i f f e r e n t c o n d i t i o n s . The HFM used i n the present i n v e s t i g a t i o n was processed at 2.81kg/cm of steam pressure f o r 40 minutes with continuous a g i t a t i o n . These p r o c e s s i n g c o n d i t i o n s seem to be s u f f i c i e n t f o r reasonably good c h i c k performance. The c o n c e n t r a t i o n of h i s t i d i n e (1.84%) i n HFM was much higher than those r e p o r t e d p r e v i o u s l y ; as a r e s u l t , i t was not the l i m i t i n g AA even though i t was only 76% a v a i l a b l e to the c h i c k s . T h i s f i n d i n g was d i f f e r e n t from those rep o r t e d by other r e s e a r c h e r s (McCasland and Richardson, 1966; Moran, J r . et a l . , 1966; Wessels, 1972; Baker ejt a l . , 1981), who repor t e d that h i s t i d i n e was one of the l i m i t i n g AAs i n HFM. Moreover, Smith (1968) found that the a v a i l a b i l i t y of h i s t i d i n e was 0% using a c h i c k bioassay with c r y s t a l l i n e AA standard r e f e r e n c e d i e t . The d i f f e r e n c e i n f i n d i n g s of the present study and those of the 117 other workers may be a t t r i b u t e d to the p r o c e s s i n g c o n d i t i o n s and/or the contamination with a small amount of blood which i n c r e a s e d the h i s t i d i n e content of the HFM. The a v a i l a b i l i t y of l y s i n e ( 8 2 % ) i n HFM was much higher than the value of 5.3% re p o r t e d by Smith (1968) using a chi c k b i o a s s a y . D i f f e r e n t r e s u l t s were obtained by Nordheim and Coon * (1984) using c h i c k bioassay and d i g e s t e d l y s i n e techniques. These authors found that the percent l y s i n e a v a i l a b i l i t y of HFM from c h i c k bioassay and di g e s t e d l y s i n e analyses ranged from 64 to 75% and 77 to 89%, r e s p e c t i v e l y . Much higher values f o r l y s i n e a v a i l a b i l i t y were obtained by the TNBS and FDNB chemical procedures. The mean percent l y s i n e a v a i l a b i l i t y determined by the TNBS and FDNB procedures were 92 and 114%, r e s p e c t i v e l y (Nordheim and Coon, 1984). A lower value of 72.5% a v a i l a b i l i t y was reported by M o r r i s and B a l l o u n (1973b) using the TNBS assay. The d i f f e r e n c e i n f i n d i n g s of the present study and those of the other workers might be due to d i f f e r e n t p r o c e s s i n g c o n d i t i o n s of the HFM and/or d i f f e r e n t assay techniques. Moran, J r . &t_ al_.(1966) suggested that the chemical methods might be o v e r r a t i n g the l y s i n e a v a i l a b i l i t y of HFM which have extremely imbalanced AA contents and i n f e r i o r p r o t e i n d i g e s t i b i l i t y . The a v a i l a b i l i t y of c y s t i n e and methionine i n HFM was 79 and 84%, r e s p e c t i v e l y . These values were s i g n i f i c a n t l y lower than those r e p o r t e d by Burgos e_t a_l. (1974) and Ki r b y e_t aJ.. (1978). The values found by these authors were a l l g r e a t e r than 93%. On 118 the other hand, the a v a i l a b i l i t y of c y s t i n e and methionine was r e p o r t e d to be both 65% i n the study of E l Boushy and Roodbeen (1984) . Much lower v a l v e s of 30 to 60% were obtained by Papadopoulos e_t a l . (1985). The a v a i l a b l e c y s t i n e content i n HFM i n t h i s study was c a l c u l a t e d to be 3.2% which i s higher than the values of 2.92 and 2.89% obtained from the standard curve method and s l o p e - r a t i o assessment, r e s p e c t i v e l y (Baker e_t a l . , 1981). The d i f f e r e n c e i n f i n d i n g s might be due to the p r o c e s s i n g methods used f o r the p r o d u c t i o n of HFM. Eggum (1970) r e p o r t e d that the c y s t i n e content of raw f e a t h e r p r o t e i n was reduced by about 50% a f t e r d i f f e r e n t p r o c e s s i n g procedures and t