UBC Theses and Dissertations

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UBC Theses and Dissertations

The effect of enzyme and mild hydrothermal treatment on the nutritive value of barley grain and canola… Gill, Catherine Suzanne 1997

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T H E E F F E C T O F E N Z Y M E A N D M I L D H Y D R O T H E R M A L T R E A T M E N T O N T H E N U T R I T I V E V A L U E O F B A R L E Y G R A I N A N D C A N O L A M E A L F O R D A I R Y C A T T L E by C A T H E R I N E S U Z A N N E G I L L B . S c . (Agr . ) , T h e U n i v e r s i t y o f B r i t i sh C o l u m b i a , 1 9 9 4 A T H E S I S S U B M I T T E D IN P A R T I A L F U L F I L L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F M A S T E R O F S C I E N C E in T H E F A C U L T Y O F G R A D U A T E S T U D I E S D e p a r t m e n t o f A n i m a l S c i e n c e W e a c c e p t th is t h e s i s a s c o n f o r m i n g to t h e r e q u i r e d ^ s j a n d a r d T H E U N I V E R S I T Y O F B R I T I S H C O L U M B I A F e b r u a r y , 1 9 9 7 © C a t h e r i n e S u z a n n e G i l l , 1 9 9 7 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of /JM'W/ti- SC^/E^C^ The University of British Columbia Vancouver, Canada Date J^A^O^'&k/ / ^ ? DE-6 (2788) ABSTRACT H i g h i n g r e d i e n t c o s t s a n d a c o m p e t i t i v e m a r k e t h a v e p r o m o t e d s u p p o r t fo r r e s e a r c h b y t h e f e e d i ndus t r y to i m p r o v e t he ut i l i za t ion o f p ro te in a n d e n e r g y f e e d s t u f f s s u c h a s b a r l e y a n d c a n o l a m e a l . T h e o b j e c t i v e o f th is r e s e a r c h w a s to d e t e r m i n e t h e feas ib i l i t y o f u s i n g e n z y m e a n d mi ld h y d r o t h e r m a l t r e a t m e n t to i m p r o v e t he nut r i t iona l v a l u e o f b a r l e y g ra i n a n d c a n o l a m e a l fo r u s e by da i r y ca t t le . In e x p e r i m e n t 1, b a r l e y w h i c h w a s s u b j e c t e d to p - g l u c a n a s e , p r o t e a s e a n d x y l a n a s e e n z y m e s a n d mi ld h y d r o t h e r m a l t r ea tmen t , w a s e v a l u a t e d u s i n g 4 8 h in vitro d ry ma t te r d i s a p p e a r a n c e ( I V D M D ) , in vitro t rue d iges t ib i l i t y ( I V T D ) , a c i d d e t e r g e n t f iber ( A D F ) a n d neu t ra l d e t e r g e n t f i be r ( N D F ) a n a l y s e s . A n u m b e r o f i n te rac t i ons ( P < 0 .05 ) w e r e o b s e r v e d fo r t he d iges t ib i l i t y p a r a m e t e r s . R e s u l t s o f f i be r a n a l y s e s w e r e i n c o n c l u s i v e . O v e r a l l , t h e a d d i t i o n o f x y l a n a s e w a s f o u n d to b e d e t r i m e n t a l a n d t he mi ld h y d r o t h e r m a l t r e a t m e n t d id not h a v e a n e f fec t o n e n z y m e act iv i ty . T h e add i t i on of p r o t e a s e a n d / o r p - g l u c a n a s e to b a r l e y s u g g e s t po ten t ia l a p p l i c a t i o n that m a y b e fu r ther i n v e s t i g a t e d . In e x p e r i m e n t 2 , c a n o l a m e a l w a s s u b j e c t e d to p r o t e a s e , x y l a n a s e e n z y m e s a n d mi ld h y d r o t h e r m a l t r ea tmen t . T r e a t m e n t s w e r e e v a l u a t e d u s i n g I V D M D , I V T D , A D F a n d N D F . S i g n i f i c a n t i n t e rac t i ons ( P < 0 .05) w e r e o b s e r v e d fo r I V D M D , I V T D , ADF a n d NDF. T h e add i t i on o f m o i s t u r e s ign i f i can t l y i n c r e a s e d ( P < 0 .05 ) I V D M D a n d I V T D . P r o t e a s e a l s o e l i c i t ed a n e f fec t ( P < 0 .05) o n the NDF c o n t e n t . F r o m th is e x p e r i m e n t , s i m i l a r c o n c l u s i o n s to t h o s e in e x p e r i m e n t 1 w e r e d r a w n : n e i t h e r x y l a n a s e n o r mi ld h e a t t r e a t m e n t w a s e f fec t i ve in imp rov i ng d igest ib i l i t y . H o w e v e r , t he a d d i t i o n o f p r o t e a s e to c a n o l a m e a l at a n i n te rmed ia te l eve l o f a p p l i c a t i o n i m p r o v e d it 's d iges t ib i l i t y s ign i f i can t l y ( P < 0 .05 ) . F i b e r a n a l y s e s r e s u l t e d in a l a r g e n u m b e r o f i n t e rac t i ons w h i c h p r o d u c e d v a r i a b l e r e s p o n s e s . E x p e r i m e n t 3 e v a l u a t e d t h e e f f ec t s o f t he t r e a t m e n t s o n t he f e rmen ta t i on c h a r a c t e r i s t i c s o f c a n o l a m e a l u s i n g t he in vitro g a s p r o d u c t i o n t e c h n i q u e . R a t e of g a s p r o d u c t i o n w a s s ign i f i can t l y ( P < 0 .05 ) a f f e c t e d by p r o t e a s e , w h e r e a s , t he l eng th o f t h e l a g p h a s e w a s s ign i f i can t l y a f f e c t e d b y t he in te rac t i on b e t w e e n p r o t e a s e a n d x y l a n a s e ( P < 0 .05 ) . In g e n e r a l , t he t r e a t m e n t s i n c r e a s e d ra te a n d d e c r e a s e d l a g t i m e . V o l a t i l e fatty a c i d ( V F A ) a n a l y s e s r e v e a l e d a s ign i f i can t i n te rac t i on ( P < 0 . 0 5 ) b e t w e e n p r o t e a s e a n d x y l a n a s e . T h e r e w a s a pos i t i ve l i n e a r / r e l a t i onsh ip ( P < 0 . 0 5 ) b e t w e e n t h e l eve l o f p r o t e a s e a p p l i c a t i o n a n d t he rat io of a c e t a t e to p r o p i o n a t e . P r o t e a s e a l s o h a d a n e f fec t ( P < 0 .05 ) o n t he rat io of bu ty ra te to p r o p i o n a t e . X y l a n a s e a l s o s ign i f i can t l y ( P < 0 .05 ) bo th a c e t a t e a n d buty ra te p r o d u c t i o n re la t i ve to p r o p i o n a t e . R e s u l t s f r o m e x p e r i m e n t 3 i n d i c a t e d the a p p l i c a t i o n o f p r o t e a s e at h i g h e r l e v e l s w a s b e n e f i c i a l in t e r m s o f i m p r o v i n g the nutr i t ional v a l u e o f c a n o l a m e a l . F r o m the w o r k c o n d u c t e d in th is t h e s i s , it m a y b e c o n c l u d e d , in t h e c a s e o f bo th b a r l e y a n d c a n o l a m e a l , tha t x y l a n a s e w a s d e t r i m e n t a l to d iges t ib i l i t y a n d that mi ld h y d r o t h e r m a l t r e a t m e n t d id not i m p r o v e e n z y m e ac t i v i t i es . M a n y e n z y m e t r e a t m e n t s i n c r e a s e d t he p ropo r t i on of f iber , s u g g e s t i n g a shi f t in f e e d c o m p o s i t i o n . T h e resu l t s o f th is s t u d y s u g g e s t po ten t ia l a p p l i c a t i o n s o f t he t r e a t m e n t o f b a r l e y w i th p r o t e a s e o r a c o m b i n a t i o n o f p r o t e a s e a n d p - g l u c a n a s e e n z y m e s . T r e a t m e n t o f c a n o l a m e a l w i th p r o t e a s e b e t w e e n t he l e v e l s o f 0 . 0 1 % a n d 0 . 0 5 % (vol / wt) r e s u l t e d in t h e g r e a t e s t i m p r o v e m e n t in it's iii nutr i t ive v a l u e for d a i r y ca t t le . T h e in vitro w o r k p r e s e n t e d in th is t h e s i s d e m o n s t r a t e s t h e po ten t i a l i m p r o v e m e n t s w i th e n z y m e t e c h n o l o g y in t he f e e d i n g qua l i t y o f b a r l e y a n d c a n o l a m e a l f e d to d a i r y ca t t le . iv TABLE OF CONTENTS ABSTRACT ii TABLE OF CONTENTS v LIST OF TABLES ix LIST OF FIGURES... xi ACKNOWLEDGMENTS xiii 1.0 INTRODUCTION 1 1.1 E X O G E N O U S E N Z Y M E S 3 1.1.1 Sources of Enzymes 3 1.1.2 Mode of Action 4 1.1.3 Monogastric Success 5 1.1.3.1 An t i -Nu t r i t i ona l P r o p e r t i e s o f C e r e a l s 6 1.1.3.2 p - g l u c a n a s e s 7 1.1 .3 .3 X y l a n a s e s 8 1.1.3.4 P r o t e a s e s 9 1.1.4 Ruminant Research with Enzymes 9 1.1.4.1 S o u r c e s o f V a r i a t i o n in R e s u l t s 10 1 .1 .4 .2 F i b ro l y t i c E n z y m e s 11 1.1.4.2. i ) F o r a g e s 11 1.1.4.2.H) C o n c e n t r a t e s 12 1.1 .4 .3 O t h e r E n z y m e s a n d E n z y m e C o m b i n a t i o n s 13 1.1.4.3.1) F o r a g e s 1 3 1.1.4.3. i i ) C o n c e n t r a t e s 13 1.1.5 Feed Processing and Enzyme Applications 14 1.2 IN VITRO T E C H N I Q U E S 16 1.2.1 Apparent Digestibility: Tilley and Terry In Vitro Technique 18 1.2.2 True Digestibility 19 1.2.3 Enzymatic Method 19 1.2.4 Rumen Fluid Inoculum 2 0 1.2.4.1 A n i m a l D ie t 2 0 1.2.4.2 R u m e n F l u i d C o l l e c t i o n 21 1 .2 .4 .3 I n o c u l u m P r e p a r a t i o n 21 1.2.5 Advantages of In Vitro Techniques 2 2 1.2.6 Disadvantages of In Vitro Techniques 2 3 1.2.7 Kinetic Descriptions of Feed Degradation 2 3 1.2.7.1 In Situ T e c h n i q u e 2 3 1.2.8 The Gas Production Technique 2 5 1.2.8.1 A d v a n t a g e s o f t he G a s P r o d u c t i o n T e c h n i q u e 2 7 v 1.2.8.2 D i s a d v a n t a g e s o f t h e G a s P r o d u c t i o n T e c h n i q u e 2 8 1.2 .8 .3 T h e F e r m e n t a t i o n P r o c e s s o f C a r b o h y d r a t e s in t h e R u m e n 2 8 1.2.8.4 E s t i m a t i o n o f G a s V o l u m e 3 0 1.2 .8 .5 U s e o f t he G a s P r o d u c t i o n T e c h n i q u e in t he E v a l u a t i o n o f C o n c e n t r a t e s 31 1.2.8.6 K i n e t i c A n a l y s i s o f G a s D a t a 3 2 1.3 O B J E C T I V E S O F T H E T H E S I S 3 3 1.4 R E F E R E N C E S 3 5 2.0 THE EFFECT OF ENZYME AND MILD HYDROTHERMAL TREATMENT ON THE IN VITRO DIGESTIBILITY OF BARLEY GRAIN 46 2.1 A B S T R A C T 4 6 2 . 2 I N T R O D U C T I O N 4 8 2 . 3 M A T E R I A L S A N D M E T H O D S 51 2.3.1 Feed Treatments 51 2 . 3 . 2 Processing Samples 5 2 2.3.3 Chemical Analyses 5 3 2 .3 .4 Animals and Feeding 5 4 2.3.5 Rumen Fluid Inoculum 54 2 . 3 . 6 In Vitro Procedure 5 5 2.3.7 Statistical Analysis 5 5 2 .4 R E S U L T S A N D D I S C U S S I O N : 5 6 2.4.7 IVDMD 5 6 2 . 4 . 2 IVTD 6 0 2.3.3 ADF 6 3 2.4.4 NDF 6 4 2 . 5 C O N C L U S I O N 6 6 2 .6 R E F E R E N C E S 6 8 3.0 THE EFFECT OF ENZYME AND MILD HYDROTHERMAL TREATMENT ON THE IN VITRO DIGESTIBILITY OF CANOLA MEAL 92 3.1 A B S T R A C T 9 2 3.2 I N T R O D U C T I O N 9 4 3 .3 M A T E R I A L S A N D M E T H O D S 9 5 3.3 .7 Feed Treatments 9 5 3 . 3 . 2 Processing of Feed Treatments 9 5 3 . 3 . 3 Chemical Analyses 9 6 3.3.4 Animals and Feeding 9 6 3.3.5 Rumen Fluid Inoculum 9 6 3 . 3 . 6 In vitro Procedure 9 6 3 . 3 . 7 Statistical Analysis 9 6 3.4 R E S U L T S A N D D I S C U S S I O N 9 7 3 .4 .7 IVDMD 9 7 3.4.2 IVTD 1 0 0 3.4.3 ADF 1 0 2 3.4.4 NDF 104 vi 3 . 5 C O N C L U S I O N S 1 0 5 3 . 6 R E F E R E N C E S 1 0 7 4.0 THE EFFECT OF ENZYME AND MILD HYDROTHERMAL TREATMENT ON THE IN VITRO GAS PRODUCTION IN CANOLA MEAL 124 4 . 1 A B S T R A C T 1 2 4 4 . 2 I N T R O D U C T I O N 1 2 6 4 . 3 M A T E R I A L S A N D M E T H O D S 1 2 8 4.3.1 Feed Treatments 1 2 8 4.3.2 Processing of Feed Treatments 1 2 8 4.3.3 Chemical Analyses 1 2 9 4.3.4 Animals and Feeding 1 2 9 4.3.5 Rumen Fluid Collection 1 2 9 4.3.6 Gas Production System 1 2 9 4.3.7 Gas Measurements 1 3 0 4.3.8 Procedure for the Gas Production Technique 1 3 1 4.3.9 Data Processing 1 3 2 4.3.10 Kinetic Analysis of Gas Data 1 3 2 4.3.11 VFA Sampling and Analyses: 1 3 3 4.3.12 Statistical Analysis 1 3 4 4 . 4 R E S U L T S A N D D I S C U S S I O N 1 3 4 4.4.1 Validation and Comparison of the Gas Production System 1 3 5 4.4.2 Rate of In Vitro Gas Production 1 3 8 4.4.3 Lag Period 1 4 1 4.4.4 Volatile Fatty Acids 1 4 3 4.4.5 VFA Ratios: acetate to propionate; butyrate to propionate 1 4 5 4 . 5 C O N C L U S I O N S 1 4 7 4 . 6 R E F E R E N C E S 1 4 9 5.0 GENERAL CONCLUSIONS .' 165 5.1 B A R L E Y 1 6 5 5 . 2 C A N O L A M E A L 1 6 6 5 . 3 S U M M A R Y 1 6 7 5 . 4 R E F E R E N C E S 1 6 9 6.0 APPENDICES 170 A P P E N D I X 6 .1 T E S T O F S U B S T R A T E A C T I V I T Y 1 O F T H E E N Z Y M E S : P - G L U C A N A S E , P R O T E A S E A N D X Y L A N A S E S U P P L I E D B Y F F I ( W I L T S H I R E , U . K . ) 1 7 1 A P P E N D I X 6 . 2 C O M P O S I T I O N O F T H E C O N C E N T R A T E R A T I O N F E D T O D O N O R C O W S 1 7 2 A P P E N D I X 6 . 3 . S A S P R O G R A M U S E D To E V A L U A T E B A R L E Y D A T A 1 7 3 A P P E N D I X 6 . 4 . S A S P R O G R A M U S E D T O E V A L U A T E C A N O L A M E A L D A T A 1 7 5 A P P E N D I X 6 . 5 C A L I B R A T I O N O F T H E G A S M E A S U R E M E N T S Y S T E M ( S C H O F I E L D A N D P E L L , 1 9 9 5 ) 1 7 6 vi i A P P E N D I X 6.6. RECORDED G A S PRODUCTION V S . STOICHIOMETRICALLY CALCULATED G A S PRODUCTION FROM V F A COMPOSITION AT 6 H O F A RANDOM SELECTION O F CANOLA MEAL SAMPLES 179 A P P E N D I X 6.7. S A S PROGRAM USED T O DETERMINE G A S PRODUCTION PARAMETERS F O R CANOLA MEAL 180 A P P E N D I X 6.8 . RAW DATA FOR THE ESTIMATED G A S PRODUCTION PARAMETERS FROM CANOLA MEAL: RATE O F G A S PRODUCTION, LAG PERIOD A N D CUMULATIVE 1 8 H G A S PRODUCTION 181 vi i i LIST OF TABLES T A B L E 2 . 1 . T R E A T M E N T S I N V E S T I G A T E D IN O R D E R T O D E T E R M I N E T H E E F F E C T S O F E X O G E N O U S E N Z Y M E A P P L I C A T I O N WITH A N D W I T H O U T A D D E D M O I S T U R E A N D H E A T O N R U M E N IN VITRO D R Y M A T T E R D I S A P P E A R A N C E A N D IN VITRO T R U E DIGESTIBILITY O F B A R L E Y G R A I N 7 3 T A B L E 2 . 2 . T H E E F F E C T O F T H E D U R A T I O N O F H E A T T R E A T M E N T A N D T H E A P P L I C A T I O N O F M O I S T U R E A N D X Y L A N A S E O N T H E 4 8 H IN VITRO D R Y M A T T E R D I S A P P E A R A N C E O F B A R L E Y G R A I N (DM B A S I S ) 7 4 T A B L E 2 . 3 . T H E E F F E C T O F P - G L U C A N A S E , P R O T E A S E A N D X Y L A N A S E A P P L I C A T I O N O N T H E 4 8 H IN VITRO D R Y M A T T E R D I S A P P E A R A N C E A N D IN VITRO T R U E DIGESTIBILITY O F B A R L E Y G R A I N (DM B A S I S ) 7 5 T A B L E 2 . 4 . T H E E F F E C T O F M O I S T U R E L E V E L O N T H E 4 8 H IN W T R O T R U E DIGESTIBILITY O F B A R L E Y G R A I N (DM B A S I S ) 7 6 T A B L E 2 . 5 . T H E E F F E C T O F P - G L U C A N A S E , P R O T E A S E A N D X Y L A N A S E A P P L I C A T I O N O N T H E ADF F R A C T I O N O F B A R L E Y G R A I N (DM BASIS) 7 7 T A B L E 2 . 6 . T H E E F F E C T O F H E A T T R E A T M E N T A N D T H E A P P L I C A T I O N O F P - G L U C A N A S E A N D X Y L A N A S E O N T H E ADF F R A C T I O N O F B A R L E Y G R A I N (DM B A S I S ) 7 8 T A B L E 2 . 7 . T H E E F F E C T O F P - G L U C A N A S E A N D P R O T E A S E A P P L I C A T I O N O N T H E NDF C O N T E N T O F B A R L E Y G R A I N (DM B A S I S ) 7 9 T A B L E 2 . 8 . T H E E F F E C T O F P - G L U C A N A S E A N D X Y L A N A S E A P P L I C A T I O N O N T H E NDF F R A C T I O N O F B A R L E Y G R A I N (DM B A S I S ) 8 0 T A B L E 2 . 9 . T H E E F F E C T O F P R O T E A S E A N D X Y L A N A S E A P P L I C A T I O N O N T H E NDF C O N T E N T O F B A R L E Y G R A I N (DM B A S I S ) 81 T A B L E 3 . 1 . T R E A T M E N T S I N V E S T I G A T E D IN O R D E R T O D E T E R M I N E T H E E F F E C T S O F E X O G E N O U S E N Z Y M E A P P L I C A T I O N WITH A N D W I T H O U T A D D E D M O I S T U R E A N D H E A T O N IN VITRO D R Y M A T T E R D I S A P P E A R A N C E A N D IN VITRO T R U E DIGESTIBILITY O F C A N O L A M E A L 1 0 9 T A B L E 3 . 2 . T H E E F F E C T O F T H E ADDITION O F P R O T E A S E A N D X Y L A N A S E T O C A N O L A M E A L O N T H E 4 8 H R U M E N IN VITRO D R Y M A T T E R D I S A P P E A R A N C E O F C A N O L A M E A L (DM B A S I S ) 1 1 0 T A B L E 3 . 3 . T H E E F F E C T O F M O I S T U R E A D D E D T O C A N O L A M E A L A T T H E T I M E O F P R O C E S S I N G O N T H E 4 8 H R U M E N IN VITRO D R Y M A T T E R DIGESTIBILITY O F C A N O L A M E A L (DM B A S I S ) 1 1 1 T A B L E 3 . 4 . T H E E F F E C T O F T H E ADDITION O F P R O T E A S E A N D X Y L A N A S E T O C A N O L A M E A L O N T H E R U M E N IN VITRO T R U E DIGESTIBILITY O F C A N O L A M E A L (DM BASIS) . . . . 1 1 2 T A B L E 3 . 5 . T H E E F F E C T O F M O I S T U R E A D D E D T O C A N O L A M E A L A T T H E T I M E O F P R O C E S S I N G O N T H E R U M E N IN VITRO T R U E DIGESTIBILITY O F C A N O L A M E A L (DM B A S I S ) 1 1 3 T A B L E 3 . 6 . T H E E F F E C T O F T H E D U R A T I O N O F H E A T T R E A T M E N T A N D T H E A P P L I C A T I O N O F P R O T E A S E A N D X Y L A N A S E T O C A N O L A M E A L O N T H E ADF F R A C T I O N O F C A N O L A M E A L (DM B A S I S ) 1 1 4 T A B L E 3 . 7 . T H E E F F E C T O F M O I S T U R E , P R O T E A S E A N D X Y L A N A S E A P P L I C A T I O N T O C A N O L A M E A L O N T H E ADF F R A C T I O N O F C A N O L A M E A L (DM B A S I S ) 1 1 5 ix T A B L E 3 . 9 . T H E E F F E C T O F M O I S T U R E ADDITION T O C A N O L A M E A L A T T H E T I M E O F P R O C E S S I N G O N T H E NDF F R A C T I O N IN C A N O L A M E A L (DM B A S I S ) 1 1 7 T A B L E 4 . 1 . T R E A T M E N T S I N V E S T I G A T E D IN O R D E R T O D E T E R M I N E T H E E F F E C T S O F E X O G E N O U S E N Z Y M E A P P L I C A T I O N WITH A N D W I T H O U T A D D E D M O I S T U R E A N D H E A T O N IN VITRO G A S P R O D U C T I O N P A R A M E T E R S A N D VFA A N A L Y S E S O F C A N O L A M E A L 1 5 2 T A B L E 4 . 2 . T H E E F F E C T O F T H E P R O T E A S E A P P L I C A T I O N O N T H E IN VITRO R A T E O F G A S P R O D U C T I O N O F C A N O L A M E A L 1 5 3 T A B L E 4 . 3 . T H E E F F E C T O F P R O T E A S E A N D X Y L A N A S E A P P L I C A T I O N O N T H E L E N G T H O F T H E L A G P H A S E IN VITRO O F C A N O L A M E A L 1 5 4 T A B L E 4 . 4 . T H E E F F E C T O F P R O T E A S E A N D X Y L A N A S E A P P L I C A T I O N O N T H E 6 H / N VITRO VFA P R O D U C T I O N O F C A N O L A M E A L 1 5 5 T A B L E 4 . 5 . T H E E F F E C T O F P R O T E A S E A P P L I C A T I O N O N T H E 6 H IN VITRO R A T I O O F A C E T A T E T O P R O P I O N A T E A N D B U T Y R A T E T O P R O P I O N A T E O F C A N O L A M E A L 1 5 6 T A B L E 4 . 