h i s r e d u c t i o n was probably a t t r i b u t e d to the c o n v e r s i o n of c y s t i n e to l a n t h i o n i n e . T h i s was s u b s t a n t i a t e d by the r e p o r t of Papadopoulos ej; a l . (1985) , who showed a decrease i n c y s t i n e and an i n c r e a s e i n l a n t h i o n i n e content i n HFM with i n c r e a s e d p r o c e s s i n g time. Moreover, Wheeler and Latshaw (1980) re p o r t e d that there was a 95% c o n v e r s i o n of l a n t h i o n i n e s u l f o n e to c y s t e i c a c i d at 110°C. T h i s might i n t e r f e r e with the c a l i b r a t i o n of the a c t u a l amount of c y s t i n e i n samples i n the present study, s i n c e the c y s t i n e content was determined from the amount of c y s t e i c a c i d obtained f o l l o w i n g a c i d h y d r o l y s i s at 110°C f o r 16 hours a f t e r performic a c i d o x i d a t i o n . The BM sample used i n t h i s study contained a high l e v e l of crude p r o t e i n and was r i c h i n l y s i n e , h i s t i d i n e and l e u c i n e but was low i n methionine, c y s t i n e , a r g i n i n e and i s o l e u c i n e . The AAA 119 (92%) and the a v a i l a b l e l y s i n e content (8.1%) i n BM are s i g n i f i c a n t l y higher than those i n HFM. However, the r a t i o of i s o l e u c i n e : l e u c i n e (1:13) i n BM i s irabalanced when compared to those i n HFM (1:1.8), SBM (1:1.5), and MM (1:2). Even with the supplementation of a r g i n i n e , i s o l e u c i n e , methionine and c y s t i n e , there were sig n s of AA imbalance imposed by the high l e v e l of l e u c i n e (2.43%) i n the d i e t c o n t a i n i n g 15% BM. T h i s AA imbalance may a l s o be accentuated to a l e s s e r extent by the low a v a i l a b l e AA content of BM when a l l the SBM was re p l a c e d by BM i n the 15% BM d i e t which was formulated a c c o r d i n g to the chemical composition of BM. N e v e r t h e l e s s , r e s u l t s i n t h i s study show t h a t BM can be i n c o r p o r a t e d i n a soy-wheat-corn based d i e t at a l e v e l of 4%. T h i s i s supported by the l i t e r a t u r e of Squibb and Braham (1955) i n which BM was found to be the most e f f e c t i v e source of l y s i n e when fed at 2 to 4% i n c h i c k r a t i o n s . Wisman ejt a l . (1958) a l s o showed that BM could be i n c o r p o r a t e d to provide 3% CP of the t o t a l 20% CP i n c h i c k s t a r t e r d i e t s . Furthermore, Mathur and Ahmed (1971) r e p o r t e d that BM at 2.5% d i e t a r y l e v e l was a good source of supplementary l y s i n e f o r c h i c k s . Recent s t u d i e s of Omar e_t a_l. (1985) showed that the 3-week performance of b r o i l e r c h i c k s fed d i e t s c o n t a i n i n g 4% BM was not d i f f e r e n t from those fed a corn-soy b a s a l d i e t . On the other hand, the l a t t e r authors found a s i g n i f i c a n t growth depression with b i r d s fed 8% BM and no improvement was obtained with other a r g i n i n e or potassium supplementation. T h i s might be caused by an imbalance of the branched-chain AA as s t a t e d p r e v i o u s l y . 120 A higher d i e t a r y l e v e l of BM (6%) as a source of l y s i n e was rep o r t e d to be s a t i s f a c t o r y i n t y p i c a l E gyptian b r o i l e r r a t i o n ( G a l a l ejt a_l. , 1977). The reason that the l a t t e r workers could use a higher l e v e l of BM than the present study and that of Omar et a l . (1985) may be due to the d i f f e r e n c e i n compositions of b a s a l d i e t s used. The b a s a l d i e t used by G a l a l e_t al_. (1977) was composed of corn, r i c e bran, cottonseed meal, fababean meal, corn g l u t e n meal and ground oats, whereas corn-soy based d i e t s were used i n the present i n v e s t i g a t i o n and that of Omar e_t a l . (1985). The p o t e n t i a l b e n e f i