6 . T H E E F F E C T O F X Y L A N A S E A P P L I C A T I O N O N T H E 6 H IN VITRO R A T I O O F A C E T A T E T O P R O P I O N A T E (A:P) A N D B U T Y R A T E T O P R O P I O N A T E (B:P) O F C A N O L A M E A L 1 5 7 X LIST OF FIGURES FIGURE 2.1. L A B O R A T O R Y S C A L E HYDROTHERMAL C O O K E R 82 FIGURE 2.2. T H E E F F E C T O F T H E ADDITION O F MOISTURE, XYLANASE (X) (% V O L / WT) AND DURATION OF HEAT T R E A T M E N T (55°C) ON T H E IN VITRO DRY MATTER DISAPPEARANCE O F BARLEY GRAIN 83 FIGURE 2.3. T H E E F F E C T O F P - G L U C A N A S E , XYLANASE (X) AND P R O T E A S E ( P ) APPLICATION (% V O L / WT) ON T H E IN VITRO DRY MATTER DISAPPEARANCE OF BARLEY GRAIN 84 FIGURE 2.4. Q U A D R A T I C RELATIONSHIP B E T W E E N L E V E L O F MOISTURE AND IN VITRO T R U E DIGESTIBILITY OF BARLEY GRAIN (Y = 87.75 + 0.07*MOISTURE L E V E L -0.0014*MOISTURE L E V E L 2 : N = 4, R 2 = 0.998, S . E . = 0.018) 85 FIGURE 2.5. T H E E F F E C T OF P - G L U C A N A S E , P R O T E A S E ( P ) AND X Y L A N A S E (X) APPLICATION (% V O L / WT ) ON T H E IN VITRO T R U E DIGESTIBILITY OF BARLEY GRAIN 86 FIGURE 2.6. T H E E F F E C T OF P - G L U C A N A S E , P R O T E A S E ( P ) AND X Y L A N A S E APPLICATION (% V O L / WT) ON T H E C O N T E N T OF ACID D E T E R G E N T FIBER IN BARLEY GRAIN 87 FIGURE 2.7. T H E E F F E C T O F P - G L U C A N A S E AND P R O T E A S E ( P ) APPLICATION (% V O L / WT) AND T H E DURATION OF HEAT T R E A T M E N T S (55°C) ON T H E C O N T E N T OF ACID D E T E R G E N T FIBER IN B A R L E Y GRAIN 88 FIGURE 2.8. T H E E F F E C T O F T H E ADDITION O F P - G L U C A N A S E AND P R O T E A S E (% V O L / WT) ON T H E C O N T E N T O F NEUTRAL D E T E R G E N T FIBER IN BARLEY GRAIN 89 F I G U R E 2.9. T H E E F F E C T O F T H E ADDITION O F P - G L U C A N A S E AND X Y L A N A S E (% VOL / WT ) ON T H E C O N T E N T O F NEUTRAL D E T E R G E N T FIBER IN BARLEY GRAIN 90 FIGURE 2.10. T H E E F F E C T O F T H E ADDITION OF P R O T E A S E AND X Y L A N A S E (% VOL / WT) ON T H E C O N T E N T OF NEUTRAL D E T E R G E N T FIBER IN BARLEY GRAIN 91 FIGURE 3.1 T H E E F F E C T O F T H E ADDITION OF XYLANASE AND P R O T E A S E (%VOL / WT) ON T H E 48 H IN VITRO DRY MATTER DISAPPEARANCE OF C A N O L A MEAL 118 FIGURE 3.2. Q U A D R A T I C RELATIONSHIP B E T W E E N MOISTURE L E V E L AND T H E 48 H IN VITRO DRY MATTER DISAPPEARANCE OF CANOLA MEAL (Y = 78.62 + 0.165* MOISTURE L E V E L - 0.003* MOISTURE L E V E L 2 , N = 4, R 2 = 0.998, S . E . = 0.105). ...119 FIGURE 3.3. T H E E F F E C T OF T H E ADDITION OF P R O T E A S E AND XYLANASE (% V O L / WT) ON T H E R U M E N IN VITRO T R U E DIGESTIBILITY OF CANOLA MEAL 120 F I G U R E 3.4. Q U A D R A T I C RELATIONSHIP B E T W E E N MOISTURE L E V E L (% V O L / WT) AND T H E R U M E N IN VITRO T R U E DIGESTIBILITY O F CANOLA MEAL (Y = 87.25 + 0.073* MOISTURE L E V E L - 0.002* MOISTURE L E V E L 2 : N = 4, R 2 = 0.991, S . E . 0.0355) 121 FIGURE 3.5. T H E E F F E C T O F T H E APPLICATION OF P R O T E A S E ( P ) , X Y L A N A S E (X) (% V O L / WT) A N D DURATION OF HEAT T R E A T M E N T ON T H E C O N T E N T OF ACID D E T E R G E N T FIBER IN C A N O L A MEAL 122 FIGURE 3.6. T H E E F F E C T O F T H E APPLICATION OF MOISTURE, P R O T E A S E ( P ) AND X Y L A N A S E (X) (% V O L / WT) ON T H E C O N T E N T OF ACID D E T E R G E N T FIBER IN C A N O L A MEAL 123 FIGURE 4.1. T H E COMPUTERIZED GAS PRODUCTION S Y S T E M (ADAPTED FROM P E L L AND S C H O F I E L D , 1993) 158 xi F I G U R E 4.2. C U S T O M I Z E D E R L E N M E Y E R F L A S K S WITH S A M P L I N G P O R T C O N T A I N I N G L U E R L O C K A N D T U B I N G A P P A R A T U S F O R V F A S A M P L I N G 159 F I G U R E 4.3. G A S P R O D U C T I O N P R O F I L E F O R A S A M P L E O F C A N O L A M E A L O V E R A 22 H I N C U B A T I O N P E R I O D WITH R U M E N F L U I D . 160 F I G U R E 4.4. T H E E F F E C T O F T H E A D D I T I O N O F P R O T E A S E O N T H E R A T E O F R U M E N IN VITRO G A S P R O D U C T I O N O F C A N O L A M E A L I N C U B A T E D WITH R U M E N FLUID : 161 F I G U R E 4.5. T H E E F F E C T O F T H E ADDITION O F P R O T E A S E A N D X Y L A N A S E (% V O L / W T ) O N T H E L E N G T H O F T H E L A G P H A S E O F C A N O L A M E A L I N C U B A T E D IN VITRO WITH R U M E N F L U I D 162 F I G U R E 4.6. T H E E F F E C T O F T H E ADDITION O F X Y L A N A S E A N D P R O T E A S E (% V O L / W T ) O N T H E P R O D U C T I O N O F VFAS IN C A N O L A M E A L F O L L O W I N G A6H/N VITRO I N C U B A T I O N WITH R U M E N FLUID 163 F I G U R E 4.7. L I N E A R R E L A T I O N S H I P B E T W E E N L E V E L O F P R O T E A S E A P P L I C A T I O N A N D T H E R A T I O O F A C E T A T E T O P R O P I O N A T E IN C A N O L A M E A L F O L L O W I N G A 6 H IN V I T R O I N C U B A T I O N W I T H R U M E N F L U I D (Y = 17 .55*PROTEASE L E V E L + 2.851: N = 4, R 2 = 0.839, S . E . = 0.17) 164 xi i ACKNOWLEDGMENTS I w o u l d l ike to e x p r e s s m y s i n c e r e t h a n k s to m y s u p e r v i s o r , Dr . J . A . S h e l f o r d o f t h e D e p a r t m e n t o f A n i m a l S c i e n c e at U . B . C . fo r h i s g u i d a n c e a n d c o n t i n u o u s s u p p o r t t h r o u g h o u t th is pro jec t . I w o u l d a l s o l ike to t h a n k Dr . M . A . G . v o n K e y s e r l i n g k o f t he D e p a r t m e n t o f A n i m a l S c i e n c e at U . B . C . fo r h e r c h e e r f u l e n c o u r a g e m e n t a n d i n v a l u a b l e a s s i s t a n c e in e n s u r i n g t he s u c c e s s of th is p ro jec t a n d th is t h e s i s . M a r y L o u S w i f t o f P r o F o r m F e e d s Inc. ( C h i l l i w a c k , B . C . ) i s g ra te fu l l y a c k n o w l e d g e d fo r in i t ia t ing th is u n i q u e pro jec t a n d fo r h e r input into t he t h e s i s . T h a n k s is a l s o e x t e n d e d to Dr . J . W . Ha l l o f t he P a c i f i c A g r i - F o o d R e s e a r c h C e n t e r ( S u m m e r l a n d , B . C . ) fo r h i s a s s i s t a n c e a n d p a t i e n c e in t he s ta t i s t i ca l a n a l y s e s a n d in te rp re ta t ion o f t he d a t a . I w o u l d a l s o l ike to t h a n k Dr . J o h n B a a h for h is s u p p o r t . T h a n k y o u to Dr . L. J . F i s h e r o f t he P a c i f i c A g r i c u l t u r a l R e s e a r c h S t a t i o n ( A g a s s i z , B . C . ) f o r h i s c o n s t r u c t i v e c o m m e n t s a n d e n c o u r a g e m e n t t h r o u g h o u t t he wr i t ing o f t he t h e s i s . I w o u l d a l s o l ike to t h a n k t h e o t h e r m e m b e r s o f m y c o m m i t t e e , Dr . R . M . Ta i t o f t he A n i m a l S c i e n c e D e p a r t m e n t at U . B . C . a n d Dr . J . V a n d e r s t o e p o f t he D e p a r t m e n t o f F o o d S c i e n c e at U . B . C . fo r the i r he lp fu l c o m m e n t s o n t h e t h e s i s . T h e e x p e r t i s e o f G i l l e s G a l z i i s g ra te fu l l y a c k n o w l e d g e d in put t ing t o g e t h e r t he g a s p r o d u c t i o n s y s t e m . T h a n k s is e x t e n d e d to L e s l i e R i c h a r d s o n w h o a s s i s t e d in t h e init ial s t a g e s o f t he e x p e r i m e n t a n d to S a r a M u i r fo r he r a s s i s t a n c e in se t t i ng u p t he g a s e x p e r i m e n t a n d to t he t e c h n i c i a n s at U . B . C . a n d P r o F o r m F e e d s Inc., w h o a s s i s t e d in l a b o r a t o r y a n a l y s e s . T h e S o u t h C a m p u s f a r m staf f at U . B . C . is a l s o t h a n k e d fo r the i r h e l p in m a i n t a i n i n g t he a n i m a l s for t h i s e x p e r i m e n t . T h a n k y o u to a l l o f m y f r i e n d s a n d c o l l e a g u e s at U . B . C . w h o e n s u r e d m y life a s a g r a d u a t e s t u d e n t w a s a n e n j o y a b l e l e a r n i n g e x p e r i e n c e . F i n a n c i a l a s s i s t a n c e fo r th is r e s e a r c h w a s p r o v i d e d b y B . C . S c i e n c e C o u n c i l - T e c h n o l o g y B . C . w i th P r o F o r m F e e d s a s t he Indust r ia l pa r tne r . C S . G i l l w a s s u p p o r t e d by a n N S E R C Indust r ia l S c h o l a r s h i p w i th P r o F o r m F e e d s a l s o a s t h e i ndus t r i a l pa r tne r . T h e e n z y m e s u s e d in th i s p ro jec t w e r e s u p p l i e d by F i n n F e e d s In te rna t iona l (Wi l t sh i re , U . K . ) . F e e d w a s s u p p l i e d by P r o F o r m F e e d s Inc. ( C h i l l i w a c k , B . C . ) . F i n a l l y , I w o u l d e s p e c i a l l y l ike to t h a n k m y h u s b a n d R i c k fo r h is l o v e , p a t i e n c e a n d s u p p o r t t h r o u g h o u t th is wo rk . T h e c o m p l e t i o n o f th i s t h e s i s w a s p o s s i b l e b e c a u s e o f t he c o n t i n u o u s e n c o u r a g e m e n t f r o m N i n a , m y f am i l y a n d m y h u s b a n d R i c k . xi i i CHAPTER 1 1.0 INTRODUCTION F e e d c o m p a n i e s in Br i t i sh C o l u m b i a ( B . C . ) a r e c h a l l e n g e d w i th s u p p l y i n g da i r y p r o d u c e r s w i th a f f o r d a b l e , h i gh qua l i t y f e e d s t u f f s . T w o m a j o r d e v e l o p m e n t s that h a v e a f f e c t e d t h e f e e d m a n u f a c t u r i n g , da i r y a n d o t h e r l i v e s t o c k i ndus t r i es in r e c e n t y e a r s a r e t h e N o r t h A m e r i c a n F r e e T r a d e A g r e e m e n t a n d t he r e c e n t l o s s of rail s u b s i d i e s . T h e major i ty o f c o n c e n t r a t e s f e d to r u m i n a n t s in B . C . a r e t r a n s p o r t e d f r o m o t h e r p r o v i n c e s a n d t h e U n i t e d S t a t e s at a s ign i f i can t c o s t fo r u s e by B . C . f a r m e r s . F e e d r e p r e s e n t s a p p r o x i m a t e l y 6 0 % o f d a i r y f a r m c o s t s , w i th g ra in a c c o u n t i n g fo r 3 0 % o f th is e x p e n s e in t h e a r e a o f t he L o w e r F r a s e r V a l l e y ( B a r k e r a n d C h e a t l e y , 1 9 9 2 ) . T h e r e f o r e , f e e d c o m p a n i e s a r e c o n s t a n t l y c h a l l e n g e d to s u p p l y f e e d s w h i c h a r e i n e x p e n s i v e a n d ef f ic ient ly u s e d b y da i r y ca t t le . In a r e a s l i ke t h e L o w e r F r a s e r V a l l e y r e g i o n o f B . C . a n d E u r o p e , w h e r e i n t e n s i v e m o n o g a s t r i c a n i m a l p r o d u c t i o n is p r e v a l e n t , e x o g e n o u s e n z y m e s h a v e p l a y e d a n i n c r e a s i n g ro le in r e d u c i n g t he e x c r e t i o n o f po l lu t ing nu t r ien ts a n d o p t i m i z i n g t h e u s e o f f e e d r e s o u r c e s . It h a s b e e n r e p o r t e d that 8 0 % o f t he w o r l d ' s pou l t ry d i e t s a r e n o w e n z y m e t r ea ted ( T r e a c h e r a n d H u n t , 1 9 9 6 ) . T h e f e e d i ndus t r y h a s p r imar i l y f o c u s e d o n t he u s e o f e x o g e n o u s e n z y m e s to i m p r o v e t h e nutr i t ive v a l u e o f i n g r e d i e n t s in s w i n e a n d pou l t ry d i e t s . T h e C a n a d i a n f e e d i ndus t r y h a s a p p l i e d e n z y m e s to pou l t ry d i e t s s i n c e t he la te 1 9 8 0 ' s ( L e s l i e , 1 9 9 5 ) . I m p r o v e m e n t s in f e e d u t i l i za t ion a s a resu l t o f e n z y m e s u p p l e m e n t a t i o n h a v e 1 h e l p e d p r o d u c e r s r e m a i n c o m p e t i t i v e in t he m a r k e t p l a c e . R e s e a r c h into t he u s e of e n z y m e s in r u m i n a n t d i e t s w a s br ie f ly i n v e s t i g a t e d t h r o u g h t h e 1 9 6 0 ' s , h o w e v e r , resu l t s w e r e t o o i n c o n s i s t e n t to w a r r a n t fu r ther i n v e s t i g a t i o n , resu l t i ng in t he l ack of a p p l i c a t i o n s ( C h e s s o n a n d A u s t i n , 1 9 9 6 ) . M a j o r a d v a n c e s in e n z y m e t e c h n o l o g y a n d t he i n c r e a s e d c o s t s o f l i v e s t o c k p r o d u c t i o n h a v e r e n e w e d in te res t into t he u s e o f e n z y m e s in r u m i n a n t d i e t s . T w o m a j o r f e e d s i n c o r p o r a t e d into d a i r y ca t t le d ie t s in W e s t e r n C a n a d a a r e b a r l e y a n d c a n o l a m e a l , h o w e v e r t he l e v e l s o f i n c l u s i o n of t h e s e f e e d s a r e l im i ted b e c a u s e h igh f i be r c o n t e n t d e c r e a s e s the i r e n e r g y v a l u e . T h e s u p p l e m e n t a t i o n o f t h e s e f e e d s t u f f s w i th e x o g e n o u s e n z y m e s h a s t he po ten t ia l to a l te r t h e f e r m e n t a t i o n pro f i le o f t he c a r b o h y d r a t e f rac t ion a n d i m p r o v e the d iges t ib i l i t y o f t h e s e f e e d s . T h e in tent o f th is t h e s i s p ro jec t w a s to i n v e s t i g a t e t he feas ib i l i t y o f i m p r o v i n g t h e nut r i t iona l v a l u e o f c a n o l a m e a l a n d b a r l e y f o r d a i r y ca t t l e u s i n g e n z y m e t e c h n o l o g y a n d mi ld h y d r o t h e r m a l t r ea tmen t . 2 1.1 EXOGENOUS ENZYMES 1.1.1 Sources of Enzymes E n z y m e s u s e d in t he f e e d indus t r y a r e pr imar i l y d e r i v e d f r o m s o u r c e s u s e d by t he f o o d a n d b e v e r a g e i n d u s t r i e s ( C h e s s o n a n d A u s t i n , 1 9 9 6 ) . E n z y m e p r e p a r a t i o n s a r e g e n e r a l l y not s p e c i f i c but c o n t a i n a n a r r a y o f e n z y m e ac t iv i t ies ( C a m p b e l l a n d B e d f o r d , 1 9 9 2 ) . A l t h o u g h t he f o o d a n d b e v e r a g e i ndus t r i es requ i re g r e a t e r spec i f i c i t y in the i r e n z y m e r e q u i r e m e n t s , e n z y m e s p r e p a r e d fo r the f e e d i ndus t r y a r e ra re l y r e q u i r e d to b e s o s p e c i f i c in na tu re . T h e d e v e l o p m e n t o f a n e n z y m e c o m p l e m e n t to a s p e c i f i c i ng red ien t r e q u i r e s a c a r e f u l m a t c h b e t w e e n e n z y m e a n d s u b s t r a t e a s w e l l a s c o n s i d e r a t i o n o f s t o r a g e a n d p r o c e s s i n g c o n d i t i o n s in o r d e r to o b t a i n t he d e s i r e d r e s p o n s e . S o u r c e s o f e n z y m e s i n c l u d e t h o s e f r om fung i ( e .g . Trichoderma a n d Aspergillus s p p . ) , b a c t e r i a ( e .g . Bacillus s p p . ) a n d y e a s t s ( e .g . Saccharomyces cerevisiae). C o m m e r c i a l e n z y m e p r o d u c t s o r " e n z y m e c o c k t a i l s " u s u a l l y c o n t a i n a mix tu re of s p e c i f i c e n z y m e ac t i v i t i es ( N e w m a n , 1 9 9 4 ) . E n z y m e " c o c k t a i l s " , h a v e b e e n g e n e r a l l y m o r e s u c c e s s f u l in a n i m a l f e e d i n g s y s t e m s t h a n a s i n g l e e n z y m e act iv i ty ( M u l d e r e t a l . , 1 9 9 1 ; C h e s s o n , 1 9 9 3 ) . K n o w l e d g e o f t he t y p e o f e n z y m e , w h e t h e r a p p l i e d a l o n e o r a s a " c o c k t a i l " , i t 's spec i f i c i t y a n d m o d e o f a c t i o n is c r u c i a l in o r d e r to t a k e a d v a n t a g e o f b e n e f i c i a l e f f ec t s a n d m i n i m i z e n e g a t i v e e f f ec t s t he e n z y m e m a y h a v e o n t h e s u b s t r a t e ( A m a d o , 1 9 9 3 ) . 3 1.1.2 Mode of Action I m p r o v e m e n t s o b s e r v e d w i th e x o g e n o u s e n z y m e s , w i th bo th m o n o g a s t r i c a n d r u m i n a n t s p e c i e s , i n c l u d e i n c r e a s e d f e e d ut i l i za t ion a n d r e d u c e d va r i a t i on in the ava i lab i l i t y o f nu t r ien ts in f e e d s t u f f s . It h a s b e e n s u g g e s t e d that e n z y m e s a id in the d i s r u p t i o n o f c o m p l e x p lan t ce l l w a l l s w h i c h c a u s e s in t race l l u la r nu t r ien ts , s u c h a s s t a r c h a n d p r o t e i n , to b e r e l e a s e d ( G r u p p e n , 1 9 9 6 ; C h e s s o n , 1 9 9 3 ) . B y t he s a m e m e c h a n i s m , a n e n z y m e " c o c k t a i l " is m o r e e f fec t i ve in d e g r a d i n g t he ce l l matr ix ( G r u p p e n , 1 9 9 6 ) . F o r e x a m p l e , t he s y n e r g i s t i c e f fec t o f s e v e r a l c a r b o h y d r a s e s w a s s h o w n to e n h a n c e t h e ra te o f h y d r o l y s i s a n d p r o d u c e t he d e s i r e d p r o d u c t s w h e n a p p l i e d to w h e a t a n d s o r g h u m f lour , s u n f l o w e r a n d p a l m - k e r n e l m e a l s (Dus te rho f t et a l . , 1 9 9 3 ) . T h e i m p r o v e d d i g e s t i o n o f f i be r o b s e r v e d w i th s o m e e n z y m e s u p p l e m e n t e d r u m i n a n t d i e t s m a y a l s o b e a resu l t o f i m p r o v e d c o l o n i z a t i o n of f e e d pa r t i c l es by r u m e n m i c r o b e s ( C h e n g et a l . , 1 9 9 5 ) . H o w e v e r , r esu l t s w i th f ibro ly t ic e n z y m e s u p p l e m e n t s a r e i n c o n s i s t e n t . W h e n c a l v e s w e r e f e d a n a l fa l fa a n d o r c h a r d g r a s s h a y d ie t w h e r e a ce l l u lo l y t i c e n z y m e w a s s p r a y e d o n t o t he g ra in s u p p l e m e n t , t he a n i m a l s s h o w e d a n u n e x p e c t e d l a c k o f r e s p o n s e ( L e a t h e r w o o d et a l . , 1 9 6 0 ) . O n t h e o t h e r h a n d , a m u l t i e n z y m e s u p p l e m e n t a t i o n f e d to c a l v e s i m p r o v e d g a i n a n d f e e d e f f i c i e n c i e s ( M o d y a n o v a n d Z e l ' n e r , 1 9 8 3 ) . G r a h a m a n d P e t t e r s s o n ( 1 9 9 2 ) f o u n d that s e v e r a l e n z y m e s w e r e requ i r ed in o r d e r to o b t a i n a p r o d u c t i o n r e s p o n s e in b ro i le r c h i c k s f e d a b a r l e y b a s e d diet . T h i s e f fec t w a s a t t r ibu ted to t he i n c r e a s e d ava i lab i l i t y o f i n t race l l u la r nu t r ien ts . A n e n z y m e " c o c k t a i l " c o n s i s t i n g o f p - g l u c a n a s e , c e l l u l a s e a n d x y l a n a s e a l s o r esu l t ed in 4 t he r e l e a s e o f m o r e i n t race l l u la r nu t r ien ts t h a n w h e n p - g l u c a n a s e a l o n e w a s a p p l i e d to b a r l e y ( M u l d e r e t a l . , 1 9 9 1 ) . 