t of combining HFM and BM was i n v e s t i g a t e d i n t h i s study. The abnormal r a t i o of the branched-c h a i n AA i n BM may hinder the b e n e f i c i a l e f f e c t of mixing HFM and BM. Maximum c h i c k performance was obtained only with 10% HFM. A comparably good performance was obtained with a combination of 2.5% BM and 7.5% HFM i n c h i c k s t a r t e r d i e t s . A s i m i l a r f i n d i n g was reported by Koci ejt jal_. (1983), who showed that a 2:1 mixture of HFM and BM could r e p l a c e 50 and 100% of the animal p r o t e i n i n b r o i l e r s t a r t e r and f i n i s h e r d i e t s , r e s p e c t i v e l y . From the above f i n d i n g s , i t would seem f e a s i b l e to i n c o r p o r a t e 4% BM or a combination of 2.5% BM and 7.5% HFM i n ch i c k s t a r t e r d i e t s as long as the market and the p r i c e of BM and HFM are comparable and economically f e a s i b l e . Future research i n the development of a r a p i d and economical method to separate the d i f f e r e n t f r a c t i o n s of blood before being processed i n t o BM could i n c r e a s e the u t i l i z a t i o n of BM i n p o u l t r y f e e d i n g . Higher 121 l e v e l s of BM could a l s o be i n c o r p o r a t e d i n d i e t s with i n g r e d i e n t s of low l e u c i n e content. There were s i g n i f i c a n t p o s i t i v e c o r r e l a t i o n s between v o l u n t a r y a l p h a - c e l l u l o s e i n t a k e s (Xg) and AA e x c r e t i o n (Ymg) i n 11 out of the 16 AAs i n the present study (Table 24). S i m i l a r f i n d i n g s were re p o r t e d by Raharjo and F a r r e l l (1984), who showed that s i g n i f i c a n t i n c r e a s e s i n l y s i n e , threonine and i s o l e u c i n e outputs with i n c r e a s i n g d i e t a r y r i c e h u l l s . Regression equations obtained from the r e l a t i o n s h i p s between a l p h a - c e l l u l o s e i n t a k e s and AA e x c r e t i o n s were used to c o r r e c t f o r the TAAE i n two AAA s t u d i e s . No s i g n i f i c a n t d i f f e r e n c e s i n AAA f o r HFM and SBM were obtained with the REC, IEC and AEC methods (Table 27). Moreover, comparable r e s u l t s i n AAA f o r HFM and SBM were obtained with the REC and LEC method (Table 42). These f i n d i n g s showed that the REC method i s r e l i a b l e f o r the c a l i b r a t i o n of TAAE i n AAA assay. There were s i g n i f i c a n t l y g r e a t e r amounts of AAs i n e x c r e t a from a 30-hour f e c a l c o l l e c t i o n p e r i o d as compared to those from an 8-hour p e r i o d (Table 41). Mucoproteins c h a r a c t e r i s t i c a l l y c o n t a i n a high p r o p o r t i o n of s e r i n e , t h r e o n i n e , p r o l i n e , glucosamine and galactosamine. The enzymes t r y p s i n , chymotrypsin and pepsin are a l s o r i c h i n s e r i n e and threonine as repo r t e d by S a l t e r and F u l f o r d (1974). The s i m i l a r p r o p o r t i o n a l i n c r e a s e s of a l l AAs i n e x c r e t a from the 8-hour to 30-hour f e c a l c o l l e c t i o n p e r i o d suggests that the higher l e v e l s of AA e x c r e t i o n from the 30-hour p e r i o d were not c o n t r i b u t e d mainly from endogenous s e c r e t i o n s (enzymes and mucoproteins). These high l e v e l s of AAs 122 could r e s u l t from the e x c r e t i o n s of m i c r o b i a l c e l l s . Moreover, the long d u r a t i o n (30 hours) of f e c a l c o l l e c t i o n might impose a s t r e s s to the b i r d s , thus r e s u l t i n g i n an i n c r e a s e i n c e l l shedding from the GIT. Despite the i n c r e a s e i n AA e x c r e t i o n s i n b i r d s from an 8-hour to a 30-hour f e c a l c o l l e c t i o n p e r i o d (Table 41), comparable r e s u l t s i n AAA f o r HFM and SBM were obtained from the two ECM. These f i n d i n g s showed that an 