1.1.3 Monogastric Success T h e s u c c e s s f u l a p p l i c a t i o n o f e x o g e n o u s e n z y m e s m a y b e i l lus t ra ted by r e s e a r c h c o n d u c t e d u s i n g m o n o g a s t r i c a n i m a l s . S e v e r a l r e v i e w s o n t h e u s e of e n z y m e s in m o n o g a s t r i c f e e d s s u m m a r i z e i m p r o v e m e n t s in a n i m a l p e r f o r m a n c e w i th t he u s e o f p - g l u c a n a s e s , p e n t a s o n a s e s ( x y l a n a s e s ) a n d o t h e r e n z y m e s ( C a m p b e l l a n d B e d f o r d , 1 9 9 2 ; C h e s s o n , 1 9 9 3 ; G e r a e r t et a l . , 1 9 9 6 ) . E n z y m e s h a v e b e e n s h o w n to : d e s t r o y ant i -nu t r i t iona l f a c t o r s , a s s i s t in t he b r e a k d o w n o f b o n d s not h y d r o l y z e d b y t h e a n i m a l ' s e n d o g e n o u s e n z y m e c o m p l e m e n t , fu r ther e n h a n c e t he e n d o g e n o u s e n z y m e ac t i v i t i es o f t he a n i m a l a n d t h e r e b y i n c r e a s e t he nutr ient a n d e n e r g y ava i l ab i l i t y o f a l t e rna t i ve f e e d s t u f f s ( C h e s s o n , 1 9 9 3 ; C l o s e , 1 9 9 6 ) . It h a s b e e n r e p o r t e d tha t e n z y m e s r e d u c e t h e var iab i l i t y o f a p p a r e n t m e t a b o l i z a b l e e n e r g y ( A M E ) v a l u e s a s s o c i a t e d w i th c e r e a l s ( G e r a e r t et a l . , 1 9 9 6 ) . I m p r o v e m e n t s in f e e d e f f i c i ency a n d g r o w t h ra tes h a v e b e e n repo r t ed w i th pou l t ry a n d s w i n e that w e r e f e d e n z y m e s u p p l e m e n t e d d ie t s ( M a r q u a r d t et a l . , 1 9 8 7 ; R o t t e r et a l . , 1987 ) . W i t h c h i c k s , i n c r e a s e d ra te o f g a i n , e f f i c i ency o f f e e d u t i l i za t ion , A M E a n d t he d igest ib i l i ty o f D M , fat a n d p ro te in h a v e a l s o b e e n r e p o r t e d ( M a r q u a r t , 1 9 9 4 ) . T h e p r ima ry g o a l s o f t he f e e d i ndus t r y i nvo l v ing , cu r ren t e n z y m e r e s e a r c h a r e m a i n l y d i r e c t e d t o w a r d s m o n o g a s t r i c s a n d a r e to r e m o v e o r d e s t r o y t he e f fec ts o f an t i -nu t r i t iona l f ac to r s ( ie. N S P s ) , i n c r e a s e o v e r a l l d iges t ib i l i t y o f t he f e e d a n d t he b ioava i l ab i l i t y o f s p e c i f i c 5 nut r ien ts a n d d e c r e a s e nut r ient po l lu t ion ( N e w m a n , 1 9 9 4 ) . F o r e x a m p l e , i m p r o v e m e n t s in t h e d iges t ib i l i t y o f t h e n o n - s t a r c h p o l y s a c c h a r i d e s ( N S P ) in c e r e a l s h a v e b e e n s h o w n to r e d u c e gu t c o n t e n t v i s c o s i t y , t h e r e b y i n c r e a s i n g t he ava i lab i l i t y o f nu t r ien ts a n d i m p r o v i n g a n i m a l p e r f o r m a n c e ( C a m p b e l l a n d B e d f o r d , 1 9 9 2 ; C h e s s o n , 1 9 9 3 ) . 1.1.3.1 Anti-Nutritional Properties of Cereals T h e c o n t i n u e d i n c r e a s e in f e e d p r i c e s a n d t he d e m a n d to m e e t i n c r e a s i n g nut r i t iona l r e q u i r e m e n t s o f p r o d u c t i o n a n i m a l s h a v e r e s u l t e d in l a rge r a m o u n t s of d i f ferent c e r e a l s n o w b e i n g i n c l u d e d in m o n o g a s t r i c d i e t s . C e r e a l s m a y c o n t a i n a l a rge p ropo r t i on o f N S P s w h i c h a r e p o l y m e r s o f s i m p l e s u g a r s a n d i n c l u d e c e l l u l o s e , h e m i c e l l u l o s e , p e c t i n s a n d o l i g o s a c c h a r i d e s . M o n o g a s t r i c a n i m a l s d o no t p o s e s s t h e e n d o g e n o u s e n z y m e s to b r e a k d o w n N S P s . In t he d i g e s t i v e t rac t , t h e s e c o m p o u n d s b e c o m e s o l u b l e t h e r e f o r e con t r i bu t i ng to a n ant i -nut r i t iona l e f fect . T h e s e s o l u b l e N S P s i n c r e a s e t h e v i s c o s i t y o f d i g e s t a w h i c h resu l t s in a r e d u c t i o n in t he ra te of f e e d p a s s a g e , f e e d i n t a k e a n d t h e e f f i c i ency o f f e e d u t i l i za t ion ; al l o f w h i c h a r e re f lec ted in p o o r a n i m a l p e r f o r m a n c e ( B e d f o r d , 1 9 9 3 ) . T h e resu l t i ng i m p a i r e d gut func t ion r e d u c e s t h e ava i l ab i l i t y o f nu t r ien ts s u c h a s g l u c o s e , fat a n d p ro te in ( F e n g l e r a n d M a r q u a r d t , 1 9 8 8 ; B e d f o r d , 1 9 9 3 ) . I n c r e a s e d v i s c o s i t y o f t he gu t a l s o c a u s e s "s t i cky d r o p p i n g s " , a cond i t i on a f fec t i ng pou l t ry w h i c h c a n resu l t in p o o r litter qua l i t y ( C a m p b e l l a n d B e d f o r d , 1992 ) . M e a t qua l i t y m a y b e a f f e c t e d if b ro i le r b i rds d e v e l o p b r e a s t d a m a g e a n d h o c k 6 p r o b l e m s ( C a m p b e l l a n d B e d f o r d , 1 9 9 2 ) . P o u l t r y d i e t s tha t c o n t a i n a l a rge p ropo r t i on o f N S P s h a v e a l o w e r A M E c o n t e n t w h i c h is n e g a t i v e l y c o r r e l a t e d wi th c o n t e n t o f s o l u b l e p o l y s a c c h a r i d e s ( a r a b i n o x y l a n s ) in t he d ie t ( A n n i s o n , 1991 ) . T h e ant i -nu t r i t iona l e f f e c t s o f N S P s m a y b e r e d u c e d w i th d ie ta r y e n z y m e s u p p l e m e n t a t i o n w h i c h wi l l b e d i s c u s s e d fur ther . S p e c i f i c e n z y m e s o f in te res t fo r u s e in m o n o g a s t r i c f e e d s i n c l u d e x y l a n a s e s for w h e a t in add i t i on to p - g l u c a n a s e s a n d c e l l u l a s e s fo r b a r l e y a n d o a t s ( M a r q u a r d t , 1 9 9 4 ) . H o w e v e r , t he i n c r e a s e in p r o d u c t i o n resu l t i ng f r o m t h e u s e o f e n z y m e s s u c h a s p - g l u c a n a s e a n d x y l a n a s e , c a n n o t b e s o l e l y e x p l a i n e d b y t he d e s t r u c t i o n of t he g e l f o r m i n g p o l y s a c c h a r i d e s ( C h e s s o n , 1 9 9 3 ) . I m p r o v e m e n t s in nut r ient a b s o r p t i o n a n d g r o w t h ra tes m a y a l s o b e a t t r ibu ted to t h e i m p r o v e d c o n d i t i o n s w i th in t he g a s t r o i n t e s t i n a l t ract ( C h e s s o n , 1 9 9 3 ) . 1.1.3.2 p-glucanases A l a r g e po r t i on o f t he e n d o s p e r m ce l l w a l l s o f c e r e a l s is c o m p o s e d of p-g l u c a n s a n d a r a b i n o x y l a n s ( G r a h a m et a l . , 1 9 8 9 ) . In b a r l e y a n d o a t s , t he ma jo r ce l l wa l l p o l y s a c c h a r i d e s a r e t he p - g l u c a n s w h i c h b e c o m e s o l u b l e a n d i n c r e a s e the v i s c o s i t y o f d i g e s t a in t h e gu t t h u s a f fec t i ng pou l t ry a n d to a l e s s e r d e g r e e s w i n e ( C a m p b e l l a n d B e d f o r d , 1 9 9 2 ) . p - g l u c a n s a r e s o l u b l e b e c a u s e t he P-(1,4) b a c k b o n e is i n t e r s p e r s e d w i th P-(1,3) l i n k a g e s b e t w e e n t he g l u c o s e p o l y m e r s p r e v e n t i n g inter-m o l e c u l a r b o n d i n g . T h e a d d i t i o n o f p - g l u c a n a s e to pou l t ry d i e t s h y d r o l y z e s the 7 s o l u b l e m i x e d l i n k e d p - g l u c a n s w h i c h m i n i m i z e s the i r an t i -nu t r i t iona l e f f ec t s s u c h a s c a u s i n g v i s c o u s d i g e s t a in t he gut . T h e i n c r e a s e in t h e p r o d u c t i o n r e s p o n s e of b ro i l e rs f e d a b a r l e y d ie t s u p p l e m e n t e d w i th p - g l u c a n a s e c a n b e e x p l a i n e d by the i n c r e a s e d ava i l ab i l i t y o f i n t race l l u la r nu t r ien ts resu l t i ng f r o m t h e l o s s o f t he integr i ty o f t he ce l l w a l l ( H e s s e l m a n a n d A m a n , 1 9 8 6 ) . 1.1.3.3 Xylanases L i k e p - g l u c a n s , a r a b i n o x y l a n s a r e s o l u b l e a n d m a y l e a d to s i m i l a r an t i -nut r i t iona l e f f ec t s a s d e s c r i b e d a b o v e if p r e s e n t in s i gn i f i can t a m o u n t s in poul t ry d ie t s . A r a b i n o x y l a n s a r e t he m a j o r ce l l wa l l c o m p o n e n t s o f r ye , w h e a t a n d tr i t icale a n d a m i n o r c o m p o n e n t in b a r l e y . T h e i r e f f ec t s c a n b e e l i m i n a t e d by s u p p l e m e n t i n g t h e d ie t w i t h x y l a n a s e to a i d in t h e s o l u b i l i z a t i o n a n d c l e a v a g e o f t h e p o l y s a c c h a r i d e s ( G r u p p e n et a l . , 1 9 9 3 ) . H o w e v e r t he act iv i ty o f t he e n z y m e m a y b e l imi ted if t he x y l a n c h a i n is h igh l y subs t i t u t ed w i th g l u c u r o n i c a c i d r e s i d u e s o r a r a b i n o s e m o l e c u l e s (B ie l y , 1 9 8 5 ) . T h e u s e o f x y l a n a s e s h a s f o c u s e d o n imp rov i ng d ie ta r y A M E v a l u e s a n d i n c r e a s i n g t he d iges t ib i l i t y o f r ye , w h e a t a n d b a r l e y in bro i le r d i e t s ( A n n i s o n a n d C h o c t , 1 9 9 1 ; K a o m a et a l . , 1 9 9 6 ) . It is n o w e s t a b l i s h e d that x y l a n a s e a p p l i e d to rye d i e t s r e d u c e s t he i n c i d e n c e o f " s t i cky d r o p p i n g s " a n d i m p r o v e s p e r f o r m a n c e in pou l t ry ( P e t t e r s s o n a n d A m a n , 1 9 8 8 , 1 9 8 9 ; T e i t g e et a l . , 1 9 9 1 ; B e d f o r d a n d C l a s s e n , 1 9 9 2 ) . 8 1.1.3.4 Proteases P r o t e a s e s m a y b e u s e d to i n c r e a s e p ro te in ava i l ab i l i t y a n d e l i m i n a t e pro te in ant i -nu t r i t iona l f a c t o r s a n d po ten t ia l a l l e r g e n i c p ro te i ns ( C l a s s e n , 1 9 9 6 ) . A l ow d iges t ib i l i t y o f p ro te in s o u r c e s s u c h a s s o y b e a n m e a l a n d c a n o l a m e a l by pou l t ry r esu l t s in r e d u c e d f e e d u t i l i za t ion . T h e s u p p l e m e n t a t i o n of c a n o l a m e a l wi th p ro teo ly t i c e n z y m e s h a s b e e n s h o w n to i m p r o v e it 's d iges t ib i l i t y by pou l t ry ( B e d f o r d a n d M o r g a n , 1 9 9 5 ) . 1.1.4 Ruminant Research with Enzymes T h e s u c c e s s e s o b s e r v e d w i th m o n o g a s t r i c s h a v e r e n e w e d in teres t in the a p p l i c a t i o n s o f e x o g e n o u s e n z y m e in r um inan t d i e t s . T h e u s e o f e x o g e n o u s e n z y m e s m a y bene f i t r u m i n a n t nutr i t ion by r e d u c i n g t h e cu r ren t l im i ta t ions o n the d i g e s t i o n p r o c e s s e s in t h e r u m e n t h e r e b y e n h a n c i n g a n i m a l p e r f o r m a n c e . A l t h o u g h t he a d d i t i o n o f e x o g e n o u s e n z y m e s to f e e d s t u f f s h a s i n d i c a t e d that t h e r e is the po ten t ia l to i m p r o v e e f f i c i e n c i e s a n d p e r f o r m a n c e , the i r a p p l i c a t i o n s a r e ma in l y l imi ted to t h e e n s i l i n g p r o c e s s ( M o d y a n o v a n d Z e l ' n e r , 1 9 8 3 ) . In terpreta t ion of r esu l t s f r o m e n z y m e r e s e a r c h is di f f icul t s i n c e t h e y a r e i n c o n s i s t e n t a n d t he m o d e of a c t i o n o f t he e n z y m e s in t he r u m i n a n t a n i m a l is l a rge l y u n k n o w n ( B e a u c h e m i n a n d R o d e , 1 9 9 6 ) . T h e po ten t ia l u s e o f a l t e rna te f e e d s t u f f s a n d g o a l s that a r e d i r e c t e d t o w a r d s m a x i m i z i n g nut r ient e f f i c i e n c i e s , jus t i f ies fu r ther i n ves t i ga t i on into t he u s e of e n z y m e s w i th r u m i n a n t f e e d i n g s y s t e m s ( C h e s s o n a n d A u s t i n , 1 9 9 6 ) . 9 1.1.4.1 Sources of Variation in Results T h e v a r i a t i o n in t he r e s p o n s e s that h a v e b e e n f o u n d w i th e n z y m e s u s e d in r u m i n a n t f e e d i n g s y s t e m s h a v e b e e n a t t r ibu ted to a n u m b e r o f d i f ferent f ac to rs . S o u r c e s o f va r i a t i on i n c l u d e : t y p e a n d a g e o f a n i m a l , d ie t c o m p o s i t i o n , e n z y m e t y p e , l eve l a n d c o m b i n a t i o n o f e n z y m e s u s e d , e n z y m e stab i l i ty a n d m e t h o d o f a p p l i c a t i o n ( B e a u c h e m i n a n d R o d e , 1 9 9 6 ) . T h e r e s p o n s e o f s t e e r s f e d a l fa l fa , t imo thy h a y o r s i l a g e b a s e d d ie ts s u p p l e m e n t e d w i th c e l l u l a s e a n d x y l a n a s e w a s f o u n d to b e d e p e n d e n t o n t he f o r a g e s o u r c e a n d t h e l eve l o f e n z y m e a p p l i e d ( B e a u c h e m i n et a l . , 1 9 9 5 ) . S u b s t a n t i a l i m p r o v e m e n t s in a v e r a g e da i l y g a i n ( A D G ) w e r e f o u n d w h e n t he e n z y m e s w e r e a d d e d to t h e a l fa l fa a n d t imo thy h a y d i e t s , h o w e v e r , o n l y a m i n o r e f fec t w a s o b s e r v e d w h e n t h e e n z y m e w a s a d d e d to b a r l e y s i l a g e . In a n o t h e r s t u d y w h e r e a f ib ro ly t ic e n z y m e w a s a p p l i e d to b a r l e y s i l a g e at v a r i o u s l e v e l s a n d f e d to c a l v e s , the i n c r e a s e in f ina l w e i g h t w a s l i near l y d e p e n d e n t o n t he l eve l o f e n z y m e ; w h e r e a s , A D G , f e e d c o n v e r s i o n rat io ( F C R ) a n d d ry ma t te r i n t ake (DMI ) al l r e m a i n e d u n a f f e c t e d ( T r e a c h e r e t a l . , 1 9 9 6 ) . A la ter s t u d y c o n d u c t e d b y t he s a m e r e s e a r c h e r s r epo r t ed that w h e n f i n i sh i ng ca t t le w e r e f e d a total m i x e d ra t ion c o n s i s t i n g of 7 0 % rye g r a s s s i l a g e a n d 3 0 % b a r l e y g r a i n w h i c h h a d b e e n t r ea ted w i th a f ibro ly t ic e n z y m e , A D G i n c r e a s e d w i thou t a c o r r e s p o n d i n g i n c r e a s e in i n t ake ( T r e a c h e r et a l . , 1 9 9 6 ) . S p e c u l a t i o n o n t h e m o d e o f a c t i o n b y t h e a u t h o r s r e s u l t e d in s u g g e s t i o n s that i n c r e a s e d e n z y m e act iv i ty w a s n e c e s s a r y wi th s i l a g e d i e t s . T h i s t heo ry w a s s u p p o r t e d b y u n p u b l i s h e d r e s e a r c h c o n d u c t e d b y t he s a m e l a b o r a t o r y w h e r e f eed lo t 10 a n i m a l s w e r e f e d a c o r n s i l a g e d ie t tha t w a s s u p p l e m e n t e d w i th a n e n z y m e ( B e a u c h e m i n a n d R o d e , 1 9 9 6 ) . T h e i n c r e a s e s in A D G a n d F C R w e r e f o u n d to c o r r e s p o n d w i th t h e i n c r e a s e s in t he a p p l i c a t i o n rate o f t he f ibro ly t ic e n z y m e c o m p l e m e n t . 1.1.4.2 Fibrolytic Enzymes 1.1.4.2.i) Forages T h e major i t y o f e n z y m e r e s e a r c h c o n d u c t e d in t he r u m i n a n t f ie ld h a s c o n c e n t r a t e d o n t h e f i b r o u s f rac t ion o f f e e d s t u f f s w h i c h is l e s s d i g e s t i b l e in the r u m e n ; w i th t h e o b j e c t i v e b e i n g to o p t i m i z e f e e d ut i l i za t ion by t he en t i re a n i m a l . F o r e x a m p l e , t h e a p p l i c a t i o n o f f ib ro ly t ic e n z y m e s h a s b e e n s h o w n to i n c r e a s e the d iges t ib i l i t i es o f bo th d r y ma t t e r ( D M ) a n d f ibe r ( G r a i n g e r a n d S t r o u d , 1 9 6 0 ; V a n W a l l e g h a m et a l . , 1 9 6 4 ) . T h i s m a y e x p l a i n t he i m p r o v e m e n t s in A D G a n d F C R o b s e r v e d in s t e e r s that w e r e f e d a f o r a g e b a s e d d ie t s u p p l e m e n t e d w i th e x o g e n o u s e n z y m e s ( G a l i e v et a l . , 1 9 8 2 ; c i t ed b y B e a u c h e m i n a n d R o d e , 1 9 9 6 ) . I n c r e a s e s in mi lk p r o d u c t i o n a n d D M I w e r e f o u n d w h e n da i r y ca t t le w e r e f e d e i t he r h a y o r ba r l ey wi th s i l a g e d i e t s in w h i c h t he f o r a g e ra t ion w a s s u p p l e m e n t e d w i th c e l l u l a s e a n d x y l a n a s e e n z y m e s ( L e w i s et a l . , 1 9 9 5 ; S t o k e s a n d Z h e n g , 1 9 9 5 ) . T h e i n c r e a s e s in mi lk p r o d u c t i o n a n d D M I , h o w e v e r , m a y not b e a s a resu l t o f i m p r o v e d D M nor neu t ra l d e t e r g e n t f i be r ( N D F ) d iges t ib i l i t i es but m a y b e a s a resu l t o f i n c r e a s e d rate of p a s s a g e o f f e e d ( L e w i s et a l . , 1 9 9 5 ) . I m p r o v e m e n t s in t he A D G a n d F C R of s t e e r s w e r e a l s o o b s e r v e d w h e n a n u n s p e c i f i e d e n z y m e w a s a p p l i e d to c o r n s i l a g e 11 ra t i ons ( R o v i c s a n d E l y , 1 9 6 2 ) . I n c r e a s e s in r u m i n a l in vitro a n d in situ D M a n d N D F d iges t ib i l i t i es w e r e o b s e r v e d w h e n f ibro ly t ic e n z y m e s w e r e a d d e d to d ry f o r a g e s h o w e v e r , w h e n e n z y m e s w e r e a d d e d to f r e s h o r w i l ted f o r a g e , n o e f fec ts w e r e o b s e r v e d ( F e n g et a l . , 1 9 9 2 a ) . In con t ras t , s t e e r s f e d f r e s h , w i l t ed o r d ry g r a s s s u p p l e m e n t e d w i th f ib ro ly t i c e n z y m e s a l l e l i c i t ed a p o s i t i v e g r o w t h r e s p o n s e a l t h o u g h i n c r e a s e s in D M I a s we l l a s in D M a n d N D F t o t a f t ract d iges t ib i l i t i es w e r e o n l y f o u n d w i th d ry g r a s s s u p p l e m e n t e d w i th e n z y m e s ( F e n g et a l . , 1 9 9 2 b ) . 1.1.4.2.H) Concentrates G r a i n s s u c h a s b a r l e y a n d s o r g h u m a n d o i l s e e d s s u c h a s c a n o l a m e a l c o n t a i n h i g h a m o u n t s o f f i be r a n d w o u l d b e e x p e c t e d to r e s p o n d to f ib ro ly t ic e n z y m e s u p p l e m e n t a t i o n h o w e v e r , s t u d i e s in th is a r e a a r e l im i ted . In a r e c e n t s t u d y the e f fec t s o f x y l a n a s e o n b a r l e y g r a i n w e r e i n v e s t i g a t e d . It w a s repo r t ed that h i g h e r x y l a n a s e l e v e l s i m p r o v e d f e e d e f f i c i ency o f s t e e r s w h e r e a s l o w e r x y l a n a s e l e v e l s h a d n o e f fec t ( B e a u c h e m i n et a l . , 1 9 9 6 ) . 12 1.1.4.3 Other Enzymes and Enzyme Combinations 1 . 1 . 4 . 3 J ) Forages A n a c t i v e a r e a o f r e s e a r c h h a s b e e n t h e i n v e s t i g a t i o n o f t h e e f f ec t s o f a f ibro ly t ic e n z y m e u s e d in c o n j u n c t i o n wi th a n a m y l a s e a n d / o r p r o t e a s e a p p l i e d to f o r a g e s in o r d e r to e n h a n c e the i r d iges t ib i l i t y a n d i m p r o v e a n i m a l p e r f o r m a n c e . E a r l y r e s e a r c h w i th b e e f ca t t l e f e d e i t he r c o r n o r oa t s i l a g e f o u n d n o c h a n g e s in DMI a l t h o u g h bo th g a i n a n d f e e d e f f i c i e n c i e s i n c r e a s e d w i th a m y l a s e , p r o t e a s e a n d c e l l u l a s e e n z y m e s u p p l e m e n t a t i o n ( B u r r o u g h s et a l . , 1 9 6 0 ) . P e r r y et a l . ( 1966 ) f ed ca t t le a c o r n o r c o r n s i l a g e d ie t w i th t he s a m e f ibro ly t ic e n z y m e s a s u s e d by B u r r o u g h s et a l . ( 1 9 6 0 ) a n d repo r t ed b e n e f i c i a l e f fec ts o n d iges t ib i l i t y . H o w e v e r , P e r r y et a l . ( 1 9 6 6 ) o b s e r v e d n o r e s p o n s e in t he d iges t ib i l i t y o f t he f e e d w h e n p ro teo ly t i c e n z y m e s w e r e a p p l i e d to a s i m i l a r d iet . In c o n t r a s t to bo th o f t h e s e latter s t u d i e s , i n c r e a s e s in D M d iges t ib i l i t i es h a v e b e e n r e p o r t e d w i th f o r a g e s w i th the add i t i on o f e i t he r a f u n g a l ( R a l s t o n et a l . , 1 9 6 2 ) a n d bac te r i a l p r o t e a s e ( R u s t et a l . , 1 9 6 5 ) . 1.1.4.3M) Concentrates I m p r o v e m e n t s in g a i n a n d f e e d e f f i c i e n c i e s o f s t e e r s a n d he i f e rs w e r e o b s e r v e d w h e n e n z y m e s c o n t a i n i n g p ro teo ly t i c , a m y l o l y t i c a n d f ibro ly t ic ac t iv i t ies w e r e a d d e d to h i g h g r a i n r a t i ons ( B u r r o u g h s et a l . , 1 9 6 0 ; C l a r k e t a l . , 1 9 6 1 ; R o v i c s a n d E l y , 1 9 6 2 ) . S i m i l a r r esu l t s w e r e p r o d u c e d w i th a n u n s p e c i f i e d e n z y m e s u p p l e m e n t a p p l i e d to c o r n g r a i n a n d c o r n s i l a g e f e d to s t e e r s ( W e i c h e n t h a l et a l . , 1 3 1 9 9 6 : c i t ed b y B e a u c h e m i n a n d R o d e , 1 9 9 6 ) . E n z y m e s u p p l e m e n t a t i o n o f c o r n g ra i n d i e t s is e x p e c t e d to e l u c i d a t e a r e s p o n s e a s t h e r u m e n m i c r o b e s g a i n a c c e s s to a n d h y d r o l y z e a d d i t i o n a l s u b s t r a t e s . C o r n p ro te in a n d s t a r c h g r a n u l e s h a v e b e e n s h o w n to b e s u r r o u n d e d by a p ro te in mat r i x w h i c h l e n d s r e s i s t a n c e to m ic rob ia l c o l o n i z a t i o n a n d d i g e s t i o n ( M c A l l i s t e r et a l . , 1 9 9 0 ) . H o w e v e r , t he r e s p o n s e to e n z y m e s u p p l e m e n t a t i o n o f c o r n b a s e d ra t i ons h a s b e e n i n c o n s i s t e n t . T h e s a m e e n z y m e c o m p l e m e n t a n d d ie t o f s i m i l a r c o m p o s i t i o n to t he o n e u s e d by B u r r o u g h s et a l . ( 1 9 6 0 ) , w a s f e d to c a l v e s a n d d id not e l ic i t a n y r e s p o n s e in f e e d e f f i c i e n c y a l t h o u g h t h e r e w e r e i m p r o v e m e n t s in D M I a n d A D G w h e n the c a l v e s w e r e f e d a n e n z y m e s u p p l e m e n t e d h igh c o r n g ra i n d ie t ( P e r r y et a l . , 1966 ) . In con t ras t , s e v e r a l s t u d i e s i nvo l v i ng f e e d l o t s t e e r s f e d c o r n g r a i n s u p p l e m e n t e d wi th a n e n z y m e " c o c k t a i l " o f p r o t e a s e a n d a m y l a s e , f a i l ed to y ie ld r e s p o n s e s in A D G or f e e d e f f i c i e n c i e s ( P e r r y et a l . , 1 9 6 0 ; W a r d et a l . , 1 9 6 0 ; W i n g a n d W i l c o x , 1960 ) . S o m e s t u d i e s h a v e a l s o y i e l d e d resu l t s f r o m e n z y m e r e s e a r c h that w e r e i n c o n c l u s i v e ( T h e u r e r e t a l . , 1 9 6 3 , N e l s o n a n d C a t r o n , 1 9 6 0 ) . 1.1.5 Feed Processing and Enzyme Applications R e c e n t r e s e a r c h w i th m o n o g a s t r i c a n i m a l s h a s i n v e s t i g a t e d t he p o s s i b l e m a r r i a g e o f e n z y m e s a n d f e e d p r o c e s s i n g w h e r e t he e n z y m e s w e r e a p p l i e d pos t p r o c e s s i n g . T h e r m a l p r o c e s s e s s u c h a s e x t r u d i n g , s t e a m ro l l ing a n d pe l le t ing a l o n e h a v e b e e n s h o w n to i m p r o v e f e e d ut i l i za t ion b y i n c r e a s i n g d iges t ib i l i t y a n d imp rov i ng p r o d u c t i o n p e r f o r m a n c e in pou l t ry ( B e d f o r d , 1 9 9 3 ) . F o r e x a m p l e , s u b j e c t i n g 14 s o r g h u m g r a i n to d r y ro l l ing h a s b e e n s h o w n to g i v e pos i t i ve r e s p o n s e s in t e r m s o f D M I , A D G a n d F C R ( B o y l e s et a l . , 1 9 9 2 ) . H o w e v e r , h e a t t r e a t m e n t i n c r e a s e s t he so lub i l i t y o f N S P s in m a n y c e r e a l s a n d the i r ant i -nut r i t iona l e f f ec t s m a y c o u n t e r a c t a n y bene f i t o f t h e p r o c e s s i n g t r e a t m e n t ( C h e s s o n a n d A u s t i n , 1 9 9 6 ) . T h i s e f fec t c a n b e r e m o v e d b y s u p p l e m e n t i n g t he d ie t w i th e x o g e n o u s e n z y m e s ( B e d f o r d et a l . , 1 9 9 1 ) . C o m b i n i n g p r o c e s s i n g a n d e n z y m e t e c h n o l o g i e s m a y bene f i t d i g e s t i o n by a c t i n g s y n e r g i s t i c a l l y in bo th m o n o g a s t r i c a n d r u m i n a n t a n i m a l s . In t h e lat ter c a s e , resu l t s f r o m s t u d i e s i nves t i ga t i ng t he c o m b i n e d e f f ec t s o f e n z y m e s a n d p r o c e s s i n g t r e a t m e n t s a r e m o r e i n c o n s i s t e n t re la t i ve to t h o s e o b t a i n e d w i th m o n o g a s t r i c s . T h e a p p l i c a t i o n o f h y d r o t h e r m a l a n d e n z y m e t r e a t m e n t s to c e r e a l g r a i n s h a s b e e n br ief ly i n v e s t i g a t e d w i th v a r y i n g resu l t s . D a i r y c o w s f e d e n z y m e t r ea ted d ry ro l led o r s t e a m ro l led s o r g h u m s h o w e d i m p r o v e d d iges t i b i l i t i es o f d ry ma t te r ( D M ) , o r g a n i c ma t te r ( O M ) , c r u d e pro te in ( C P ) a n d N D F f r a c t i o n s , w i thou t a n y a d v e r s e e f f ec t s o n mi lk p r o d u c t i o n ( C h e n et a l . , 1 9 9 5 ) . K r a u s e et a l . ( 1 9 8 9 ) r e p o r t e d that s t e e r s f e d s t e a m f l a k e d s o r g h u m that w a s t r ea ted w i th a n u n s p e c i f i e d e n z y m e h a d i n c r e a s e d D M I a n d a n e v e n g r e a t e r r e s p o n s e in A D G , h o w e v e r , t h e r e w e r e n o i m p r o v e m e n t s in f e e d e f f i c i ency . In the s a m e s t u d y , w h e n d ry ro l led s o r g h u m w a s a l s o t r ea ted w i th a n u n s p e c i f i e d e n z y m e a n d f e d to s t e e r s , e v e n g r e a t e r i n c r e a s e s w e r e re f l ec ted in D M I a n d A D G wi th a l a rge i m p r o v e m e n t in f e e d e f f i c i ency . In c o n t r a s t to t he a b o v e , d iges t ib i l i t y w a s not i m p r o v e d in a s t u d y w h e r e s t e e r s w e r e f e d a p r o c e s s e d a n d e n z y m e t rea ted s o r g h u m d ie t ( R i c h a r d s o n et a l . , 1 9 9 0 ) . 15 T h e a b o v e r e v i e w s u m m a r i z e s m a n y o f t he resu l t s f r o m r e s e a r c h wi th e x o g e n o u s e n z y m e s a p p l i e d to d ie t s f e d to r u m i n a n t a n i m a l s . T h e major i ty o f the e x p e r i m e n t s w e r e a n i m a l s t u d i e s , a l t h o u g h a f e w s t u d i e s i n v o l v e d in vitro a n a l y s e s o f e n z y m e t r e a t e d f e e d . In vitro a s s a y s h a v e b e e n s h o w n to b e a n i n e x p e n s i v e a l t e rna t i ve to a n i m a l t r ia ls a n d i n v a l u a b l e in e v a l u a t i n g o f a l a rge n u m b e r of s a m p l e s . T h e w o r k c o n d u c t e d fo r th is t h e s i s r e q u i r e d in vitro a n a l y s e s a s a p r e l i m i n a r y s t e p to i nves t i ga t i ng a l a rge n u m b e r o f s a m p l e s a n d t h e in te rac t i ve e f fec ts o f e n z y m e s a n d mi ld h y d r o t h e r m a l t r e a t m e n t o f b a r l e y a n d c a n o l a m e a l . 1.2 IN VITRO TECHNIQUES In vitro t e c h n i q u e s d e s i g n e d fo r f e e d e v a l u a t i o n p u r p o s e s h a v e g a i n e d w i d e a c c e p t a n c e a s c o s t e f fec t i ve a l t e r n a t i v e s to a n i m a l b a s e d r e s e a r c h w h i c h is o f ten e x p e n s i v e , l a b o r i n t e n s i v e a n d l imi ted in t he n u m b e r o f s a m p l e s that m a y b e a s s e s s e d at o n e t i m e . T h e major i ty o f in vitro m e t h o d s that a r e s p e c i f i c to e v a l u a t i n g r um inan t f e e d s t u f f s i n v o l v e t he i n c u b a t i o n o f a s a m p l e o f t he f e e d w i th r u m e n f lu id i n o c u l u m . T h e s e t e c h n i q u e s a r e u s u a l l y b a s e d o n a m e a s u r e o f e n d po in t d i g e s t i o n o r t he a p p e a r a n c e o f m e t a b o l i c p r o d u c t s o v e r t ime . C o m p r e h e n s i v e r e v i e w s de ta i l i ng t h e h is to ry , d e v e l o p m e n t a n d m e t h o d o l o g y o f in vitro r um inan t f e e d e v a l u a t i o n t e c h n i q u e s a r e g i v e n by J o h n s o n ( 1 9 6 3 ; 1 9 6 6 ) . O n e o f t h e s e r e v i e w s p r e s e n t s t he p i o n e e r i n g r e s e a r c h c o n d u c t e d b e t w e e n 1 8 8 2 a n d 1 8 8 8 in the a r e a of s c i e n c e w h i c h i n v e s t i g a t e d t h e f e r m e n t a t i o n o f c e l l u l o s e u s i n g r u m e n f lu id f r o m a n ox . A n o t h e r s i gn i f i can t con t r i bu t i on to t he e v o l u t i o n o f in vitro t e c h n i q u e s w a s the 16 d e t e r m i n a t i o n o f t h e m i n e r a l c o m p o s i t i o n o f s h e e p s a l i v a ( M c D o u g a l l ' s , 1 9 4 8 ) . T h i s d i s c o v e r y e n a b l e d t he c h e m i c a l r e p r o d u c t i o n o f s a l i v a a n d t h e d e v e l o p m e n t of the bu f fe r s o l u t i o n w h i c h i s st i l l in u s e t o d a y ( J o h n s o n , 1 9 6 6 ) . In 1 9 6 3 , T i l l e y a n d T e r r y d e v e l o p e d a n in vitro t e c h n i q u e to e v a l u a t e f o r a g e d iges t ib i l i t y w h i c h h a s b e c o m e the s t a n d a r d . 17 1.2.1 Apparent Digestibility: Tilley and Terry In Vitro Technique T h e T i l l e y a n d T e r r y ( 1 9 6 3 ) in vitro t e c h n i q u e is a t w o s t a g e m e t h o d tha t p r o v i d e s a n e s t i m a t e o f t h e a p p a r e n t d iges t ib i l i t y o f a feeds tu f f . T h e t e c h n i q u e i n v o l v e s t h e i n c u b a t i o n o f a f eeds tu f f w i th r u m e n f lu id i n o c u l u m fo r 4 8 h, f o l l o w e d by a 4 8 h i n c u b a t i o n in a n a c i d p e p s i n s o l u t i o n . T h i s p r o c e d u r e s i m u l a t e s rum inan t d i g e s t i o n w h e r e t h e f e e d is f irst f e r m e n t e d by m i c r o b e s in t he r u m e n , re fe r red to a s the f irst s t a g e , a n d t h e n s u b j e c t e d to p o s t r u m i n a l d i g e s t i o n , t he s e c o n d s t a g e . A n e n d po in t d r y ma t t e r d iges t ib i l i t y ( I V D M D ) o r o r g a n i c ma t t e r d iges t ib i l i t y ( O M D ) m e a s u r e m e n t is o b t a i n e d . T h e o n l y d i f f e r e n c e b e t w e e n I V D M D a n d O M D is e s s e n t i a l l y tha t t h e lat ter is c o r r e c t e d fo r a s h c o n t e n t w h i c h is a s s u m e d to b e ind iges t i b l e in th is s y s t e m . I V D M D r e p r e s e n t s t he f rac t i on o f t he f e e d that is d i g e s t i b l e in t h e r u m e n b y m i c r o b e s a n d i n c l u d e s bo th t he f i b r o u s a n d ce l l s o l u b l e f r ac t i ons . T h e u n d i g e s t e d p lan t r e s i d u e h o w e v e r , is c o n t a m i n a t e d w i th s o m e u n d i g e s t e d m i c r o b i a l m a t e r i a l . T h i s t e c h n i q u e h a s b e e n s u c c e s s f u l b e c a u s e of the h igh c o r r e l a t i o n b e t w e e n t h e in vitro r esu l t s a n d in vivo a p p a r e n t d igest ib i l i ty m e a s u r e m e n t s ( V a n S o e s t , 1 9 8 2 ) . A l t h o u g h t he m e t h o d w a s o r ig ina l l y d e v e l o p e d to i m p r o v e e s t i m a t e s o f in vivo f o r a g e d igest ib i l i t y , it h a s a l s o b e e n s u c c e s s f u l l y u s e d w i th c o n c e n t r a t e s . F o r e x a m p l e , a p p a r e n t I V D M D o f w h e a t w a s f o u n d to b e we l l c o r r e l a t e d ( R 2 = 0 .83 ) w i th in vivo D M D m e a s u r e m e n t s ( A d e s o g a n et a l . , 1995 ) . 18 1.2.2 True Digestibility G o e r i n g a n d V a n S o e s t ( 1 9 7 0 ) m o d i f i e d t he s e c o n d s t e p o f t he T i l l ey a n d T e r r y m e t h o d to a r r i ve at a n e s t i m a t e o f " t rue d iges t ib i l i t y " ( I V T D ) . T h e t rue d iges t ib i l i t y t e c h n i q u e i n v o l v e s i n c u b a t i n g f e e d s a m p l e s w i th r u m e n f lu id for 4 8 h a n d t h e n subs t i t u t i ng t h e a c i d p e p s i n i n c u b a t i o n wi th a neu t ra l d e t e r g e n t f i be r r i nse . T h i s p r o c e d u r e r e s u l t s in t h e r e m o v a l o f al l ce l l s o l u b l e s w h i c h , in e s s e n c e , r e p r e s e n t s e n d po in t d i g e s t i o n . 1.2.3 Enzymatic Method A d iges t ib i l i t y m e a s u r e m e n t c a n a l s o b e o b t a i n e d u s i n g a n e n z y m a t i c m e t h o d . S e v e r a l e n z y m a t i c m e t h o d s a r e a v a i l a b l e ( V a n S o e s t , 1 9 9 4 ) . F o r e x a m p l e , a p r o c e d u r e d e v e l o p e d b y J o n e s a n d H a y w a r d ( 1 9 7 5 ) i n v o l v e s i n c u b a t i n g the f e e d s a m p l e in a n a c i d p e p s i n s o l u t i o n f o l l o w e d b y a s e c o n d i n c u b a t i o n w i th a f u n g a l c e l l u l a s e . T h e r e is l e s s p r e c i s i o n w i th th is m e t h o d b e c a u s e r u m e n m i c r o b e s p r o d u c e a va r ie t y o f e n z y m e s a n d m a y a d a p t to a va r ie t y o f d i f ferent s u b s t r a t e s ( V a n S o e s t , 1 9 8 2 ) . F o r a r e v i e w o f s e v e r a l e n z y m a t i c m e t h o d s t h e r e a d e r is re fe r red to N o c e k (1988 ) . E n z y m a t i c t e c h n i q u e s d i f fer f r o m o t h e r in vitro t e c h n i q u e s b e c a u s e t h e y d o no t r e q u i r e t h e u s e o f r u m e n f lu id a n d t h u s t h e m a i n t e n a n c e o f d o n o r a n i m a l s a n d a n a n a e r o b i c e n v i r o n m e n t . 19 1.2.4 Rumen Fluid Inoculum 1.2.4.1 Animal Diet L i n d b e r g ( 1 9 8 5 ) br ie f ly d i s c u s s e d a n u m b e r o f f a c t o r s w h i c h i n f l u e n c e e s t i m a t e s o f r u m e n f e r m e n t a t i o n by v a r i o u s in vitro t e c h n i q u e s . S o m e of t h e s e f a c t o r s i n c l u d e : so lub i l i t y , e n d p r o d u c t a c c u m u l a t i o n , c o n t i n u o u s f e r m e n t a t i o n , p ro teo ly t i c e n z y m e s , o p t i m a l p H a n d t he b a s a l d ie t f e d to t he a n i m a l . F i b e r s o u r c e in the d ie t h a s a l s o b e e n s h o w n to a f fec t t he resu l t s o f in vitro e x p e r i m e n t s ( C h e r n e y et a l . , 1 9 9 3 ) . T h e rat io o f c o n c e n t r a t e to f o r a g e a f fec ts t he r u m e n m i c r o b i a l popu la t i on a s t he v a r i o u s m i c r o o r g a n i s m s h a v e d i f ferent af f in i t ies fo r s u b s t r a t e s ( R u s s e l a n d B a l d w i n , 1 9 7 9 ) a n d t h e p r o d u c t s o f f e r m e n t a t i o n v a r y a c c o r d i n g l y ( O w e n s a n d G o e t s c h , 1 9 8 8 ) . R u m e n f lu id d o n o r a n i m a l s s h o u l d t h e r e f o r e b e m a i n t a i n e d o n a b a l a n c e d ra t ion w i th s i m i l a r d i e ta r y c o m p o n e n t s a s to b e t e s t e d in vitro ( W a r n e r , 1 9 5 6 ; B o w i e , 1 9 6 2 ) . T h e d i u rna l va r i a t i on in r u m e n f lu id act iv i ty c a n b e m i n i m i z e d by i n c r e a s i n g t h e f e e d i n g f r e q u e n c y ( W a r n e r , 1 9 6 5 ) a n d c o n d u c t i n g c o l l e c t i o n s at s i m i l a r t i m e s . T h e re ten t ion o f r u m e n d i g e s t a is d e c r e a s e d w i th i n c r e a s e d f e e d i n g f r e q u e n c y w h i c h m a y b e r e l e v a n t w h e n a p p l y i n g t he in situ t e c h n i q u e ( W a r n e r , 1 9 8 1 ) . 2 0 1.2.4.2 Rumen Fluid Collection S o m e s t u d i e s r e c o m m e n d c o l l e c t i n g r u m e n f lu id a f e w h o u r s a f ter f e e d i n g to e n s u r e a m ix tu re o f bac te r i a l s p e c i e s ( M o o r e et a l . , 1 9 6 2 ) . It h a s b e e n repo r ted that p o s t f e e d i n g c o l l e c t i o n o f r u m e n f lu id r e d u c e s t he va r i a t i on b e t w e e n i n c u b a t i n g s a m p l e s in vitro (T i l ley a n d T e r r y , 1 9 6 3 ) . O t h e r s t u d i e s , i n c l u d i n g s o m e invo l v ing the g a s p r o d u c t i o n s y s t e m a d v o c a t e c o l l e c t i n g r u m e n f lu id pr io r to f e e d i n g to e n s u r e l ow r u m e n f lu id ac t iv i ty in b l a n k s ( M e n k e et a l . , 1 9 7 9 ; M e n k e a n d S t e i n g a s s , 1988 ) . R e c e n t s t u d i e s i n v o l v i n g t he in vitro g a s p r o d u c t i o n s y s t e m h o w e v e r , h a v e reve r ted to c o l l e c t i n g t h e r u m e n f lu id a f ter f e e d i n g w h e n bac te r i a l ac t iv i ty is a s s u m e d to b e at it 's m a x i m u m ( P e l l a n d S c h o f i e l d , 1 9 9 3 ; S c h o f i e l d a n d P e l l , 1 9 9 5 ) . 1.2.4.3 Inoculum Preparation T h e major i ty o f r u m e n f lu id i n o c u l u m p r e p a r a t i o n s u s e d in t he first in vitro s t u d i e s w e r e c o m p r i s e d o f r u m e n f lu id w h i c h h a d b e e n s t r a i n e d t h r o u g h c h e e s e c l o t h a n d a d d e d to a bu f fe r a n d m i n e r a l m ix tu re ( Q u i n , 1 9 3 8 , 1 9 4 3 ; H u n g a t e et a l . , 1 9 5 5 ; P e a r s o n a n d S m i t h , 1 9 4 3 : c i t ed by J o h n s o n , 1 9 6 6 ) , a l t h o u g h a f e w r e s e a r c h e r s u s e d u n s t r a i n e d r u m e n f lu id ( L o u w et a l . , 1 9 4 9 ; B u r r o u g h s et a l . , 1 9 5 0 ) . A d h e s i o n o f b a c t e r i a to t h e p lan t ce l l wa l l i n c r e a s e s l inear ly u p to 9 h ( L a t h a m et a l . , 1 9 7 8 ) . It h a s b e e n s u g g e s t e d that a p p r o x i m a t e l y 7 0 - 8 0 % o f t he r u m e n m i c r o b e s a r e a s s o c i a t e d w i th t h e pa r t i cu la te ma t te r in t h e r u m e n w h e r e a s s t r a i n e d r u m e n f luid a l o n e a c c o u n t s fo r 2 0 - 3 0 % o f m i c r o b i a l ac t iv i ty ( C h e n g , K . - J . ; p e r s o n a l c o m m u n i c a t i o n ) . In 21 m a n y s t u d i e s , t h e m i x i n g o f r u m e n f lu id in a b l e n d e r to d i s l o d g e m i c r o b e s a t t a c h e d to f e e d pa r t i c l es h a s p r o v e n to b e b e n e f i c i a l in o b t a i n i n g re l i ab le e s t i m a t e s o f in vitro d i g e s t i o n c o e f f i c i e n t s . H o w e v e r , s o m e r e s e a r c h s u g g e s t s tha t b l e n d i n g a f fec ts t he v iab i l i ty o f b a c t e r i a a n d d e s t r o y s p r o t o z o a ( C r a i g et a l . , 1 9 8 4 ) . In t he g a s p r o d u c t i o n s y s t e m , p r e l i m i n a r y s t u d i e s c o n d u c t e d b y P e l l a n d S c h o f i e l d ( 1 9 9 3 ) i n d i c a t e d that b l e n d e d a n d u n b l e n d e d r u m e n f lu id g a v e s i m i l a r resu l t s . U n b l e n d e d r u m e n f lu id is p re fe r red s i n c e b l e n d i n g h a s b e e n s h o w n to i n c r e a s e t h e e r ro r in g a s p r o d u c t i o n m e a s u r e m e n t s b y i n c r e a s i n g t he a m o u n t o f s m a l l f e e d pa r t i c l es in t he f luid a n d e x p o s i n g t h e m i c r o b e s to m o r e o x y g e n ( P e l l a n d S c h o f i e l d , 1 9 9 3 ) . 