8-hour f e c a l c o l l e c t i o n p e r i o d was s u f f i c i e n t f o r the c a l i b r a t i o n of TAAE and TAAF f o r the AAA assay used i n the present i n v e s t i g a t i o n . On the other hand, other workers rep o r t e d that more than 24 hours were needed f o r the i n t e s t i n a l c l e a r a n c e of the undigested AAs from HFM and SBM i n b i r d s ( S i b b a l d , 1979a; K e s s l e r and Thomas, 1981; Parsons et a l . , 1982a) . The d i f f e r e n c e i n f i n d i n g s of the present study and those of the other workers might be a t t r i b u t e d to the age of the b i r d s used. The e f f e c t of d i f f e r e n t l e v e l s of p r o t e i n supplements on AAA was i n v e s t i g a t e d i n the present study. There appeared to be a r e l a t i o n s h i p between PS and PL i n one t r i a l and some i n g r e d i e n t s might be a f f e c t e d more than o t h e r s . However, no v a l i d c o n c l u s i o n could be drawn from the two experimental t r i a l s , s i n c e there were no c o n s i s t e n t trends obtained from these r e s u l t s . The s i g n i f i c a n t d i f f e r e n c e s i n AAA r e s u l t i n g from v a r y i n g l e v e l s of p r o t e i n i n c l u s i o n s were probably the r e s u l t of s e v e r a l f a c t o r s i n c l u d i n g experimental d e s i g n . N e v e r t h e l e s s , h i g h e s t AAA values were obtained at the 20% crude p r o t e i n l e v e l s . 123 SUMMARY AND CONCLUSIONS A s e r i e s of experiments was conducted with growing c h i c k s on the e v a l u a t i o n of HFM and BM as p r o t e i n supplements f o r p o u l t r y . HFM processed at 2.81kg/cm of steam pressure f o r 40 minutes with continuous a g i t a t i o n , c o n t a i n s a high l e v e l of CP (89%). R e l a t i v e to the requirements of c h i c k s , HFM is' r i c h i n c y s t i n e , t h r e o n i n e , a r g i n i n e and i s o l e u c i n e , but i s d e f i c i e n t i n l y s i n e and methionine. R e s u l t s i n d i c a t e that the AAA i n HFM ranged from 76% f o r h i s t i d i n e to 92% f o r i s o l e u c i n e , and with a mean AAA value of 87%. With methionine and l y s i n e supplementation, up to 37% of the d i e t a r y CP could be s u p p l i e d by HFM i n c h i c k s t a r t e r r a t i o n s . BM c o n t a i n s a high l e v e l of CP (95%) and i s r i c h i n l y s i n e , h i s t i d i n e and l e u c i n e but i s low i n methionine, c y s t i n e , a r g i n i n e and i s o l e u c i n e . Even with a higher mean AAA (92%) and a v a i l a b l e l y s i n e content (8.1%) , BM could not be u t i l i z e d as e f f i c i e n t as HFM by the growing c h i c k s . With a r g i n i n e , i s o l e u c i n e , methionine and c y s t i n e supplementation, only up to 4% BM could be i n c o r p o r a t e d i n t o c h i c k s t a r t e r d i e t s . The r a t i o of i s o l e u c i n e : l e u c i n e i n BM i s 1:13 and t h i s might be the c a u s a t i v e f a c t o r f o r the poor c h i c k performance i n higher l e v e l s of i n c l u s i o n of BM i n c h i c k s t a r t e r d i e t s . T h i s AA imbalance e f f e c t was a l s o shown i n a subsequent experiment. A combination of 2.5% BM and 7.5% HFM i n c h i c k s t a r t e r r a t i o n s produced reasonably good c h i c k performance. However, higher p r o p o r t i o n s BM r e s u l t e d i n 124 decreases i n feed c o n v e r s i o n e f f i c i e n c y . There were p o s i t i v e c o r r e l a t i o n s between v o l u n t a r y a l p h a -c e l l u l o s e i n t a k e s and AA e x c r e t i o n s i n 11 out of the 16 AAs s t u d i e d . Regression equations obtained from these r e l a t i o n s h i p s were used f o r the c o r r e c t i o n of TAAE i n the AAA assay, and r e s u l t s showed that i t i s r e l i a b l e f o r the e s t i m a t i o n of TAAE. Moreover, values of AAA f o r both HFM and SBM were not a f f e c t e d by d i f f e r e n t d u r a t i o n s of f e c a l c o l l e c t i o n . E i g h t hours were s u f f i c i e n t f o r the c o l l e c t i o n of ex c r e t a i n AAA t r i a l s with the growing c h i c k s . There seems to be a r e l a t i o n s h i p between PS and PL on AAA and some i n g r e d i e n t s might be a f f e c t e d more than the o t h e r s . However, no c o n s i s t e n t trend could be e s t a b l i s h e d from the two experimental t r i a l s i n the present i n v e s t i g a t i o n . The above f i n d i n g s would suggest that 10% HFM or 4% BM can be used s i n g l y as p r o t e i n supplements f o r the growing c h i c k s . A l s o , HFM and BM should be used i n p o u l t r y r a t i o n s on the b a s i s of the a v a i l a b l e AAs. Fu r t h e r r e s e a r c h should be conducted to a l l e v i a t e the e f f e c t s of high c y s t i n e and l e u c i n e content i n HFM and BM, r e s p e c t i v e l y , i n the growing c h i c k s . The REC method i s r e l i a b l e f o r the c o r r e c t i o n of TAAE f o r the e s t i m a t i o n of AAA i n the growing b i r d s . 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P o u l t r y N u t r i t i o n , Beekbergen, PTJDOC, Wageningen, pp. 97-101. T i t u s , H.W., T . C B y e r l y , N.R. E l l i s , and R.B. N e s t l e r , 1936. E f f e c t of packing house by-products i n the d i e t of c h i c k e n , on the pr o d u c t i o n and h a t c h e a b i l i t y of eggs. J . Agr. Res. 53:453-465. Tsang, S.T.L., E.L. McKee, C P . Andrews, C E . Winslade, R.L. Steinhauser, and H.A. Windsor, 1963. The u t i l i z a t i o n of hydrolyzed p o u l t r y f e a t h e r s i n i s o n i t r o g e n o u s and i s o c a l o r i c b r o i l e r r a t i o n s . P o u l t r y S c i . 42:1369-1372. Udea, H., S. Yabuta, H. Yokota, and I. Ya s a k i , 1981. Involvement of feed i n t a k e and feed u t i l i z a t i o n i n the growth r e t a r d a t i o n of c h i c k s given the e x c e s s i v e amounts of l e u c i n e , l y s i n e , p h e n y l a l a n i n e or methionine. Nutr. Rep. I n t . 24:135-144. W a l l i s , I . , and D. Balnave, 1983. A comparison of d i f f e r e n t d r y i n g techniques f o r energy and amino a c i d analyses of p o u l t r y e x c r e t a . Br. P o u l t r y S c i . 24:255-260. 136 Ward, J.M., J r . , R.A. McNabb, and F.M.A. McNabb, 1975. The e f f e c t s of changes i n d i e t a r y p r o t e i n and water a v a i l a b i l i t y on u r i n a r y n i t r o g e n compounds i n the r o o s t e r , G a l l u s  domesticus - 1. Urine flow and the e x c r e t i o n of u r i c a c i d and ammonia. Comp. Biochem. P h y s i o l . 51A:165-169. Wessels, J.P.H., 1972. A study of the p r o t e i n q u a l i t y of d i f f e r e n t f e a t h e r meals. P o u l t r y S c i . 51:537-541. Wheeler, K.B., and J.D. Latshaw, 1980. E v a l u a t i o n of the s u l f u r amino a c i d and l a n t h i o n i n e content of f e a t h e r meal. P o u l t r y S c i . 59:1672. (A b s t r . ) W i l d e r , O.H.M., P.C. Ostby, and B.R. Gregory, 1955. The use of chicken f e a t h e r meal i n feeds. P o u l t r y S c i . 34:518-524. Wisman, E.L., C.E. Holmes, and R.W. Engel, 1958. U t i l i z a t i o n of p o u l t r y by-products i n p o u l t r y r a t i o n s . P o u l t r y S c i . 37:834-838. 