1.2.5 Advantages of In Vitro Techniques T h e g r e a t e s t a d v a n t a g e s o f in vitro t e c h n i q u e s u s e d to s i m u l a t e a n i m a l p e r f o r m a n c e is tha t t h e y a r e l e s s i n t e n s i v e a n d c o s t l y t h a n a n i m a l s t u d i e s . T h e s e m e t h o d s h a v e a l s o b e e n s h o w n to b e u s e f u l in s c r e e n i n g a l a rge n u m b e r o f s a m p l e s a n d g o o d c o r r e l a t i o n s h a v e b e e n o b t a i n e d w h e n c o m p a r e d w i th in vivo r esu l t s ( V a n S o e s t , 1 9 8 2 ) . A l t h o u g h c h e m i c a l m e t h o d s m a y b e f a s t e r a n d h a v e be t te r rep l i ca t i on , t he in vitro m e t h o d s m o r e a c c u r a t e l y r e p r e s e n t b i o l o g i c a l real i ty . 2 2 1.2.6 Disadvantages of In Vitro Techniques A l l o f t h e a b o v e in vitro m e t h o d s , w i th t h e e x c e p t i o n o f t h e e n z y m a t i c m e t h o d , requ i re t h e m a i n t e n a n c e o f c a n n u l a t e d a n i m a l s to s e r v e a s r u m e n f lu id d o n o r s . It is th is la t ter po in t w h i c h c r e a t e s o n e o f t he g r e a t e s t c h a l l e n g e s s u r r o u n d i n g t h e s e t y p e s o f s t u d i e s , t he m a i n t e n a n c e o f a n a n a e r o b i c e n v i r o n m e n t in t h e r u m e n f lu id . It is i m p e r a t i v e that a n a n a e r o b i c e n v i r o n m e n t b e m a i n t a i n e d in o r d e r to e n s u r e s u c c e s s o f t h e e x p e r i m e n t . T h e d e t e r m i n a t i o n o f a p p a r e n t o r t rue d iges t ib i l i t i es r e q u i r e s l o n g p e r i o d s o f t i m e a n d i n v o l v e s m a n y p r e p a r a t o r y s t e p s . A ma jo r d r a w b a c k to t h e s e m e t h o d s is that n o in fo rma t ion o n t h e k i ne t i c s o f d e g r a d a t i o n m a y b e o b t a i n e d u n l e s s l e n g t h y a n d l a b o r i n t e n s i v e t i m e c o u r s e i n c u b a t i o n s a r e c o n d u c t e d . 1.2.7 Kinetic Descriptions of Feed Degradation 1.2.7.1 In Situ Technique T h e in situ b a g t e c h n i q u e w a s f irst d e v e l o p e d b y Q u i n et a l . , ( 1 9 3 8 ) (c i ted by J o h n s o n , 1 9 6 6 ) a n d a l l o w s fo r a t h o r o u g h d e s c r i p t i o n o f t he f e r m e n t a t i o n prof i le o f a feeds tu f f t h e r e b y e n a b l i n g t h e d e t e r m i n a t i o n o f t h e ra te a n d e x t e n t o f d i s a p p e a r a n c e o f a feeds tu f f . F e e d s t u f f s a r e c o n t a i n e d w i th in s m a l l n y l o n b a g s w h i c h a r e s u s p e n d e d a n d i n c u b a t e d in t h e r u m e n fo r v a r i o u s p e r i o d s o f t i m e ( M e h r e z a n d O r s k o v , 1 9 7 7 ; O r s k o v , 1 9 8 2 ) . T h e d i s a p p e a r a n c e o f ma te r i a l f r o m the b a g i n d i c a t e s a p p a r e n t D M D ( M e h r e z a n d O r s k o v , 1 9 7 7 ) . T h i s t e c h n i q u e is c o m m o n l y u s e d to 2 3 d e t e r m i n e f r ac t i ona l r a tes o f p ro te in d e g r a d a t i o n e m p l o y i n g t h e e q u a t i o n s o f O r s k o v a n d M c D o n a l d ( 1 9 7 9 ) . A l t h o u g h t h e m e t h o d m o s t c l o s e l y r e s e m b l e s t h e r u m e n e n v i r o n m e n t ( B r o d e r i c k e t a l . , 1 9 8 8 ) , t h e m e t h o d h a s a l s o b e e n c r i t i c i zed fo r s e v e r a l t e c h n i c a l p r o b l e m s . T h e in situ m e t h o d is i n t e n s i v e , r e q u i r e s t h e m a i n t e n a n c e o f c a n n u l a t e d a n i m a l s a n d o n l y a f e w f e e d s a m p l e s m a y b e i n c u b a t e d at o n e t i m e . In a d d i t i o n , f ine f e e d p a r t i c l e s m a y b e lost t h r o u g h t he p o r e s o f t he b a g s w h i c h a r e a s s u m e d to b e part o f t h e i m m e d i a t e l y s o l u b l e f rac t ion but m a y not in fac t h a v e b e e n d i g e s t e d ( B r o d e r i c k e t a l . , 1 9 8 8 ) . T h e e n t r a n c e o f f e e d pa r t i c l es into t he n y l o n b a g ( N o c e k , 1 9 8 5 ) a n d b a c t e r i a l c o n t a m i n a t i o n o f t h e s a m p l e ( N o c e k , 1 9 8 7 ) h a v e b e e n s h o w n to af fec t d e g r a d a b i l i t y v a l u e s o f f e e d s . M i c r o b i a l c o n t a m i n a t i o n is s m a l l w i th p ro te in s u p p l e m e n t s , a n d h igh l y d i g e s t i b l e f o r a g e s ( M e h r e z a n d O r s k o v , 1 9 7 7 ; V a r v i k k o a n d L i n d b e r g , 1 9 8 5 ) . H o w e v e r , w i th rap id l y f e r m e n t a b l e c e r e a l s s u c h a s ba r l ey a n d poo r l y d i g e s t e d f o r a g e s , c o n t a m i n a t i o n c o u l d b e s ign i f i can t ( V a r v i k k o a n d L i n d b e r g , 1 9 8 5 ) . It s h o u l d b e n o t e d tha t t h e p r e s e n c e o f m i c r o b i a l ma t t e r wi l l no t g rea t l y a f fec t D M d e g r a d a b i l i t y m e a s u r e m e n t s but wi l l i n f l u e n c e n i t r ogen s t u d i e s ( V a r v i k k o a n d L i n d b e r g , 1 9 8 5 ) . In e v a l u a t i n g t he f e r m e n t a t i o n k i ne t i cs o f f e e d s t u f f s t he g r e a t e s t d i s a d v a n t a g e o f th is t e c h n i q u e is t he a s s u m p t i o n that t he s o l u b l e f rac t ion o f a feeds tu f f is en t i re l y d e g r a d a b l e ( B r o d e r i c k et a l . , 1 9 8 8 ) . T h e " t rue" f e r m e n t a t i o n pro f i le o f a feeds tu f f , h o w e v e r , c a n b e d e s c r i b e d u s i n g t h e r u m e n in vitro g a s p r o d u c t i o n t e c h n i q u e . 2 4 1.2.8 The Gas Production Technique T h e k i n e t i c s o f d i g e s t i o n o f a feeds tu f f c a n b e d e s c r i b e d w i th t he u s e o f t he g a s p r o d u c t i o n t e c h n i q u e f irst d e v e l o p e d by M e n k e et a l . ( 1 9 7 9 ) . T h e g a s p roduc t i on t e c h n i q u e i n v o l v e s t h e i n c u b a t i o n o f a feeds tu f f w i th r u m e n f lu id i n o c u l u m a n d a s f e r m e n t a t i o n p r o c e e d s , t h e g a s p r o d u c e d is r e c o r d e d at s p e c i f i c t i m e s . G a s p r o d u c t i o n is r e l a t e d to the d iges t ib i l i t y a n d m a y b e u s e d w i th o t h e r p a r a m e t e r s to e s t i m a t e t h e m e t a b o l i z a b l e e n e r g y v a l u e o f a feeds tu f f f e d to r u m i n a n t a n i m a l s ( M e n k e et a l . , 1 9 7 9 ; M e n k e a n d S t e i n g a s s , 1 9 8 8 ) . A n e s t i m a t e o f t h e e n e r g y v a l u e of a f eeds tu f f is o b t a i n e d b y c o m b i n i n g t h e resu l t s o f p r o x i m a t e a n a l y s i s s u c h a s c r u d e p ro te i n , c r u d e fat , c r u d e f i be r a n d c r u d e a s h , w i th t h o s e o b t a i n e d f r om g a s p r o d u c t i o n s t u d i e s ( M e n k e a n d S t e i n g a s s , 1 9 8 8 ) . G a s p r o d u c t i o n a n d in situ d e g r a d a b i l i t y w e r e bo th f o u n d to b e we l l c o r r e l a t e d w i th t he r a t e s o f f e r m e n t a t i o n fo r w h e a t ( A d e s o g a n et a l . , 1 9 9 5 ) . H o w e v e r , resu l t s m a y d e p e n d o n t h e t y p e o f f eeds tu f f a s t h e r e w a s n o s ign i f i can t co r re l a t i on b e t w e e n g a s p r o d u c t i o n a n d t he in situ o r in vivo d e g r a d a b i l i t i e s in t he s t u d i e s c o n d u c t e d by P i v a et a l . , ( 1 9 8 8 ) w h i c h i n v o l v e d h a y , s i l a g e a n d g r a s s . T h i s lat ter s t u d y a l s o d e t e r m i n e d that n i t r ogen f r ee ex t rac t ( N F E ) a n d A D F h a d t he g r e a t e s t i n f l uence o n f e e d f e r m e n t a t i o n . A s t r o n g l i nea r r e l a t i onsh ip b e t w e e n N D F d i s a p p e a r a n c e a n d g a s p r o d u c t i o n h a s b e e n s h o w n to ex i s t w i th f o r a g e s (Pe l l a n d S c h o f i e l d , 1993 ) . B l u m m e l a n d O r s k o v ( 1 9 9 3 ) f o u n d a g o o d co r re l a t i on b e t w e e n in vitro g a s p r o d u c t i o n a n d t h e n y l o n b a g d e g r a d a b i l i t y o f f e e d w h i c h w a s c l o s e l y re la ted to f e e d in take a n d g r o w t h ra te in ca t t le . 2 5 M e a s u r e m e n t o f t h e g a s p r o d u c e d f r o m r u m e n m i c r o o r g a n i s m s in b a t c h c u l t u r e s w a s f irst c o n d u c t e d b y M e n k e a n d E h r e n s v a r d ( 1 9 7 4 ) (c i ted by T h e o d o r o u et a l . , 1 9 9 4 ) . M e n k e et a l . ( 1 9 7 9 ) m o n i t o r e d t he g a s a c c u m u l a t i o n o f a feeds tu f f i n c u b a t e d in g l a s s s y r i n g e s w i th r u m e n f lu id i n o c u l u m . A s f e r m e n t a t i o n p r o g r e s s e d , g a s p r o d u c t i o n c a u s e d t h e s y r i n g e p l u n g e r to r i se a n d e n a b l e d a d i rec t m e a s u r e m e n t o f g a s v o l u m e . T h i s a u t h o r r e p o r t e d a h igh co r re l a t i on b e t w e e n the a c c u m u l a t i o n o f g a s e o u s p r o d u c t s a n d f e e d d e g r a d a t i o n ( M e n k e et a l . , 1 9 7 9 ) . T h e g a s p r o d u c t i o n t e c h n i q u e w a s fu r ther re f ined by M e n k e a n d S t e i n g a s s (1988 ) . V o l u m e t r i c r e c o r d i n g s o f g a s p r o d u c e d f r o m m i c r o b i a l f e r m e n t a t i o n w e r e first a c c o m p l i s h e d u s i n g a m a n u a l m e t h o d w h e r e t he g a s v o l u m e w a s d e t e r m i n e d by v i s u a l l y r e a d i n g t h e m o v e m e n t o f t he s y r i n g e p l u n g e r . R e c e n t t e c h n o l o g i c a l a d v a n c e s h a v e a l l o w e d fo r t h e c h a n g e s in g a s v o l u m e s to b e e l ec t r on i ca l l y m e a s u r e d by r e c o r d i n g t h e c h a n g e s in p r e s s u r e . T h e o r o d o u et a l . ( 1 9 9 4 ) m e a s u r e d g a s p r o d u c t i o n b y u s i n g a l ight emi t t ing d i o d e v o l t m e t e r a n d e l e c t r o n i c p r e s s u r e t r a n s d u c e r s that w e r e m a n u a l l y i nse r ted into f i xed v o l u m e f e r m e n t a t i o n f l a s k s . A s g a s a c c u m u l a t e d w i th in t he f l a s k s , the p r e s s u r e r e q u i r e d to s t a b i l i z e t h e s y s t e m to a t m o s p h e r i c p r e s s u r e w a s d e t e r m i n e d at s p e c i f i e d t i m e i n te rva l s . T h i s t e c h n i q u e is b a s e d o n t he a s s u m p t i o n that m ic rob ia l d i g e s t i o n w o u l d b e i n f l u e n c e d by t he a c c u m u l a t i o n o f g a s in t he s y s t e m . It h a s b e e n recen t l y s h o w n that if p r e s s u r e is m a i n t a i n e d w i th in t h e r a n g e o f 0 to 0 .6 a t m o s p h e r e s , f e r m e n t a t i o n wi l l not b e a f f e c t e d (Pe l l a n d S c h o f i e l d , 1 9 9 3 ) . 2 6 A m o r e s o p h i s t i c a t e d c o m p u t e r i z e d s y s t e m to o b t a i n g a s p r o d u c t i o n resu l ts w a s d e v e l o p e d b y P e l l a n d S c h o f i e l d ( 1 9 9 3 ) . In th is s y s t e m , e l e c t r o n i c p r e s s u r e t r a n s d u c e r s w e r e c o n n e c t e d to f e r m e n t a t i o n f l a s k s a n d i n t e r f aced w i th a d a t a l o g g e r a n d c o m p u t e r ( P e l l a n d S c h o f i e l d , 1 9 9 3 ) . A s g a s a c c u m u l a t e d in a f i xed v o l u m e c o n t a i n e r , t h e resu l t i ng p r e s s u r e w a s r e c o r d e d at d e t e r m i n e d t i m e in te rva ls . A c a l i b r a t i o n e q u a t i o n is n e c e s s a r y in o r d e r to d e t e r m i n e t h e r e l a t i o n s h i p b e t w e e n p r e s s u r e i n c r e a s e s a n d v o l t a g e r e a d i n g s . T h e d a t a c a n b e e a s i l y t r a n s f e r r e d to a s p r e a d s h e e t w h i c h e n a b l e s c o r r e c t i o n s for b l a n k s a n d e a s y m a n i p u l a t i o n o f the d a t a . 1.2.8.1 Advantages of the Gas Production Technique T h e m a i n a d v a n t a g e o f t he g a s p r o d u c t i o n t e c h n i q u e is it m e a s u r e s the a p p e a r a n c e o f m e t a b o l i c p r o d u c t s a n d t h u s it re f lec ts t he " t rue" f e r m e n t a t i o n of a feeds tu f f . It h a s b e e n s h o w n that t he o b s e r v e d f e r m e n t a t i o n pro f i le is r e p r e s e n t a t i v e o f t he en t i re s a m p l e ( H u n t i n g t o n , 1 9 9 5 ) . T h e g a s p r o d u c t i o n t e c h n i q u e d o e s not r equ i r e s e p a r a t i o n o f t h e d i g e s t e d a n d u n d i g e s t e d c o m p o n e n t s u n l i k e m a n y o t h e r in vitro m e t h o d s ( M e n k e et a l . , 1 9 7 9 ) . F u r t h e r m o r e , bo th t he s o l u b l e a n d i n s o l u b l e c a r b o h y d r a t e f r a c t i o n s p r o d u c e g a s a s t h e y a r e m e t a b o l i z e d to vo la t i l e fatty a c i d s ( V F A ) b y r u m e n m i c r o o r g a n i s m s ( H u n t i n g t o n , 1 9 9 5 ) . T h e p r o c e d u r e a l s o r equ i r es a s m a l l s a m p l e s i z e w h i c h is re f l ec ted in t he u s e o f l imi ted a m o u n t s of l abo ra to ry s u p p l i e s . 2 7 1.2.8.2 Disadvantages of the Gas Production Technique T h e r e a r e s e v e r a l s h o r t c o m i n g s a s s o c i a t e d w i th t he g a s p r o d u c t i o n t e c h n i q u e . F i rs t l y , t h e c o m p u t e r i z e d t e c h n i q u e is a c o s t l y i n v e s t m e n t b e c a u s e it r e q u i r e s h i gh l y s o p h i s t i c a t e d e q u i p m e n t . S e c o n d l y , a s t a n d a r d f eeds tu f f m u s t b e i n c u b a t e d w i th in e a c h run in o r d e r to c o r r e c t fo r t he va r i a t i on in r u m e n f lu id act iv i ty b e t w e e n r u n s if n e c e s s a r y ( M e n k e a n d S t e i n g a s s , 1 9 8 8 ) . Th i r d l y , m i c r o b i a l c e l l s in t he r u m e n f lu id con t r i bu te to g a s p r o d u c t i o n a s t h e y d i e a n d a r e f e r m e n t e d to V F A s by o t h e r m i c r o o r g a n i s m s . T h i s lat ter po in t is o f pa r t i cu la r i m p o r t a n c e w h e n shor t i n c u b a t i o n s a r e u s e d s i n c e it h a s b e e n s h o w n that V F A s a r e a p o o r i nd i ca t i on of f e r m e n t a t i o n ra tes a n d ex ten t o f d i g e s t i o n ( V a n S o e s t , 1 9 8 2 ) . L a s t l y , t he f e r m e n t a t i o n o f r ead i l y f e r m e n t a b l e f e e d s t u f f s s u c h a s b a r l e y g r a i n , resu l t s in the p r o d u c t i o n o f h i g h a m o u n t s o f p r o p i o n i c a c i d w h i c h d o e s no t con t r i bu te to g a s p r o d u c t i o n , t h u s requ i r i ng c a r e f u l i n te rp re ta t ion o f g a s d a t a . 1.2.8.3 The Fermentation Process of Carbohydrates in the Rumen. D i e t a r y c a r b o h y d r a t e s i n c l u d e s o l u b l e s u g a r s , s t a r c h a n d f iber . S o l u b l e s u g a r s a r e rap id l y f e r m e n t e d in t he r u m e n w h e r e t he t y p e o f s u g a r d e t e r m i n e s the ra te o f f e r m e n t a t i o n . I n c r e a s i n g t he l e v e l s o f t h e s e s o l u b l e s u g a r s in t he d ie t resu l t s in t he p r o d u c t i o n o f h i gh a m o u n t s o f p r o p i o n i c a c i d t h u s d e c r e a s i n g r u m e n p H ( H u n g a t e , 1 9 6 6 ) . T h e d e c r e a s e in f i be r d i g e s t i o n a s s o c i a t e d w i th l o w r u m e n p H m a y b e c a u s e d b y a shi f t in t he t y p e s o f m i c r o b e s f r o m ce l l u lo l y t i c to a m y l o l y t i c p o p u l a t i o n s ( M a n n a n d O r s k o v , 1 9 7 5 ) . S t r u c t u r a l c a r b o h y d r a t e s a r e c o m p r i s e d of 2 8 c e l l u l o s e a n d h e m i c e l l u l o s e f r ac t i ons . S t a r c h , p e c t i n s a n d p o l y u r o n i d s a r e m o r e q u i c k l y d i g e s t e d t h a n f i be r c o m p o n e n t s a l t h o u g h m o r e s l o w l y f e r m e n t e d b y r u m e n m i c r o o r g a n i s m s t h a n t he s o l u b l e s u g a r s ( V a n S o e s t , 1 9 8 2 ) . D i e t a r y c a r b o h y d r a t e s a r e m e t a b o l i z e d by r u m e n m i c r o o r g a n i s m s to ob ta in a s o u r c e o f e n e r g y . T h e m a i n e n d p r o d u c t s o f t he a n a e r o b i c f e r m e n t a t i o n p r o c e s s a r e t he sho r t c h a i n vo la t i l e fat ty a c i d s ( V F A ) : a c e t a t e , p r o p i o n a t e , a n d bu ty ra te , a n d t he g a s e s : c a r b o n d i o x i d e ( C 0 2 ) a n d m e t h a n e ( C H 4 ) ( W o l i n , 1 9 7 5 ) . R u m i n a n t s ut i l ize V F A s a s a m a j o r s o u r c e o f e n e r g y , fo r e x a m p l e , f o r a g e d i e t s s u p p l y 5 0 - 8 5 % of the m e t a b o l i z a b l e e n e r g y ( O w e n s a n d G o e t s c h , 1 9 8 8 ) , h o w e v e r , g a s p r o d u c e d a s a resu l t o f c a r b o h y d r a t e m e t a b o l i s m c o n s t i t u t e s a m a j o r l o s s o f e n e r g y ( H u n g a t e , 1 9 6 6 ) . T h e e q u a t i o n s o f W o l i n ( 1 9 6 0 ) a r e p r e s e n t e d b e l o w to i l lust rate the p r o d u c t i o n o f g a s d u r i n g t h e p r o d u c t i o n o f V F A s ( E q u a t i o n s 1, 2 a n d 3) . E x c e s s h y d r o g e n p r o d u c e d d u r i n g t he p r o c e s s c o m b i n e s w i th C 0 2 to p r o d u c e C H 4 ( E q u a t i o n 4 ) . F r o m t h e s e e q u a t i o n s it c a n b e d e t e r m i n e d that in t he p r o d u c t i o n of V F A s , a c e t a t e a n d bu t y ra te pa r t i c i pa te d i rec t l y in g a s p r o d u c t i o n w h e r e a s p r o p i o n a t e d o e s not . C 3 H 6 0 3 + H 2 0 C H 3 C O O H + C 0 2 + 2 H 2 ( ace ta te ) ( E q u a t i o n 1) C 3 H 6 0 3 + H 2 => C H 3 C H 2 C O O H + H 2 0 (p rop iona te ) ( E q u a t i o n 2) 2 C 3 H 6 0 3 ^ C H 3 C H 2 C H 2 C O O H + 2 C 0 2 + 2 H 2 (bu ty ra te) ( E q u a t i o n 3) C 0 2 + 4 H 2 => C H 4 + 2 H 2 0 ( E q u a t i o n 4 ) 2 9 T h e r e is i n c r e a s e d e n e r g y e f f i c i e n c y w h e n t he p ropo r t i on o f p r o p i o n a t e i n c r e a s e s re la t i ve to a c e t a t e a n d bu ty ra te . T h i s is , in par t , a s a resu l t o f a d e c r e a s e in e n e r g y l o s s t h r o u g h d e c r e a s e d g a s p r o d u c t i o n , p r imar i l y m e t h a n e ( V a n S o e s t , 1994 ) . M e t h a n e is p r o d u c e d b y r u m e n m e t h a n o g e n i c b a c t e r i a w h i c h m e t a b o l i z e C 0 2 a n d H 2 v i a t he p r o d u c t i o n o f f o r m a t e ( O w e n s a n d G o e s t c h , 1 9 8 8 ) . F r o m the s t o i c h i o m e t r i c e q u a t i o n s o f H u n g a t e ( 1 9 6 6 ) a n d t h e e q u a t i o n s o f W o l i n ( 1 9 6 0 ) , g a s p r o d u c t i o n c a n b e e s t i m a t e d f r o m t h e V F A c o m p o s i t i o n o f t he r u m e n f lu id . 