137 APPENDIX 138 Table 1. A n a l y s i s of v a r i a n c e f o r growth parameters i n Experiment I Source Mean square df Feed consumption Weight gain Feed/gain Treatment E r r o r 7 24 14841 3389 12626 17.32 ** 0.0050 0.0008 P<0.01 Table 2. A n a l y s i s of v a r i a n c e f o r growth parameters i n Experiment I I Source df Mean square Feed consumption Weight gain Feed/gain Treatment E r r o r 7 24 225193 8283 ** 89189 6270 ** 0.0390 0.0132 ** P<0.01. kP<0.05. Table 3. A n a l y s i s of v a r i a n c e f o r growth parameters i n Experiment I I I Source Mean square df Feed consumption Weight gain Feed/gain Treatment E r r o r 7 24 2482 3723 3702 1470 0.0066 0.0020 139 Table 4. A n a l y s i s of v a r i a n c e f o r amino a c i d a v a i l a b i l i t y i n Experiment I V 1 Amino a c i d Degree of freedom  PS PL PSxPL E r r o r PS Mean square PL PSxPL E r r o r A l a n i n e 3 2 6 12 60 A r g i n i n e 3 2 6 12 79 A s p a r t i c a c i d 3 2 6 12 263 Glutamic a c i d 3 2 6 12 148 H i s t i d i n e 3 2 6 12 399 I s o l e u c i n e 3 2 6 12 170 Leucine 3 2 6 12 50 Lys i n e 3 2 6 12 253 Ph e n y l a l a n i n e 3 2 6 12 11 P r o l i n e 3 2 6 12 96 Seri n e 3 2 6 12 83 Threonine 3 2 6 12 96 T y r o s i n e 3 2 6 12 72 V a l i n e 3 2 6 12 56 Average 3 2 6 12 97 ** ** ** ** ** ** ** ** ** ** ** ** ** ** 11 7 30 13 61 41 18 20 29 76 ** ** ** ** ** 23 ** 1.33 2.67 8.47 6.73 1.09 7.37 0.85 31.00 4.32 3.88 4.98 5.50 4.55 1.59 2.94 ** 1, 3, 2, 4, 3, 4, 2, 2, 3, 4, 2, 3, 6, 2, 88 01 66 03 27 75 17 95 33 24 68 86 38 13 2.03 ** PS = p r o t e i n source; PL = p r o t e i n l e v e l . P<0.01. * P<0.05. 140 Table 5. A n a l y s i s of v a r i a n c e dry matter (DM) i n t a k e DM output i n Experiment f o r the of the V, Part r e l a t i o n s h i p between N-free d i e t and f e c a l 1 Source SS df MS F value Regression 80.4 1 80.4 24.4** D e v i a t i o n s 100.1 30 3.3 T o t a l 180.5 31 5.8 P<0.01. Table 6. A n a l y s i s of v a r i a n c e dry matter (DM) i n t a k e output i n Experiment V, f o r the of the N-Part 1 r e l a t i o n s h i p between -f r e e d i e t and f e c a l N Source SS df MS F value Regression 302.8 1 302.8 0.06 D e v i a t i o n s 156193.8 30 5206.5 T o t a l 156496.6 31 5048.3 141 Table 7. A n a l y s i s of v a r i a n c e of the r e l a t i o n s h i p s between amino a c i d e x c r e t i o n (Ymg) and a l p h a - c e l l u l o s e i n t a k e (Xg) from a N-free d i e t i n Experiment V, Part 1 Amino a c i d Degree of freedom Mean square Regression D e v i a t i o n s Regression D e v i a t i o n s A l a n i n e ! 30 87 8+ 24 3 A r g i n i n e 1 30 60 2 23 .5 A s p a r t i c a c i d 1 30 340 .8+ 92 .1 C y s t i n e 1 30 17 .5 9 .2 Glutamic a c i d 1 30 350 > 5 160 .0 H i s t i d i n e 1 30 231 .9** 21 .6 I s o l e u c i n e 1 30 36 11 .3 Leucine 1 30 222. 9* 46 3 Ly s i n e 1 30 117 5* 28 0 Methionine 1 30 4 2 * 3 1 Ph e n y l a l a n i n e 1 30 107 0* 14 .6 P r o l i n e 1 30 124 .3+ 43 .0 Se r i n e 1 30 93 • 8 * 32 .4 Threonine 1 30 123 24 3 Ty r o s i n e 1 30 53 .9* 11 .0 V a l i n e 1 30 46 .4 20 .5 ** P<0.01. P<0.05. +P<0.10. 142 Table 8. A n a l y s i s of v a r i a n c e f o r amino a c i d (AA) a v a i l a b i l i t y i n Experiment V, P a r t 2 a 1 Mean square  PS(S) ECM(M) PL(L) SxM SxL MxL SxMxL E r r o r AA l d f 2df 3df 2df 3df , 6df 6df 72df A l a 592.63** 3.97 9.42* 0.25 3.13 1.40 1.02 2.33 Arg 679.31** 1.16 5.77 0.34 1.06 0.41 0.40 1.28 Asp 3872.13** 5.22 3.75 4.00 15.15* 2.60 1.34 5.02 Cys 3390.44** 2.84 12.88 1.84 23.48 6.44 1.48 5.55 Glu 2773.50** 0.69 5.23 0.44 1.25 0.84 0.85 3.28 His 4181.88** 1.03 7.33 2.16 18.10 2.03 0.35 6.84 H e 262.94** 0.81 13.50** 0.00 2.69 0.59 0.30 1.72 Leu 270.31** 3.31 9.08* 1.78 0.81 1.63 1.06 1.99 Lys 3216.63** 9.41 30.75* 3.63 1.90 4.52 3.47 8.48 Met 2102.69** 4.09 44.50** 1.69 17.94.. 4.67 4.71 7.44 Phe 202.75** 0.66 54.40* 0.19 15.65 0.44 0.37 3.00 Pro 1388.19** 0.28 9.17* 0.81 0.92 0.59 0.24 2.79 Ser 1397.44** 1.34 17.73 0.78 7.58 0.91 0.76 2.79 Thr 1448.06** 1.06 6.98.. 0-00 2.94.. 0.82 0.46 3.83 Tyr 490.88** 1.75 52.04** 0.63 22.92* 1.76 1.30 4.96 Val 638.00 2.44 13.06 0.09 3.27 1.29 0.41 2.36 Ave 1395.06 1.00 7.17 0.25 0.58 0.73 0.21 1.83 ^PS = p r o t e i n source; ECM = endogenous amino a c i d c o r r e c t i o n method; PL = p r o t e i n l e v e l . **P<0.01. *P<0.05. 