1.2.8.4 Estimation of Gas Volume Di rec t g a s p r o d u c t i o n c a n b e e x p l a i n e d by t h e a m o u n t a n d p ropo r t i ons of V F A s p r o d u c e d f r o m f e r m e n t a t i o n ( M e n k e a n d S t e i n g a s s , 1 9 8 8 ; B l u m m e l a n d O r s k o v , 1 9 9 3 ; B e u v i n k a n d S p l o e s t r a , 1 9 9 2 ; O p a t p a t a n a k i t et a l . , 1 9 9 4 ) . E a c h m m o l o f V F A p r o d u c e d f r o m the f e r m e n t a t i o n p r o c e s s a l s o r esu l t s in t he r e l e a s e o f a n ind i rec t e q u i v a l e n t m m o l o f C 0 2 f r o m the in vitro bu f fe r s o l u t i o n a s t he V F A s a r e n e u t r a l i z e d ( M e n k e a n d S t e i n g a s s , 1 9 8 8 ; B e u v i n k a n d S p l o e s t r a , 1 9 9 2 ; B l u m m e l a n d O r s k o v , 1 9 9 3 ) . Ind i rect g a s p r o d u c t i o n wi l l o c c u r a s l o n g a s t he buf fer ing c a p a c i t y o f t h e i n c u b a t i o n m e d i u m is not e x c e e d e d . D i rec t a n d ind i rec t g a s p r o d u c t i o n resu l t i ng f r o m e a c h m m o l o f a c e t a t e , p r o p i o n a t e a n d bu ty ra te h a s b e e n d e t e r m i n e d to b e 4 , 3 a n d 2 m m o l s C 0 2 , r e s p e c t i v e l y ( B e u v i n k a n d S p l o e s t r a , 1 9 9 2 ) . T h e f o l l o w i n g e q u a t i o n h a s b e e n s h o w n to d e s c r i b e t he a m o u n t of g a s d i rec t l y p r o d u c e d f r o m the v a r i o u s c a r b o h y d r a t e s o u r c e s w h e n c o n s i d e r i n g total V F A c o n t e n t a n d p r o p o r t i o n s t h e r e o f ( B e u v i n k a n d S p l o e s t r a , 1 9 9 2 ) : 3 0 mL gas = Mv mmol HAc + 2 Mv mmol HB + 0.87 Mv mmol total VFA ( E q u a t i o n 4 ) w h e r e HAc is a c e t a t e , HB is bu ty ra te a n d Mv is m o l a r g a s v o l u m e at s t a n d a r d t e m p e r a t u r e a n d p r e s s u r e (24 L * m o l 1.2.8.5 Use of the Gas Production Technique in the Evaluation of Concentrates. T h e g a s p r o d u c t i o n t e c h n i q u e h a s b e e n s u c c e s s f u l l y u s e d in t he e v a l u a t i o n of p a r a m e t e r s e s t i m a t e d f r o m t h e g a s p r o d u c t i o n o f c o n c e n t r a t e s ( M e n k e et a l . , 1 9 7 9 ; M e n k e a n d S t e i n g a s s , 1 9 8 8 , K r i s h n a m o o r t h y et a l . , 1 9 9 1 , 1 9 9 5 ; R i b e i r o et a l . , 1 9 9 0 ; B e u v i n k a n d S p l o e s t r a , 1 9 9 2 ; O p a t p a t a n a k i t et a l . , 1 9 9 4 , 1 9 9 5 ) . Q u i n (1943 ) (c i ted by J o h n s o n , 1 9 6 6 ) m e a s u r e d t he p r o d u c t i o n o f g a s f r o m c a r b o h y d r a t e s a n d f o u n d that t h e p r o d u c t s o f f e r m e n t a t i o n w e r e c l o s e l y re la ted to d i g e s t i o n in t he a n i m a l . T h e e v a l u a t i o n o f c o n c e n t r a t e s in vitro r e q u i r e s c a r e f u l m o n i t o r i n g o f the p H of t he s y s t e m . T h e f e r m e n t a t i o n o f c o n c e n t r a t e s resu l t s in h i g h e r a m o u n t s o f a c i d p r o d u c t i o n pu t t ing a g r e a t e r l o a d o n t h e buf fe r ing c a p a c i t y o f t he s y s t e m . T h e buf fe r ing c a p a c i t y o f t he s y s t e m wi l l b e a f f e c t e d if t he p H fa l l s b e l o w 6 .0 ( M e n k e et a l . , 1 9 7 9 ) . T h e p r e s e n c e o f C 0 2 in t he r u m e n s y s t e m h e l p s m a i n t a i n p H b a l a n c e by a c t i n g a s a bu f fe r in t h e f o r m o f b i c a r b o n a t e ( H C 0 3 " ) . A l l o f t h e V F A s con t r i bu te to the p ro ton p o o l . H o w e v e r , a s p r e v i o u s l y r e p o r t e d , t he f o r m a t i o n o f p r o p i o n a t e d o e s not p r o d u c e C 0 2 in a d d i t i o n to con t r i bu t i ng to t he p ro ton p o o l , bo th o f w h i c h 31 con t r i bu te to t h e d e c r e a s e in r u m e n p H s e e n in c o w s f e d h igh c o n c e n t r a t e d ie t s ( V a n S o e s t , 1 9 9 4 ) . T h e f e r m e n t a t i o n o f c o n c e n t r a t e s g e n e r a l l y r esu l t s in t he f o r m a t i o n of g r e a t e r a m o u n t s o f p r o p i o n a t e at t he e x p e n s e o f a c e t a t e p r o d u c t i o n resu l t i ng in l e s s C 0 2 a n d C H 4 p r o d u c t i o n ( M e n k e et a l . , 1 9 7 9 , M e n k e a n d S t e i n g a s s , 1 9 8 8 ) . T h i s rat io wil l v a r y d e p e n d i n g o n t he c o n t e n t o f r ead i l y f e r m e n t a b l e c a r b o h y d r a t e s in t he s a m p l e . F o r th is r e a s o n , in te rp re ta t ion o f t he f e r m e n t a t i o n p ro f i l es o f c o n c e n t r a t e s m a y b e m o r e di f f icul t t h a n that o f f o r a g e s e s p e c i a l l y w h e n c o m p a r i n g c o n c e n t r a t e s f r om d i f ferent s o u r c e s . 1.2.8.6 Kinetic Analysis of Gas Data M a t h e m a t i c a l m o d e l s p r o v i d e a m e a n s for d e s c r i b i n g t he f e r m e n t a t i o n k i ne t i cs o f v a r i o u s f e e d s t u f f s . M a n y m o d e l s h a v e b e e n d e v e l o p e d to ref lect the r e l a t i onsh ip b e t w e e n b a c t e r i a l n u m b e r s a n d s u b s t r a t e d i s a p p e a r a n c e . S i g m o i d a l m o d e l s u s e d to d e s c r i b e t h e d i g e s t i o n o f f i be r in t he r u m e n w e r e p r imar i l y d e v e l o p e d for u s e in c o n j u n c t i o n w i th d a t a o b t a i n e d f r o m the n y l o n b a g t e c h n i q u e ( O r s k o v a n d M c D o n a l d , ( 1 9 7 9 ) ; B r o d e r i c k a n d C r a i g , ( 1 9 8 0 ) ; a n d V a n S o e s t , (1982) ) . T h e on l y n o n - s i g m o i d a l m o d e l is t he e x p o n e n t i a l m o d e l d e v e l o p e d b y O r s k o v a n d M c D o n a l d ( 1979 ) . T h i s m o d e l h a s c o m e u n d e r c r i t i c i sm s i n c e it p r o d u c e s h igh root m e a n s q u a r e v a l u e s w h e n f i t ted to g a s p r o d u c t i o n d a t a b e c a u s e o f t h e a s s u m p t i o n that a f ter a d i s c r e t e l a g p e r i o d , f e e d is f e r m e n t e d at a m a x i m u m rate ( B e u v i n k a n d S p l o e s t r a , 1 9 9 2 ) . 3 2 S c h o f i e l d et a l . ( 1 9 9 4 ) fit t he s i m p l e e x p o n e n t i a l m o d e l w h i c h i n c o r p o r a t e d l a g , l og is t i c a n d bo th t h e G o m p e r t z s i n g l e a n d d u a l p o o l m o d e l s to g a s d a t a o b t a i n e d f r o m v a r i o u s s u b s t r a t e s i n c l u d i n g bac te r i a l c e l l u l o s e , a l p h a - c e l l u l o s e a n d m i x t u r e s o f t h e t w o pur i f ied s u b s t r a t e s o f f i be r d i g e s t i o n . T h e s e a u t h o r s f o u n d that a log is t i c m o d e l d e s c r i b i n g bac te r i a l g r o w t h that i n c o r p o r a t e d t h e d i f ferent f e r m e n t a t i o n r a t e s o f t he v a r i o u s c o m p o n e n t s in the i r m i x e d - f i b e r s u b s t r a t e m o s t a c c u r a t e l y d e s c r i b e d g a s p r o d u c t i o n . U s i n g g a s p r o d u c t i o n d a t a f r o m the i n c u b a t i o n o f g r a s s s i l a g e s w i th r u m e n f lu id , B e u v i n k a n d K o g u t ( 1 9 9 3 ) d e v e l o p e d a mod i f i ed G o m p e r t z e q u a t i o n to d e s c r i b e t he f e r m e n t a t i o n p r o c e s s . F r a n c e et a l . ( 1993 ) d e v e l o p e d a c o m p l e x e x p o n e n t i a l m o d e l i n c l u d i n g l ag to d e s c r i b e t he f e r m e n t a t i o n of l e a v e s f r o m t rop i ca l f o r a g e t r e e s . T h e s i m p l e e x p o n e n t i a l m o d e l wi th lag ( S c h o f i e l d et a l . , 1 9 9 4 ) is a m o d i f i c a t i o n o f t h e e q u a t i o n o f M e r t e n s a n d Lo f ten (1980 ) . K r i s h n a m o o r t h y et a l . ( 1 9 9 1 , 1 9 9 5 ) f o u n d t he m o d e l to a c c u r a t e l y ref lect the g a s p r o d u c t i o n p ro f i l es o f e n e r g y s u p p l e m e n t s a n d t rop i ca l f e e d s t u f f s . T h i s m o d e l w a s f o u n d to a c c u r a t e l y re f lec t t he g a s p r o d u c t i o n pro f i le o f c a n o l a m e a l in e x p e r i m e n t 3 . 1.3 OBJECTIVES OF THE THESIS E v a l u a t i o n o f e n z y m e a n d mi ld h y d r o t h e r m a l t r e a t m e n t o f b a r l e y a n d c a n o l a m e a l w a s c o n d u c t e d w i th a s y s t e m e m p l o y i n g t he u s e o f I V D M D a n d I V T D resu l t s (T i l ley a n d T e r r y , 1 9 6 3 ; G o e r i n g a n d V a n S o e s t , 1 9 7 0 ) . F u r t h e r e v a l u a t i o n o f the e f f ec t s o f t h e t r e a t m e n t s o n c a n o l a m e a l w a s c o n d u c t e d u s i n g t h e c o m p u t e r i z e d in 3 3 vitro g a s p r o d u c t i o n t e c h n i q u e ( P e l l a n d S c h o f i e l d , 1 9 9 3 ) . U n f o r t u n a t e l y , d u e to the la rge n u m b e r o f s a m p l e s a n d t i m e c o n s t r a i n t s , b a r l e y c o u l d not b e e v a l u a t e d to the s a m e ex ten t . T h e u s e o f t he g a s p r o d u c t i o n s y s t e m p r o v i d e d i n fo rma t ion o n t he r u m e n in vitro f e r m e n t a t i o n c h a r a c t e r i s t i c s o f c a n o l a m e a l w h e n t r ea ted wi th e n z y m e s a n d h y d r o t h e r m a l p r o c e s s i n g . T h e o b j e c t i v e s o f th is t h e s i s p ro jec t w e r e to : 1) D e t e r m i n e o p t i m a l t r e a t m e n t c o m b i n a t i o n s o f t h e e n z y m e s a n d h y d r o t h e r m a l t r e a t m e n t u s i n g I V D M D , I V T D , A D F a n d N D F v a l u e s a s i n d i c e s o f t h e nutr i t ive v a l u e o f b a r l e y fo r da i r y ca t t le . 2) D e t e r m i n e t he e f fec t s o f e n z y m e a n d h y d r o t h e r m a l t r e a t m e n t u s i n g I V D M D , I V T D , A D F a n d N D F v a l u e s a s i n d i c e s o f t h e nutr i t ive v a l u e o f c a n o l a m e a l . 3 ) F u r t h e r e v a l u a t e t h e t r e a t m e n t e f f ec t s o n t he c h a r a c t e r i s t i c s o f f e r m e n t a t i o n o f c a n o l a m e a l u s i n g a s i n d i c e s : ra te o f g a s p r o d u c t i o n , l e n g t h o f t he l a g p h a s e , t he p r o d u c t i o n o f V F A s a n d the i r p r o p o r t i o n s . 3 4 1.4 REFERENCES A d e s o g a n , A . T . , O w e n , E . a n d D. I. G i v e n s . 1 9 9 5 . A c o m p a r i s o n b e t w e e n in vitro d iges t ib i l i t y , in situ d e g r a d a b i l i t y a n d a g a s p r o d u c t i o n t e c h n i q u e fo r p red ic t i ng t h e in vivo d iges t ib i l i t y o f w h o l e c r o p w h e a t . Br i t . G r a s s l . S o c , O c c a s i o n a l s y m p o s i u m N o . 2 9 . A m a d o , R. 1 9 9 3 . E n z y m e s in f o o d a n d f e e d p r o c e s s i n g - a r e v i e w . P a g e s 5 - 1 5 in C . W e n k e a n d M . B o e s s i n g e r , e d s . E n z y m e s in A n i m a l Nut r i t i on . P r o c . 1st S y m p . , K a r t a u s e I t t ingen, S w i t z e r l a n d . A n n i s o n , G . 1 9 9 1 . R e l a t i o n s h i p b e t w e e n l e v e l s o f s o l u b l e n o n s t a r c h p o l y s a c c h a r i d e s a n d t he a p p a r e n t m e t a b o l i s a b l e e n e r g y o f w h e a t s a s s a y e d in b ro i l e r c h i c k e n s . J . A g r i c . F o o d . C h e m . , 3 9 : 1 2 5 2 - 1 2 5 6 . A n n i s o n , G . a n d M . C h o c t . 1 9 9 1 . Ant i -nu t r i t i ve ac t i v i t i es o f c e r e a l n o n - s t a r c h p o l y s a c c h a r i d e s in bro i le r d i e t s a n d s t r a t e g i e s m i n i m i z i n g the i r e f fec ts . W o r l d ' s P o u l t . S c i . J . 4 7 : 2 3 2 - 2 4 2 . B a r k e r , R . a n d B . C h e a t l e y . 1 9 9 2 . D a i r y , F r a s e r V a l l e y , S u m m e r 1 9 9 2 . P l a n n i n g for profit : A g D e x 4 1 0 - 8 1 0 . P r o v i n c e o f B . C . M in i s t r y o f A g r i c u l t u r e , F i s h e r i e s a n d F o o d . 1 9 9 2 . B e a u c h e m i n , K. A . , R o d e , L. M . a n d V . J . H . S e w a l t . 1 9 9 5 . F ib ro l y t i c e n z y m e s i n c r e a s e f i be r d iges t ib i l i t y a n d g r o w t h ra te o f s t e e r s f e d d ry f o r a g e s . C a n . J . A n i m . S c i . , 7 5 : 6 4 1 - 6 4 4 . B e a u c h e m i n , K. A . a n d L. M . R o d e . 1 9 9 6 . 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S y m p . A l l t e c h T e c h n i c a l P u b l i c a t i o n s , N i c h o l a s v i l l e , K Y . R o v i c s , J . J . a n d C . M . E l y . 1 9 6 2 . R e s p o n s e o f b e e f ca t t le to e n z y m e s u p p l e m e n t s . J . A n i m . S c i . , 2 1 : 1 0 1 2 (Abs t rac t ) . R u s s e l , J . B . a n d R. L. B a l d w i n . 1 9 7 9 . C o m p a r i s o n o f s u b s t r a t e af f in i t ies a m o n g r u m e n b a c t e r i a : a p o s s i b l e d e t e r m i n a n t o f r u m e n bac te r i a l c o m p e t i t i o n . A p p l . E n v i r o n . M i c r o b i o l . , 3 7 : 5 3 1 - 5 3 6 . R u s t , J . W . , J a c o b s e n , N . L. a n d M c G i l l i a r d A . D a n d D. K. H o t c h k i s s . 1 9 6 5 . S u p p l e m e n t a t i o n o f d a i r y ca l f d i e t s w i th e n z y m e s . II. E f f ec t o n nutr ient u t i l i za t ion a n d o n c o m p o s i t i o n o f r u m e n f lu id . J . A n i m . S c i . , 2 4 : 1 5 6 - 1 6 0 . S c h o f i e l d , P . a n d A . N . P e l l . 1 9 9 5 . Va l i d i t y o f u s i n g a c c u m u l a t e d g a s p r e s s u r e r e a d i n g s to m e a s u r e f o r a g e d i g e s t i o n in vitro: a c o m p a r i s o n i nvo l v i ng t h ree f o r a g e s . J . D a i r y S c i . , 7 8 : 2 2 3 0 - 2 2 3 8 . 4 3 S c h o f i e l d , P . , Pi t t , R . E . , a n d A . N . P e l l . 1 9 9 4 . K i n e t i c s o f f i be r d i g e s t i o n f r o m in vitro g a s p r o d u c t i o n . J . A n i m . S c i . , 7 2 : 2 9 8 0 - 2 9 9 1 . S t o k e s , M . R . a n d S . Z h e n g . 1 9 9 5 . T h e u s e o f c a r b o h y d r a s e e n z y m e s a s f e e d a d d i t i v e s fo r e a r l y l ac ta t i on c o w s . P a g e 3 5 in 2 3 r d B i a n n u a l C o n f e r e n c e o n R u m e n F u n c t i o n , C h i c a g o , IL. (Abs t rac t . T e i t g e , D. A . , C a m p b e l l , G . L , C l a s s e n , H . L . a n d P . A . T h a c k e r . 1 9 9 1 . H e a t p r e t r e a t m e n t a s a m e a n s o f i m p r o v i n g t he r e s p o n s e to d i e ta r y p e n t o s a n a s e in c h i c k s f e d r y e . C a n . J . A n i m . S c i . , 7 1 : 5 0 7 - 5 1 3 . T h e o d o r o u , M . K. , W i l l i a m s , B . A . D h a n o a , M . S . , M c A l l a n , A . B . a n d J . F r a n c e . 1 9 9 4 . A s i m p l e g a s p r o d u c t i o n m e t h o d u s i n g a p r e s s u r e t r a n s d u c e r to d e t e r m i n e t he f e r m e n t a t i o n k i ne t i c s o f r u m i n a n t f e e d s . A n i m . F e e d S c i . T e c h n o l . , 4 8 : 1 8 5 - 1 9 7 . T h e u r e r , B . , W o o d s , W . a n d W . B u r r o u g h s . 1 9 6 3 . I n f l uence o f e n z y m e s u p p l e m e n t s in l a m b fa t t en ing ra t i ons . J . A n i m . S c i . , 2 2 : 1 5 0 - 1 5 4 . T i l l ey , J . M . A . a n d R. A . T e r r y . 1 9 6 3 . A t w o - s t a g e t e c h n i q u e fo r t he in vitro d i g e s t i o n o f f o r a g e c r o p s . J . Br i t . G r a s s l . S o c , 18 : 1 0 4 - 1 1 1 . T r e a c h e r , R . a n d C . W . Hun t . 1 9 9 6 . R e c e n t d e v e l o p m e n t s in f e e d e n z y m e s for r u m i n a n t r a t i ons . P a g e s 3 7 - 5 4 in P r o c . 3 1 s t A n n . P a c . N W A n i m . Nutr . C o n f . S e a t t l e , W A , U . S . A . T r e a c h e r , R. , M c A l l i s t e r , T . A . , P o o p , J . D. , M i r , Z . , M i r , P . a n d K . - J . C h e n g . 1 9 9 6 . E f f e c t o f e x o g e n o u s c e l l u l a s e s a n d x y l a n a s e s o n f e e d u t i l i za t ion a n d g r o w t h p e r f o r m a n c e o f f e e d l o t s t e e r s . C a n . S o c A n i m . S c i . A n n u a l M e e t i n g , L e t h b r i d g e , A l b e r t a . (Abstract in press). V a n S o e s t , P . J . 1 9 8 2 . Nut r i t i ona l e c o l o g y o f t he rum inan t . O & B B o o k s Inc., C o r v a l l i s , O r e g o n . V a n S o e s t , P . J . 1 9 9 4 . Nut r i t i ona l e c o l o g y o f t he rum inan t . 2 n d e d . C o r n e l l U n i v e r s i t y P r e s s . I t haca , N Y . V a n W a l l e g h e m , P . A . , A m m e r m a n , C . B . , C h i c c o , C . F . M o o r e , J . E . a n d L. R. A r r i n g t o n . 1 9 6 4 . E n z y m e s u p p l e m e n t s a n d d iges t ib i l i t y o f p ro te in a n d e n e r g y in ra t i ons h i g h in d r i e d c i t rus pu lp . J . A n i m . S c i . , 2 3 : 9 6 0 - 9 6 2 . V a r v i k k o , T . a n d J . E . L i n d b e r g . 1 9 8 5 . M i c r o b i a l n i t r ogen in n y l o n b a g r e s i d u e s quan t i f i ed b y f e e d 1 5 N d i lu t ion . Br i t . J . Nut r . , 5 4 : 4 7 3 . 4 4 W a r d , J . K. , R i c h a r d s o n , D. a n d W . S . T s i e n . 