143 Table 9. A n a l y s i s of v a r i a n c e f o r amino a c i d (AA) a v a i l a b i l i t y i n Experiment V, Part 2b Mean square  PS(S) ECM(M) PL(L) SxM SxL MxL SxMxL E r r o r AA l d f l d f 3df l d f 3df *3df 3df 48df A l a Arg Asp Cys Glu His H e Leu Lys Met Phe Pro Ser Thr Tyr V a l Ave 371.50 427.44 2668.94 2299.28 1843.81 2897.19 179.25 188.25 2004.88 1403.53 123.88 913.56 939.56 980.56 327.94 422.50 ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** 929.50 0.88 0.00 10.09 4.78 1.31 1.00 0.50 5.69 1.25 8.31 0.38 0.25 2.94 0.56 3.13 4.56 1.38 2.92 2.10 2.93 14.63 3.94 6.53 8.06 7.63" 18.83, 33.41 38.77 5.19 12.46 5.33 39.02 6.60 4.695 ** ** ** 0.13 0.06 5.75 3.25 0.69 1.19 0.63 3.13 0.06 3.19 0.00 1.13 1.19 0.00 1.38 0.00 0.19 2.96 1.38 11.30 13.62 0.48 13.31 1.98 0.88 4.69 12.63 11.79' 0.63 6.67 18.67 2.10 0.17 1. 17 0 .92 2 .01 0. 10 0 .31 1 .20 3. 52 1 .82 5 .11 8. 26 1 .92 5 .05 1. 48 1 .40 3 .27 0. 51 0 .30 5 .77 0. 88 0 .31 1 .73 2. 85 1 .35 1 .87 5. 27 3 .56 8 .65 5. 73 8 .99 8 .66 0. 38 0 .17 2 .85 0. 71 0 .31 2 .75 1. 13 1 .23 2 .65 1. 63 0 .50 3 .39 2. 75 1 .60 5 .11 1. 73 0 .50 2 .39 0. 73 0 .44 1 .67 *PS = p r o t e i n source; ECM = endogenous amino a c i d c o r r e c t i o n method; PL = p r o t e i n l e v e l . **P<0.01. *P<0.05. 144 Table 10. A n a l y s i s of v a r i a n c e f o r amino a c i d (AA) a v a i l a b i l i t y i n Experiment V, P a r t 2c Mean square  PS(S) ECM(M) PL(L) SxM SxL MxL SxMxL E r r o r AA l d f l d f 3df l d f 3df * 3df 3df 48df Ala Arg Asp Cys Glu His H e Leu Ly s Met Phe Pro Ser Thr Tyr V a l 408.00 458.63 2659.88 2328.06 1892.56 2675.30 169.06 202.00 2233.75 1334.13 140.25 965.63 963.38 960.44 346.44 432.25 ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** 2.88 1.63 4.16 0.06 0.25 0.19 0.31 0.44 9.56 1.97 0.31 0.13 1.38 0.63 0.19 0.19 10.58 5.04' 4.48 4.17 6.02 6.64 9.63 9.98 21.52 39.98 37.27 8.81 14.60 6.48 40.50 11.46 s ** ** ** * ** ** 0.81 0.94 6.25 2.31 0.00 0.88 0.00 1.38 4.44 0.69 0.59 0.13 0.50 0.00 0.38 0.00 2.35 0.44 11.25 15.82 1.10 10.69 1.44 0.13 1.83 22.65 8.85 0.88 6.15 2.17 14.85 2.54 * 0, 0, 3, 0, 0, 2, 81 52 81 73 58 79 0.88 1.29 4.85 6.20 0.73 0.35 1.13 0.88 2.08 1.33 0.13 0.35 1.68 0.55 1.04 0.48 0.10 1.65 4.21 3.21 0.31 0.29 0.98 0.46 1.92 0.19 2.62 1.40 5.45 6.44 3.63 7.69 1. 2, 82 61 8.54 ,68 38 ,01 ,84 ,83 ,53 ,75 Ave 944.81 ** 0.06 6.85 0.00 0.54 0.75 0.21 2.12 •'"PS = p r o t e i n source; ECM = endogenous amino a c i d c o r r e c t i o n method; PL = p r o t e i n l e v e l . **P<0.01. *P<0.05. 145 Table 11. A n a l y s i s of v a r i a n c e f o r amino a c i d (AA) a v a i l a b i l i t y i n Experiment V, Part 2d Mean square PS(S) ECM(M) PL(L) SxM SxL MxL SxMxL E r r o r AA l d f l d f 3df l d f 3df 3df 3df 48df A l a Arg Asp Cys Glu His H e Leu Ly s Met Phe Pro Ser Thr Tyr V a l 405.19 472.50 2419.59 2155.44 1811.56 2793.13 177.13 152.88 2198.25 1469.88 141.31 898.38 893.25 945.81 307.94 421.81 ** ** ** ** ** ** ** ** ** *# ** ** ** ** ** ** 7, 1, 1, 3, 0, 1, 1, 50 50 38 75 94 78 38 Ave 916.56 ** 4.63 17.38 2.41 1.06 0.75 0.19 1.81 1.56 3.06 1.88 6.48 4.65' 2.65 13.43 1.46 3.48 9.81 2.54 25.69' 20.50 33.15 5.04 9.42 2.88 26.19 9.52 ** ** ** ** 3.60 0.69 2.25 2.46 1.69 2 .36 0.50 0.88 0.73 0.38 1 .24 0.00 9.03. 0.54 0.54 4 .49 0.00 18.99 10.34 1.93 5 .15 0.31 1.67 0.35 0.25 2 .94 4.41 12.63 2.84 0.27 7 .06 0.25 0.40 0.13 0.38 1 .60 0.13 1.38 0.42 0.46 1 .49 6.38 0.75 3.44 2.54 8 .25 0.75 1.89 2.13 5 .98 0.44 11.08* 0.27 0.54 2 .79 0.81 0.42 0.60 0.29 2 .62 0.13 2.94 0.35 0.27 2 .89 0.06 0.08 0.33 4 .27 0.38 13.67* 0.63 0.23 4 .26 0.06 2.19 0.69 0.60 1 .94 0.00 0.50 0.63 0.15 1 .70 PS = p r o t e i n source; ECM = endogenous amino a c i d c o r r e c t i o n method; PL = p r o t e i n l e v e l . P<0.01. *P<0.05. 