1 9 6 0 . V a l u e o f a d d e d e n z y m e p r e p a r a t i o n s in b e e f ca t t le ra t i ons . J . A n i m . S c i . , 1 9 : 1 2 9 8 (Abs t rac t ) . W a r n e r , A . C . I. 1 9 5 6 . C r i t e r i a fo r e s t a b l i s h i n g t h e va l id i t y o f in vitro s t u d i e s wi th r u m e n m i c r o o r g a n i s m s in s o - c a l l e d ar t i f ic ia l r u m e n s y s t e m s . J . G e n . M i c r o b i o l . , 1 4 : 7 3 3 . W a r n e r , A . C . I. 1 9 6 5 . F a c t o r s i n f l u e n c i n g n u m b e r s a n d k i n d s o f m i c r o o r g a n i s m s in t h e r u m e n . P a g e s 3 4 6 - 3 5 9 in R. W . D o u g h e r t y e d . , P h y s i o l o g y o f d i g e s t i o n in t h e r u m i n a n t . B u t t e r w o r t h s , W a s h i n g t o n . W a r n e r , A . C . I. 1 9 8 1 . R a t e o f p a s s a g e o f d i g e s t a t h r o u g h t h e gu t o f m a m m a l s a n d b i rds . Nut r . A b s t r . R e v . s e r . B . , 5 1 : 7 8 9 - 8 2 0 . W e i c h e n t h a l , B . , R u s h , I. a n d B . V a n Pe l t . 1 9 9 6 . A n e n z y m e - m i c r o b i a l f e e d p roduc t fo r f i n i s h i n g s t e e r s . B e e f ca t t le repor t , U n i v . N e b r a s k a - L i n c o l n , L i n c o l n , N E , U S , p p . 6 8 - 6 9 . W i n g , J . M . a n d W i l c o x , C . J . 1 9 6 0 . E f fec t o f s u p p l e m e n t a r y d i g e s t i v e e n z y m e s o n g r o w t h o f d a i r y he i f e r s . J . D a i r y S c i . , 4 3 : 1 6 5 5 - 1 6 7 5 . W o l i n , M . J . 1 9 6 0 . A t h e o r e t i c a l r u m e n f e r m e n t a t i o n b a l a n c e . J . D a i r y S c i . , 4 3 : 1 4 5 2 -1 4 5 9 . W o l i n , M . J . 1 9 7 5 . In te rac t ions b e t w e e n bac te r i a l s p e c i e s o f t he r u m e n . P a g e s 1 3 4 -1 4 8 in I. W . M c D o n a l d a n d A . C . I. W a r n e r e d s . , D i g e s t i o n a n d M e t a b o l i s m of t h e r u m i n a n t ; P r o c . IV Intl. S y m p . R u m i n a n t P h y s i o l . U n i v . o f N e w E n g l a n d P u b l . Un i t , A r m i d a l e , N S W , A u s t r a l i a . 4 5 CHAPTER 2 2.0 THE EFFECT OF ENZYME AND MILD HYDROTHERMAL TREATMENT ON THE IN VITRO DIGESTIBILITY OF BARLEY GRAIN. 2.1 ABSTRACT B a r l e y g r a i n is a m a j o r c o m p o n e n t o f lac ta t ing da i r y ca t t le d i e t s in t he L o w e r F r a s e r V a l l e y r e g i o n o f Br i t i sh C o l u m b i a a n d m u c h o f t he P a c i f i c N o r t h w e s t . T h e i n c l u s i o n o f b a r l e y i n c r e a s e s t h e e n e r g y d e n s i t y o f t h e ra t i on , h o w e v e r , i t 's h i g h f i be r c o n t e n t h a s a n e g a t i v e i m p a c t o n it 's c o n t e n t o f a v a i l a b l e e n e r g y . T h e ob jec t i ve of t he p r e s e n t s t u d y w a s to e v a l u a t e t h e feas ib i l i t y o f u s i n g e n z y m e s a n d mi ld h y d r o t h e r m a l t r e a t m e n t to i m p r o v e t h e a v a i l a b l e e n e r g y c o n t e n t o f b a r l e y g r a i n . T h e e x p e r i m e n t c o n s i s t e d o f a 4 x 4 x 4 x 2 x 2 mul t i - fac tor ia l d e s i g n w h i c h i n c l u d e d the fo l l ow ing t r e a t m e n t s : h e a t a p p l i e d at 5 5 ° C fo r 1 5 , 3 0 o r 6 0 m in p e r i o d s o r not a p p l i e d a t a l l ; m o i s t u r e a p p l i e d at 0 % , 1 0 % , 2 0 % , o r 3 0 % ; p - g l u c a n a s e (Trichoderma sp.) a p p l i e d a t 0 % , 0 . 0 0 1 % , 0 . 0 1 % o r 0 . 1 % ; p r o t e a s e (Bacillus sp.) a p p l i e d a t 0 % o r 0 . 0 0 2 5 % a n d x y l a n a s e (Trichoderma sp.) a p p l i e d at 0 % o r 0 . 0 0 1 % . S a m p l e s w e r e i n c u b a t e d in vitro to d e t e r m i n e a p p a r e n t a n d t rue d iges t ib i l i t i es a n d f ibe r c o n t e n t . Nu t r i en t c o m p o s i t i o n o f t h e b a r l e y g ra i n w a s 8 7 . 3 % D M a n d 1 2 . 7 % C P , 7 . 3 2 % A D F a n d 2 2 . 8 % N D F , o n a D M b a s i s . A s ign i f i can t th i rd o r d e r in te rac t ion ( P < 0 .01 ) o n I V D M D w a s n o t e d fo r t he du ra t i on o f t he h e a t t r ea tmen t , a n d t he 4 6 a d d i t i o n o f m o i s t u r e a n d x y l a n a s e w h e r e t r e a t m e n t s e i t he r h a d n o ef fec t o r s ign i f i can t l y d e c r e a s e d ( P < 0 .05 ) I V D M D . T h e r e w a s a l s o a s ign i f i can t i n te rac t ion ( P = 0 . 0 0 0 1 ) b e t w e e n t he e n z y m e s : p - g l u c a n a s e , p r o t e a s e a n d x y l a n a s e w h e r e bo th i n c r e a s e d a n d d e c r e a s e d I V D M D v a l u e s w e r e o b s e r v e d ( P < 0 .05 ) . A n in te rac t ion a m o n g t he e n z y m e s w a s a l s o d e t e r m i n e d to ex i s t fo r I V T D v a l u e s ( P = 0 . 0 0 0 1 ) w h e r e r esu l t s p a r a l l e l e d t h o s e o f I V D M D . A q u a d r a t i c e f fec t ( P < 0 .01 ) o f m o i s t u r e o n I V T D w a s d e s c r i b e d b y Y = 8 7 . 7 5 + 0 . 0 7 1 * m o i s t u r e - 0 . 0 0 1 3 6 * m o i s t u r e 2 . In vitro d iges t ib i l i t y r esu l t s w e r e i n c o n s i s t e n t , a s s o m e t r e a t m e n t s i n c r e a s e d a n d o the rs d e c r e a s e d d iges t ib i l i t y re la t i ve to t he con t ro l . F i b e r a n a l y s e s re f l ec ted resu l t s o b t a i n e d w i th in vitro d iges t ib i l i t i es . A D F w a s s ign i f i can t l y ( P < 0 .01 ) a f f e c t e d by the s ign i f i can t i n te rac t i on ( P < 0 .05 ) b e t w e e n p - g l u c a n a s e , p r o t e a s e a n d x y l a n a s e . T h e r e w a s a l s o a s i gn i f i can t i n te rac t i on ( P < 0 .05 ) n o t e d fo r t he d u r a t i o n o f t he h e a t t r ea tmen t , a n d t h e a p p l i c a t i o n o f p - g l u c a n a s e a n d p r o t e a s e e n z y m e s o n A D F con ten t . T w o - w a y i n t e rac t i ons w h i c h w e r e f o u n d to h a v e a s ign i f i can t e f fec t ( P < 0 .05 ) o n t h e N D F c o n t e n t o f b a r l e y i n c l u d e d : p - g l u c a n a s e a n d p r o t e a s e , p-g l u c a n a s e a n d x y l a n a s e , p r o t e a s e a n d x y l a n a s e . T h e r e s p o n s e s w e r e v a r i a b l e . X y l a n a s e w a s d e t r i m e n t a l o n t he d iges t ib i l i t y o f b a r l e y . R e s u l t s i n d i c a t e d t he add i t i on of p r o t e a s e at 0 . 0 1 % o r a c o m b i n a t i o n o f p - g l u c a n a s e a n d p r o t e a s e a r e a r e a s w o r t h y o f fu r the r r e s e a r c h w h i c h m a y i m p r o v e t h e nut r i t iona l v a l u e o f b a r l e y g ra in in r u m i n a n t f e e d i n g s y s t e m s . 4 7 2.2 INTRODUCTION B a r l e y g r a i n is t he m a j o r e n e r g y s o u r c e f e d to da i r y ca t t l e in t he L o w e r F r a s e r V a l l e y r e g i o n o f B r i t i sh C o l u m b i a a n d m a y c o m p r i s e 5 0 - 6 0 % o f t he c o n c e n t r a t e ra t ion ( P r o F o r m F e e d s Inc., p e r s o n a l c o m m u n i c a t i o n ) . It's l o w e r d i g e s t i b l e e n e r g y c o n t e n t c o m p a r e d to c o r n is par t ly a resu l t o f h i gh l e v e l s o f A D F ( 9 % A D F ) a n d N D F ( 2 1 % N D F ) w h i c h m a y l imit it 's p e r f o r m a n c e in da i r y ca t t le d i e t s . T h e b a r l e y k e r n e l c o n s i s t s o f a hu l l , e n d o s p e r m a n d e m b r y o n i c g e r m layer . T h e hul l c o n t a i n s t he major i ty o f t he f iber . T h e e n d o s p e r m is m a d e up of a n a l e u r o n e l aye r , s t a r c h y e n d o s p e r m a n d a d e p l e t e d ce l l l aye r , t he lat ter b e i n g of little nut r i t ional v a l u e to t h e r u m i n a n t a n i m a l ( N e w m a n a n d M c G u i r e , 1 9 8 5 ) . T h e a l e u r o n e l a y e r is p r imar i l y c o m p o s e d a r a b i n o x y l a n s a n d p - g l u c a n s . ( N e w m a n a n d M c G u i r e , 1 9 8 5 ) . It is w e l l e s t a b l i s h e d that p - g l u c a n s a n d a r a b i n o x y l a n s , w h e n f e d to m o n o g a s t r i c s , exh ib i t an t i -nu t r i t iona l e f f ec t s a s t h e s e a n i m a l s d o not p o s s e s s the e n z y m e s to b r e a k t h e m d o w n ( B e d f o r d , 1 9 9 3 ) . T h u s , s u p p l e m e n t a t i o n of e x o g e n o u s e n z y m e s to m o n o g a s t r i c d i e t s is i n c r e a s i n g in o r d e r to r e d u c e t he e f fec ts o f t h e s e n o n s t a r c h p o l y s a c c h a r i d e s ( N S P ) . T h i s h a s l ed to i n c r e a s e d l e v e l s of i n c l u s i o n of c e r e a l s , s u c h a s b a r l e y a n d w h e a t , in pou l t ry a n d s w i n e d i e t s w i t hou t c o m p r i m i s i n g a n i m a l p e r f o r m a n c e . T h e s e N S P s ra re ly p o s e a p r o b l e m to r u m i n a n t a n i m a l s b e c a u s e r u m e n m i c r o b e s p o s s e s s t he e n z y m e s to a s s i s t in the i r d i g e s t i o n , p-g l u c a n s , fo r e x a m p l e , a r e 9 8 % r u m e n d e g r a d a b l e ( E n g s t r o m et a l . , 1 9 9 2 ) . H o w e v e r , 4 8 in h i gh p r o d u c i n g c o w s , f e e d i n take a n d f e e d p a s s a g e rate m a y b e i n c r e a s e d to s u c h a n e x t e n t tha t a r e d u c t i o n in t he d i g e s t i o n o f s p e c i f i c f e e d c o m p o n e n t s m a y o c c u r . T h e s u c c e s s f u l a p p l i c a t i o n o f f e e d e n z y m e s to m o n o g a s t r i c f e e d i n g r e g i m e s h a s r e n e w e d in te res t in t he p o s s i b l e a p p l i c a t i o n o f t h e s e f e e d a d d i t i v e s to r um inan t f e e d i n g s y s t e m s . R e s e a r c h into t he add i t i on o f e x o g e n o u s e n z y m e s to c o n c e n t r a t e s that a r e f e d to r u m i n a n t s h a s y i e l d e d v a r i a b l e resu l t s ( re fer to C h a p . 1, s e c t . 1.1.4) ( B e a u c h e m i n a n d R o d e , 1 9 9 6 ) . E n z y m e c o m p o s i t e s c o n t a i n i n g a s p e c t r u m of d i f ferent e n z y m e s , s u c h a s t h o s e w i th p ro teo ly t i c , a m y l o l y t i c a n d f ibro ly t ic ac t i v i t ies , e l i c i ted p o s i t i v e r e s p o n s e s in t e r m s o f D M I a n d / o r A D G a n d / o r f e e d e f f i c i e n c i e s w h e n a d d e d to h i g h g r a i n r a t i ons ( B u r r o u g h s e t a l . , 1 9 6 0 ; C l a r k e t a l . , 1 9 6 1 ; R o v i c s a n d E l y , 1 9 6 2 B o y l e s e t a l . , 1 9 9 2 ; W e i c h e n t h a l et a l . , 1 9 9 6 ) . In con t ras t , a l ack of r e s p o n s e in D M I , A D G o r f e e d e f f i c i ency w a s r e p o r t e d in o t h e r s t u d i e s w h i c h i n v e s t i g a t e d t h e e f f ec t s o f m u l t i - e n z y m e p r e p a r a t i o n s a d d e d to a va r ie t y of c o n c e n t r a t e s ( W a r d et a l . , 1 9 6 0 ; W i n g a n d W i l c o x , 1 9 6 0 ; R i c h a r d s o n et a l . , 1 9 9 0 ; P e r r y et a l . , 1 9 6 6 ) . B a r l e y g r a i n , in par t i cu la r , h a s b e e n t he f o c u s o f s e v e r a l r e s e a r c h e x p e r i m e n t s . In a s t u d y w h e r e s t e e r s w e r e f e d a 6 5 % b a r l e y g ra in d ie t s u p p l e m e n t e d w i th p ro teo ly t i c a n d a m y l o l y t i c e n z y m e s , t h e r e w a s a no ted i m p r o v e m e n t in f e e d e f f i c i e n c y ( K e r c h e r , 1 9 6 6 ) . W h e n s t e e r s w e r e f e d a b a r l e y g ra in d ie t s u p p l e m e n t e d w i th x y l a n a s e , t h e r e w e r e i m p r o v e m e n t s in D M I , A D G a n d f e e d e f f i c i e n c y ( B e a u c h e m i n et a l . , 1 9 9 6 ) . 4 9 T h e a p p l i c a t i o n o f e x o g e n o u s e n z y m e s to b a r l e y a s a m e a n s o f i m p r o v i n g it 's d iges t ib i l i t y m a y i m p a c t d i g e s t i o n in s e v e r a l w a y s . F i rs t l y , i n c r e a s i n g b a r l e y d iges t ib i l i t y m a y pos i t i ve l y a f fec t t he p r o d u c t i o n o f m i c r o b i a l p ro te in by p rov id i ng g r e a t e r a m o u n t s o f a v a i l a b l e c a r b o h y d r a t e s a s a n e n e r g y s o u r c e . T h e da i r y f a r m e r s l o c a t e d in t h e L o w e r F r a s e r V a l l e y r e g i o n o f B r i t i sh C o l u m b i a re ly o n h igh qua l i t y g r a s s s i l a g e a s t h e m a i n f o r a g e s o u r c e f e d to the i r l ac ta t i ng d a i r y h e r d s . T h e s e s i l a g e s t e n d to b e h igh in s o l u b l e n i t rogen (von K e y s e r l i n g k et a l . , 1 9 9 6 ) . T h i s l a rge s u p p l y o f s o l u b l e n i t r ogen to t he r u m e n m u s t b e b a l a n c e d w i th a c o m p l e m e n t a r y s u p p l y o f r e a d i l y a v a i l a b l e c a r b o h y d r a t e s . T h i s is impor tan t in o r d e r to o p t i m i z e the u s e o f r u m e n d e g r a d a b l e n i t r ogen a n d e n e r g y s o u r c e s fo r t h e p r o d u c t i o n of m i c r o b i a l p ro te in ( K r i s h n a m o o r t h y et a l . , 1 9 9 1 ) a n d to m a x i m i z e t he s u p p l y of m i c r o b i a l p ro te in to t h e in tes t i ne ( S t o k e s et a l . , 1 9 9 1 ) . M a i n t e n a n c e o f th is b a l a n c e is a l s o e s s e n t i a l in o r d e r to r e d u c e t he e x c r e t i o n o f e x c e s s n i t r ogen a n d m a x i m i z e f e e d u t i l i za t ion ( H u n t i n g t o n , 1 9 9 5 ) . S e c o n d l y , i n c r e a s i n g t h e r u m i n a l d e g r a d a b i l i t y o f b a r l e y m a y i m p r o v e total t rac t d iges t ib i l i t y . A l t h o u g h r u m i n a l a c i d o s i s is a s s o c i a t e d w i th h i g h rumina l l y d i g e s t i b l e c e r e a l g r a i n s , L e n v e n t i n i e t a l . , ( 1 9 9 0 ) f o u n d that t he e f fec t o f l ow r u m e n p H w a s m i n i m i z e d w i th t he add i t i on o f a bu f fe r to t he ra t ion . B o t h a l o w r u m e n p H a n d a shi f t in t he r u m e n m i c r o b i a l p o p u l a t i o n m a y con t r i bu te to r e d u c e d f iber d i g e s t i o n . T h e a d d i t i o n o f f ib ro ly t ic e n z y m e s to pa r t i cu la r d i e t s m a y e n a b l e th is d i s a d v a n t a g e to b e o v e r c o m e . 5 0 T h e d iges t ib i l i t y o f b a r l e y m a y b e i m p r o v e d b y i n c r e a s i n g t h e d igest ib i l i t y o f t he s t ruc tu ra l c a r b o h y d r a t e s , t he hul l f r ac t i on in par t i cu la r , a n d by e n h a n c i n g t he a c c e s s b y r u m e n m i c r o b e s to t h e s t a r c h g r a n u l e s that a r e e m b e d d e d wi th in t he pro te in mat r i x . R e s e a r c h is w a r r a n t e d in o r d e r to fu r the r i m p r o v e t he d igest ib i l i t y (Hun t et a l . , 1 9 9 5 ) a n d s u b s e q u e n t l y t he d i g e s t i b l e e n e r g y c o n t e n t o f ba r l ey . T h e A l b e r t a B a r l e y C o m m i s s i o n ( 1 9 9 5 ) repo r t s tha t fo r e v e r y 1 % i n c r e a s e in i m p r o v e m e n t in t h e f e e d e f f i c i e n c y o f b a r l e y , t h e l i v e s t o c k i ndus t r y wi l l a c c r u e 10 mi l l ion d o l l a r s in b e n e f i t s . T h e r e f o r e , t h e o b j e c t i v e o f th is s t u d y w a s to i nves t i ga te t h e feas ib i l i t y o f u s i n g e x o g e n o u s e n z y m e s a p p l i e d in c o n j u n c t i o n w i th mi ld h y d r o t h e r m a l t r e a t m e n t in o r d e r to i n c r e a s e t he nutr i t ive v a l u e o f b a r l e y f ed to da i ry ca t t le . 2.3 MATERIALS AND METHODS 2.3.1 Feed Treatments T h e e x p e r i m e n t c o n s i s t e d o f a c o m p l e t e l y r a n d o m i z e d 4 x 4 x 4 x 2 x 2 mul t i -f ac to r ia l d e s i g n to e v a l u a t e t h e e f f ec t s o f e n z y m e s a n d mi ld h y d r o t h e r m a l t r ea tmen t o n b a r l e y g r a i n u s i n g r u m e n in vitro d r y ma t te r d i s a p p e a r a n c e ( I V D M D ) a n d r u m e n in vitro t rue d iges t ib i l i t y ( I V T D ) . B a r l e y f e e d t r e a t m e n t s u n d e r i n ves t i ga t i on w e r e the a p p l i c a t i o n o f p - g l u c a n a s e (Trichoderma sp) at 0 % , 0 . 0 0 1 % , 0 . 0 1 % o r 0 . 1 % , p r o t e a s e (Bacillus sp.) at 0 % o r 0 . 0 0 2 5 % , x y l a n a s e (Trichoderma sp.) at 0 % o r 0 . 0 0 1 % , t h e a d d i t i o n o f m o i s t u r e a t 0 % , 1 0 % , 2 0 % o r 3 0 % a n d t h e a p p l i c a t i o n o f h e a t a t 5 5 ° C fo r 0 , 1 5 , 3 0 o r 6 0 m i n p e r i o d s ( T a b l e 2 .1 ) . T r e a t m e n t l e v e l s w e r e 51 pr imar i l y b a s e d o n t h e r e c o m m e n d a t i o n s o f t he e n z y m e s u p p l i e r , F i n n F e e d s Intl. (Wi l t sh i re , U . K ) . E n z y m e s w e r e not p u r e c u l t u r e s a n d s u b s t r a t e act iv i ty o f t he e n z y m e s m a y b e f o u n d in A p p e n d i x 6 . 1 . 2.3.2 Processing Samples B a r l e y g r a i n , g r o u n d t h r o u g h a 3 m m s c r e e n w a s o b t a i n e d f r o m P r o F o r m F e e d s Inc. l o c a t e d at C h i l l i w a c k , B . C . F e e d s a m p l e s w e r e w e i g h e d a n d p l a c e d in a H o b a r t m i x e r ( m o d e l A - 2 0 0 , H o b a r t M f g . C o . L t d . , D o n M i l l s , O n t . , C a n a d a ) at w h i c h t ime m o i s t u r e a n d / o r e n z y m e s w e r e a d d e d d u r i n g m i x i n g a s r e q u i r e d for e a c h t r ea tmen t . M o i s t u r e w a s a p p l i e d w i th a m a n u a l s p r a y e r in o r d e r to fac i l i ta te e q u a l d is t r ibu t ion o v e r t h e en t i re f e e d s a m p l e . E n z y m e s w e r e d i l u ted in 5 m L of d is t i l led w a t e r a n d a p p l i e d (% v o l / wt ) v i a s y r i n g e to t h e f e e d . T r e a t m e n t s w e r e t h o r o u g h l y m i x e d fo r a n a d d i t i o n a l 5 m in at w h i c h t i m e a 4 0 0 g s a m p l e o f t h e f e e d w a s p l a c e d w i th in a 4 L g l a s s v e s s e l a n d s e a l e d for fu r ther p r o c e s s i n g in a s i m u l a t e d h y d r o t h e r m a l c o o k e r . A l a b o r a t o r y i n c u b a t o r w a s a d a p t e d to s i m u l a t e a h y d r o t h e r m a l c o o k e r by f i t t ing ro l le rs o n w h i c h t h e 4 L s e a l e d g l a s s v e s s e l s c o n t a i n i n g t he t r ea ted mate r ia l w e r e p l a c e d ( F i g u r e 2 .1 ) . T h e ro ta t ion o f t he v e s s e l s w a s s e t at 15 r p m to s i m u l a t e c o m m e r c i a l h y d r o t h e r m a l c o n d i t i o n s . ( P r o F o r m F e e d s Inc., p e r s o n a l c o m m u n i c a t i o n s ) . T h e i n c u b a t o r a n d v e s s e l s w e r e bo th p r e h e a t e d a n d m a i n t a i n e d at 5 7 ° C ± 2 ° C . F e e d s a m p l e s t r ea ted w i th 2 0 % o r 3 0 % m o i s t u r e l e v e l s t ook 5 2 a p p r o x i m a t e l y 7 m i n to a t ta in 5 5 ° C , w h e r e a s al l o t h e r s r e q u i r e d a p p r o x i m a t e l y 5 m i n . F o l l o w i n g r e m o v a l o f t he s a m p l e s f r o m the c o o k e r , i nd i v i dua l t r e a t m e n t s w e r e s p r e a d ou t (2 c m d e p t h ) o n l a r g e t r a y s a n d a i r d r i e d at a m b i e n t t e m p e r a t u r e for 3 h. D r y i n g w a s fac i l i t a ted w i th a l a r g e f a n w h i c h s i m u l a t e d t he f o r c e d a i r d r y e r s u s e d by f e e d m a n u f a c t u r i n g fac i l i t i es . S a m p l e s w e r e t h e n s e a l e d in p l as t i c b a g s a n d s t o r e d in a c h e s t f r e e z e r a t - 1 0 ° C fo r s u b s e q u e n t l abo ra to r y a n a l y s e s . 