146 Table 12. A n a l y s i s of v a r i a n c e f o r amino a c i d (AA) a v a i l a b i l i t y i n Experiment V, P a r t 2e* AA Degree of freedom PS PL PSxPL E r r o r PS Mean square PL PSxPL E r r o r A l a nine A r g i n i n e A s p a r t i c a c i d C y s t i n e Glutamic a c i d H i s t i d i n e I s o l e u c i n e Leucine L y s i n e Methionine P h e n y l a l a n i n e P r o l i n e S e r i n e Threonine T y r o s i n e V a l i n e Average 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 187 207 1461 1239 963 1389 85 121 1019 ** ** ** ** ** ** ** ** ** ** 3, 1 5, 2, 5, 5, 23 18 80 00 09 08 4.74, 9.41 10. 71 636;: 31.48 61 491 506 493 184 216 479 ** ** ** ** ** ** 21.82 4.76, 9.75" 5.66, 28.76 5.48 4.40 ** 1.25 0.63 8.21 5.50 1.06 5.92 0.54 1.06 5.47 20.10 4.79 0.65 5. 1, 92 07 11.58 1.23 0.34 2.27 1.36 6.07 6.34 3.96 6.40 1.95 2.99 8.93 10.36 3, 3, 2, 2, 6, 3, 45 15 61 95 39 20 2.09 1 ** PS = p r o t e i n source; PL = p r o t e i n l e v e l . P<0.01. * P<0.05. 147 Table 13. A n a l y s i s of v a r i a n c e f o r amino a c i d (AA) a v a i l a b i l i t y i n Experiment V, Part 2 f 1 AA Degree of freedom PS PL PSxPL E r r o r PS Mean square PL PSxPL E r r o r A l a n i n e A r g i n i n e A s p a r t i c a c i d C y s t i n e Glutamic a c i d H i s t i d i n e I s o l e u c i n e Leucine L y s i n e Methionine P h e n y l a l a n i n e P r o l i n e S e r i n e Threonine T y r o s i n e V a l i n e Average 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 184 220 1214 1064 882 1510 94 70 986 771 62 424 435 488 145 206 ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** 0.83 0.98 0.70 20.89 0.31 1.96 4.16 1.06 13.44 7.69 17.28 ** ** 1, 3, 1, 19 81 28 451 ** 12.95 2.88 1.01 2.67 1.09 4.87 10.02 0.81 7.67 1.79 1.18 2.74 1.49 6.73 ,24 ,98 22 8.77 1.33 0.26 1.75 1.05 4 3, 2, 5, 1, 15 76 58 14 52 0.75 8.36 6.96 2.25 2.35 2.69 3.83 3.83 1.58 1.25 ** LPS = p r o t e i n source; PL = p r o t e i n l e v e l . P<0.01. * P<0.05. 148 Table 14. A n a l y s i s of v a r i a n c e f o r amino a c i d (AA) a v a i l a b i l i t y i n Experiment V, Part 2g AA Degree of freedom PS PL PSxPL E r r o r PS Mean square PL PSxPL E r r o r A l a n i n e A r g i n i n e A s p a r t i c a c i d C y s t i n e Glutamic a c i d H i s t i d i n e I s o l e u c i n e Leucine L y s i n e Methionine P h e n y l a l a n i n e P r o l i n e S e r i n e Threonine T y r o s i n e V a l i n e Average 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 222 253 1205 1092 930 1288 84 83 1219 700 80 475 458 468 163 216 ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** 8.13 4.38' 2.52 2.90 1.50 4.35 5.74' 1.92 15.70 14.78 16.17 4.42 ** 5, 1, 93 72 466 ** 13.82 7.35' 3.23 1.27 0.18 4.68 10.88 1.09 5.23 1.04 0.66 0.53 5.67 4.39 0.51 1.25 1, 5, 51 18 1.45 0.39 2.97 1.44 4.84 6.55 3.29 8.98 1.69 2.22 8.14 5.00 3.32 2.88 3.08 4.71 4.68 2.30 2.16 *PS = p r o t e i n source; PL **P<0.01. = p r o t e i n l e v e l . P<0.05. 149 Table 15. A n a l y s i s of v a r i a n c e f o r amino a c i d (AA) a v a i l a b i l i t y i n Experiment VI AA Degree of freedom PS ECM PSxECM E r r o r PS Mean square ECM PSxECM E r r o r A l a n i n e A r g i n i n e A s p a r t i c a c i d C y s t i n e Glutamic a c i d H i s t i d i n e I s o l e u c i n e Leucine L y s i n e Methionine P h e n y l a l a n i n e P r o l i n e S e r i n e Threonine T y r o s i n e V a l i n e Average 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 105 89 512 550 366 1007 30 49 314 289 23 221 210 216 20 88 ** ** ** ** ** ** ** ** ** ** ** ** ** ** 200 ** 0.09 1.45 2.03. 31.36 8.28 2.81. 8.72* 0.63 17.02 8.41 13.52 17.23 6.50 0.00. 9.00 2.75 0.47 1.97 7.02 13.50 5.77 15.80 21.85 7.00 0.00 45.23 4.85 8.55 2.56 3, 4, 23 19 0.00 3.58 1.72 2.19 1.55 5.96 5.47 4.19 12.53 1.66 3.50 13.31 5.54 4.52 4, 3. 4, 20 26 83 1.89 1.90 2.66 PS = p r o t e i n source; ECM = endogenous amino a c i d c o r r e c t i o n method; PL = p r o t e i n l e v e l . *# P<0.01. *P<0.05. 

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