2.3.3 Chemical Analyses A r e p r e s e n t a t i v e s a m p l e f r o m e a c h f e e d t r e a t m e n t w a s c o l l e c t e d a n d g r o u n d t h r o u g h a 1 m m s c r e e n fo r I V D M D a n d I V T D u s i n g t he A n k o m in vitro s y s t e m ( D a i s y 1 0 0 , A n k o m c o . , Fa i rpo r t , N Y ) a n d for c h e m i c a l a n a l y s e s . A c i d d e t e r g e n t f i be r ( A D F ) a n d neu t ra l d e t e r g e n t f i be r ( N D F ) w e r e d e t e r m i n e d o n al l s a m p l e s , in d u p l i c a t e , u s i n g t h e filter b a g t e c h n i q u e ( K o m a r e k e t a l . , 1 9 9 4 ) a n d t h e A n k o m f i be r a n a l y z e r ( # F 2 0 0 , A n k o m C o . , Fa i rpo r t , N Y ) . F i l te r b a g s w e r e o f s y n t h e t i c n o n w o v e n fi l ter ma te r i a l w i th a p o r e s i z e o f 4 0 m i c r o n s (#F57 , A n k o m C o . , Fa i rpo r t , N Y ) . T h e N D F p r o c e d u r e i n c l u d e d t he u s e o f a h e a t s t a b l e a l p h a - a m y l a s e ( # F A A , A n k o m C o . , Fa i rpo r t , N Y ) a s s u g g e s t e d by V a n S o e s t et a l . , ( 1 9 9 1 ) fo r u s e in f e e d s c o n t a i n i n g h igh a m o u n t s o f s t a r c h . R e s i d u a l d ry ma t te r ( D M ) d e t e r m i n a t i o n s w e r e c o n d u c t e d by d r y i ng a r e p r e s e n t a t i v e 1 g s a m p l e at 1 0 0 ° C in a f o r c e d a i r o v e n unti l c o n s t a n t w e i g h t w a s a c h i e v e d . 5 3 2.3.4 Animals and Feeding T w o H o l s t e i n c o w s f i t ted wi th r u m i n a l c a n n u l a w e r e u s e d a s d o n o r a n i m a l s for r u m e n f lu id . T h e c o w s w e r e f e d a 6 0 % g r a s s h a y a n d 4 0 % g ra i n d ie t ( A p p e n d i x 6 .2) t w i c e da i l y a t 0 7 : 3 0 h a n d 1 6 : 0 0 h . T h e a n i m a l s w e r e h o u s e d in f r ee s ta l l s for the du ra t i on o f t h e e x p e r i m e n t . A n i m a l s w e r e c a r e d fo r a c c o r d i n g to t he g u i d e l i n e s se t ; for th b y t h e C a n a d i a n C o u n c i l o f A n i m a l C a r e ( 1993 ) . 2.3.5 Rumen Fluid Inoculum R u m e n f lu id w a s c o l l e c t e d 3 h pos t f e e d i n g f r o m bo th a n i m a l s a n d the c o l l e c t i o n s p o o l e d . R u m e n f lu id a n d s o l i d s f r o m the f i b r o u s m a t w e r e c o l l e c t e d into a p r e w a r m e d a n d air t ight c o n t a i n e r fo r t r anspo r t to t he l a b o r a t o r y w h i l e m a i n t a i n i n g a n a e r o b i c c o n d i t i o n s a s m u c h a s p o s s i b l e . A l l e q u i p m e n t w a s p r e w a r m e d to 3 9 ° C . R u m e n f lu id w a s b l e n d e d in a W a r i n g b l e n d e r at m a x i m u m s p e e d for 2 , 3 0 s e c p e r i o d s ( F a y et a l . , 1 9 8 0 ) . T h e h o m o g e n a t e w a s t h e n f i l te red t h r o u g h 5-8 m m o f g l a s s w o o l a n d 4 l a y e r s o f c h e e s e c lo th a n d t he f i l t rate u s e d a s i n o c u l u m w h e n m i x e d w i th m i n e r a l a n d bu f fe r s o l u t i o n s ( G o e r i n g a n d V a n S o e s t , 1 9 7 0 ) . 5 4 2.3.6 In Vitro Procedure I V D M D w a s d e t e r m i n e d a c c o r d i n g to t he f irst s t e p o f t he T i l l ey a n d T e r r y ( 1 9 6 3 ) r u m e n in vitro d i g e s t i o n p r o c e d u r e . A n in-vitro a p p a r a t u s ( I V 1 0 0 , D a i s y II, A n k o m C o . , Fa i rpo r t , N Y ) w a s u s e d to d e t e r m i n e I V D M D o f t he s a m p l e s ( A n k o m C o . , Fa i r po r t , N Y ; T i l l ey a n d T e r r y , 1 9 6 3 ) . T h e s y s t e m w a s m a i n t a i n e d at 3 9 . 5 ° C a n d c o n s i s t e d o f f o u r 4 L r e a c t i o n v e s s e l s w h i c h ro ta ted at 1 r p m to s i m u l a t e r u m e n c o n d i t i o n s . Q u a d r u p l i c a t e 0 . 5 g s a m p l e s w e r e w e i g h e d ou t into f i l ter b a g s (#F56 , A n k o m C o . , Fa i rpo r t , N Y ) a n d h e a t s e a l e d . S i x t e e n s a m p l e s , g r o u p e d into r e a c t i o n v e s s e l s a c c o r d i n g to e n z y m e t r ea tmen t , w e r e p l a c e d in e a c h run . A f t e r a 4 8 h i n c u b a t i o n , s a m p l e s w e r e r e m o v e d , w a s h e d in d is t i l led H 2 0 a n d d r i e d at 6 0 ° C unti l c o n s t a n t w e i g h t w a s a c h i e v e d . I V D M D w a s o b t a i n e d by d i f f e r e n c e in s a m p l e w e i g h t s . B a g a n d s a m p l e s w e r e t h e n s u b j e c t e d to N D F a n a l y s i s in o r d e r to o b t a i n t rue d igest ib i l i t y by d i f f e r e n c e f r o m the o r i g ina l s a m p l e w e i g h t ( G o e r i n g a n d V a n S o e s t , 1 9 7 0 ) . 2.3.7 Statistical Analysis Sta t i s t i ca l a n a l y s e s w e r e c o n d u c t e d u s i n g t he G e n e r a l L i n e a r M o d e l ( G L M ) p r o c e d u r e o f S ta t i s t i ca l A n a l y s i s S y s t e m ( S A S Inst i tute Inc., 1 9 9 0 ) u s i n g the p r o g r a m l i s ted in A p p e n d i x 6 . 3 , w h i c h a l s o i n c l u d e s t he m o d e l s t a t e m e n t . In the p r e s e n t s t u d y , it w a s a s s u m e d the m e a n s q u a r e e r ro r o f t he h i g h e s t o r d e r i n te rac t i on w a s r e p r e s e n t a t i v e o f t he e x p e r i m e n t a l e r ro r ( C o c h r a n a n d C o x , 1950 ) . 5 5 M e a n s e p a r a t i o n s w e r e o b t a i n e d u s i n g t h e l e a s t s q u a r e m e a n s fo r I V D M D , I V T D , A D F a n d N D F m e a s u r e m e n t s . 2.4 RESULTS AND DISCUSSION: D r y m a t t e r ( D M ) c o n t e n t o f t he u n t r e a t e d b a r l e y g r a i n s a m p l e w a s 8 7 . 3 % a n d t he l eve l o f c r u d e p ro te in ( C P ) w a s 1 2 . 7 % o f D M . A c i d d e t e r g e n t f i be r ( A D F ) w a s 7 . 3 2 % o f D M a n d neu t ra l d e t e r g e n t f i be r ( N D F ) w a s 2 2 . 8 % a s a p e r c e n t o f D M . T h e b a r l e y u s e d in th is e x p e r i m e n t h a d s i m i l a r nut r ient c o m p o s i t i o n , w i th t he e x c e p t i o n of N D F , to t h e v a l u e s c i t ed by N R C ( 1 9 8 9 ) for b a r l e y g ra i n w h i c h h a s a t yp i ca l D M c o n t e n t o f 8 8 % , C P c o n t e n t o f 1 3 . 5 % , A D F a n d N D F c o n t e n t o f 7 % a n d 1 9 % , r e s p e c t i v e l y . 2.4.1 IVDMD T h e r e w a s a s i gn i f i can t t h r e e w a y in te rac t i on ( P < 0 .05 ) n o t e d for du ra t i on of h e a t t r e a t m e n t , m o i s t u r e a n d x y l a n a s e a p p l i c a t i o n ( F i g u r e 2 .2 ) . H o w e v e r , n o t r e a t m e n t s i gn i f i can t l y i n c r e a s e d I V D M D ( P < 0 .05 ) w h e n c o m p a r e d to t he un t rea ted s a m p l e ( T a b l e 2 .2 ) . A l l t r e a t m e n t m e a n s w e r e e i t he r s i m i l a r o r l o w e r t h a n t h e I V D M D v a l u e o f 8 1 . 8 % fo r t h e con t ro l . A p r e v i o u s s t u d y repo r t ed a c o m p a r a b l e D M l o s s for b r o k e n b a r l e y f o l l ow ing a 4 8 h r u m i n a l n y l o n b a g i n c u b a t i o n o f 8 5 % (No rd in a n d C a m p l i n g , 1 9 7 6 ) . In t he p r e s e n t s t u d y , t he l o w e s t I V D M D v a l u e w a s 7 9 . 2 % w h i c h w a s o b t a i n e d w i th t he a p p l i c a t i o n o f 3 0 m i n o f h e a t at 5 5 ° C a n d 0 . 0 0 1 % x y l a n a s e . 5 6 F r o m T a b l e 2 . 2 , it is a p p a r e n t t he major i ty o f t r e a t m e n t c o m b i n a t i o n s d e c r e a s e d I V D M D re la t i ve to t h e c o n t r o l . R e s u l t s p r e s e n t e d in T a b l e 2 . 2 d o not s u p p o r t the s u g g e s t i o n tha t m o i s t u r e m a y p l a y a ro le in d e t e r m i n i n g t h e e f f i c a c y of e n z y m e s ( B e a u c h e m i n a n d R o d e , 1 9 9 6 ) . T h e resu l t s of t he p r e s e n t e x p e r i m e n t a r e dif f icult to in terpret d u e to t h e l a r g e n u m b e r o f i n t e rac t i ons p r e s e n t . F i g u r e 2 . 3 i l l us t ra tes t h e s i gn i f i can t i n te rac t i on ( P = 0 . 0 0 0 1 ) n o t e d fo r t h e a d d i t i o n o f p - g l u c a n a s e , p r o t e a s e a n d x y l a n a s e . T h e add i t i on o f x y l a n a s e d e c r e a s e d I V D M D w i th t h e e x c e p t i o n o f t he c o m b i n a t i o n o f bo th 0 . 0 0 2 5 % p r o t e a s e a n d 0 . 0 0 1 % x y l a n a s e w h e r e I V D M D i n c r e a s e d f r o m 8 2 . 4 % to 8 2 . 9 % . T h e add i t i on of p-g l u c a n a s e a l o n e d i d not s i gn i f i can t l y ( P < 0 .05 ) a f fec t I V D M D resu l t s . T h e add i t i on of P - g l u c a n a s e in c o m b i n a t i o n w i th p r o t e a s e s ign i f i can t l y i n c r e a s e d I V D M D re la t ive to t he u n t r e a t e d s a m p l e ( P < 0 .05 ) . T h e add i t i on of 0 . 1 % p - g l u c a n a s e a n d 0 . 0 0 2 5 % p r o t e a s e i n c r e a s e d I V D M D f r o m 8 2 . 4 0 % to 8 4 . 4 2 % ( T a b l e 2 .3 ) . W i t h i n e a c h leve l o f P - g l u c a n a s e a p p l i c a t i o n h o w e v e r , t he a d d i t i o n of p r o t e a s e d i d not fu r ther i n c r e a s e I V D M D ( P < 0 .05 ) . T h e a d d i t i o n o f p r o t e a s e a l o n e a l s o d i d not h a v e a pos i t i ve e f fec t o n I V D M D . T h e a d d i t i o n o f p r o t e a s e h a s b e e n p r e v i o u s l y s h o w n to i n c r e a s e t h e d e g r a d a b i l i t y o f b a r l e y s t a r c h f o l l ow ing a 2 4 h i n c u b a t i o n ( M c A l l i s t e r et a l . , 1993 ) . T h e s e r esu l t s l ed t h e a u t h o r s to s p e c u l a t e that s t ruc tu ra l c a r b o h y d r a t e s m a y a l s o p l ay a ro le in t h e l im i ted d i g e s t i o n o f b a r l e y b y r u m e n m i c r o b e s . S t a r c h g r a n u l e s a r e s t o r a g e p o l y s a c c h a r i d e s l o c k e d w i th in a p ro te in matr ix ( R o o n e y a n d P f l u g l d e r , 1 9 8 6 ) . M c A l l i s t e r et a l . , ( 1 9 9 3 ) d i s c o v e r e d t h r o u g h t he u s e of 5 7 e l e c t r o n m i c r o s c o p y that a l t h o u g h g r i nd i ng c e r e a l g r a i n s e x p o s e d t he e n d o s p e r m to e n z y m a t i c a t t ack , t h e s t a r c h g r a n u l e s r e m a i n e d e m b e d d e d w i th in t he p ro te in matr ix . T h e d i f f e r e n c e in t h e d e g r a d a b i l i t y o f c e r e a l s m a y b e par t ly a t t r ibu ted to the p r e s e n c e o f a p ro te in mat r i x w h i c h s u r r o u n d s t he s t a r c h g r a n u l e s ( M c A l l i s t e r et a l . , 1 9 9 0 a , b, c ) . S t a r c h s t ruc tu re a n d c o m p o s i t i o n , in a d d i t i o n to it 's i n te rac t ion wi th p ro te in , is impo r tan t in d e t e r m i n i n g it 's d igest ib i l i t y . T h e e f f i c i e n c y o f m i c r o b i a l c o l o n i z a t i o n h a s a l s o b e e n s u g g e s t e d a s a f ac to r in d e t e r m i n i n g t he rate a n d ex ten t o f d e g r a d a t i o n in t h e r u m e n ( M c A l l i s t e r , 1 9 9 0 b ) . E n h a n c i n g t he m ic rob ia l c o l o n i z a t i o n o f f e e d pa r t i c l es h a s b e e n s u g g e s t e d a s a m e a n s o f i m p r o v i n g f iber d iges t ib i l i t y in t h e r u m e n ( C h e n g et a l . , 1 9 9 5 ) . U n f o r t u n a t e l y , th is w a s not m e a s u r e d in t he p r e s e n t e x p e r i m e n t . W a l d o ( 1 9 7 3 ) r e p o r t e d that b a r l e y s t a r c h w a s 9 4 % rum ina l l y d e g r a d a b l e . T h i s v a l u e is h i g h e r t h a n that r e p o r t e d b y H e r r e r a - S a l d a n a et a l . ( 1 9 9 0 ) , w h o f o u n d that b a r l e y s t a r c h w a s 9 1 % r u m e n d e g r a d a b l e . T h e s e s a m e a u t h o r s d e t e r m i n e d that b a r l e y g r a i n w a s 7 8 % rum ina l l y d e g r a d a b l e , a s s u m i n g a 6 % h 1 ra te o f p a s s a g e , . T h i s la t ter v a l u e is c o m p a r a b l e to t h e 8 1 % I V D M D v a l u e o b t a i n e d in t he p r e s e n t s t u d y f o l l o w i n g a 4 8 h r u m e n in vitro i n c u b a t i o n . A l t h o u g h b a r l e y is c o n s i d e r e d to b e i n t e r m e d i a t e l y r a n k e d w i th r e g a r d s to s t a r c h a n d D M d e g r a d a b i l i t i e s w h e n c o m p a r e d to o t h e r c e r e a l g r a i n s s u c h a s o a t s , w h e a t , c o r n a n d m i lo ; d iges t ib i l i t y e x p e r i m e n t s o f ten d o no t r e c o g n i z e t h e p o o r d iges t ib i l i t y o f t he hul l f r ac t i on ( H e r r e r a - S a l d a n a et a l . , 1 9 9 0 ) . F o r e x a m p l e , o n l y 5 0 . 4 % o f t h e b a r l e y hul l d i s a p p e a r e d f o l l ow ing a 10 h i n c u b a t i o n in t h e r u m e n ( A r o n e n et a l . , 1 9 9 1 ) . In f e e d i n g a h igh p r o d u c i n g da i r y c o w 5 8 it is impo r tan t to c o n s i d e r t h e f a s t e r ra te o f r u m e n p a s s a g e w h i c h m a y n e g a t i v e l y i n f l u e n c e d iges t ib i l i t y . A s p r e v i o u s l y m e n t i o n e d , r esu l t s f r o m the t h r e e w a y i n t e rac t i ons a r e dif f icult to in terpret . A l t h o u g h s ign i f i can t l y l o w e r ( P < 0 .05 ) I V D M D v a l u e s w e r e o b t a i n e d wi th a va r i e t y o f e n z y m e c o m b i n a t i o n s , a c l e a r pa t te rn is l a c k i n g . T h e g r e a t e s t d e c r e a s e in I V D M D w a s o b t a i n e d w i th t h e c o m b i n a t i o n o f 0 . 0 0 1 % p - g l u c a n a s e a n d 0 . 0 0 1 % x y l a n a s e w h i c h r e s u l t e d in a 6 . 5 d iges t ib i l i t y uni t d e c r e a s e in I V D M D w h e n c o m p a r e d to t h e c o n t r o l , p - g l u c a n a s e a p p l i e d at 0 . 0 1 % a l o n e resu l t ed in a s i gn i f i can t d e c r e a s e o f 5 .0 d iges t ib i l i t y un i ts in I V D M D f r o m 8 2 . 4 % to 7 7 . 4 % ; w h e r e a s t he l o w e s t a n d h i g h e s t l eve l o f a p p l i c a t i o n o f p - g l u c a n a s e d id not a f fect I V D M D ( P < 0 . 0 5 ) ( T a b l e 2 .3 ) . A l t h o u g h t h e resu l t s o f th is s t u d y a r e not a s de f in i t i ve a s t he resu l t s of p r e v i o u s s t u d i e s , it a p p e a r s t h e r e m a y b e a n o p t i m a l l e v e l o f p r o t e a s e a p p l i c a t i o n o r ye t u n d e f i n e d c o m b i n a t i o n o f p - g l u c a n a s e a n d p r o t e a s e to b a r l e y g ra i n w h e r e t h e r e s h o u l d b e a r e s p o n s e in t e r m s o f d igest ib i l i t y . P a s t r e s e a r c h h a s s h o w n that n e g a t i v e r e s p o n s e s a r e p o s s i b l e if t h e e n z y m e l eve l is s u b - o p t i m a l ( K r a u s e et a l . , 1 9 9 6 c i t ed b y B e a u c h e m i n a n d R o d e , 1 9 9 6 ) . In a s t u d y c o n d u c t e d w i th s t e e r s f ed a n e n z y m e s u p p l e m e n t e d b a r l e y ra t ion , a q u a d r a t i c r e s p o n s e w a s n o t e d in t e r m s of A D G ( R o d e a n d B e a u c h e m i n , u n p u b l i s h e d d a t a ; c i ted by B e a u c h e m i n a n d R o d e , 1 9 9 6 ) . A s e c o n d s t u d y f o u n d a l i n e a r r e s p o n s e in A D G f r o m s t e e r s f e d a t e m p e r e d ro l led b a r l e y g r a i n d ie t s u p p l e m e n t e d w i th x y l a n a s e ( I w a a s a et a l . , u n p u b l i s h e d 5 9 d a t a ; c i t ed b y B e a u c h e m i n a n d R o d e , 1 9 9 6 ) . T h i s m a y a l s o b e t rue in t he p r e s e n t s t u d y w h e r e s o m e e n z y m e t r e a t m e n t s d e c r e a s e d t h e r u m e n in vitro d e g r a d a b i l i t y . W h e n K r a u s e et a l . , ( 1 9 9 6 ) (c i ted by B e a u c h e m i n a n d R o d e , 1 9 9 6 ) i n v e s t i g a t e d t h e in situ d e g r a d a b i l i t y o f b a r l e y in s t e e r s f e d a n e n z y m e s u p p l e m e n t e d b a r l e y s t r a w o r s i l a g e d ie t t he a u t h o r s r e p o r t e d i n c r e a s e d d e g r a d a b i l i t y o f b a r l e y g r a i n o n l y w i th t he s i l a g e b a s e d d i e t s . T h i s l ed to s p e c u l a t i o n that e n z y m e - s u b s t r a t e b i n d i n g m a y p l a y a ro le in i m p r o v i n g t h e o b s e r v e d d igest ib i l i t y in t he s i l a g e d i e t s ( B e a u c h e m i n et a l . , 1 9 9 6 ) . E n z y m e s u b s t r a t e b i n d i n g m a y a l s o p l a y a ro le in t h e c u r r e n t s t u d y w h e r e r e s u l t s a r e i n c o n s i s t e n t a n d t r e n d s c a n n o t b e e s t a b l i s h e d . A c o m b i n a t i o n o f a n u m b e r o f f a c t o r s m a y p r o v i d e a n e n v i r o n m e n t c o n d u c i v e o r d e t r i m e n t a l to e n z y m e a c t i o n . 2.4.2 IVTD A q u a d r a t i c r e l a t i o n s h i p ( P < 0 .01 ) w a s f o u n d b e t w e e n t he a p p l i c a t i o n o f m o i s t u r e a n d t h e I V T D o f b a r l e y g r a i n d e s c r i b e d by t h e fo l l ow ing e q u a t i o n : Y = 8 7 . 7 5 + 0 .071 * m o i s t u r e l eve l - 0 . 0 0 1 3 6 * m o i s t u r e l e v e l 2 ( F i g u r e 2 .4 ) . M e a n s f r om t r e a t m e n t w i th 2 0 % a n d 3 0 % m o i s t u r e w e r e s ign i f i can t l y h i g h e r ( P < 0 .01 ) t h a n the con t ro l but w e r e not d i f fe rent f r o m e a c h o t h e r ( T a b l e 2 .4 ) . T h e o p t i m a l l eve l o f m o i s t u r e a p p l i c a t i o n in o r d e r to m a x i m i z e I V T D , d e r i v e d f r o m t he r e g r e s s i o n e q u a t i o n w a s d e t e r m i n e d to b e 2 6 % wi th a resu l t i ng I V T D o f 8 8 . 7 % ; a s m a l l i n c r e a s e o f 0 . 9 % re la t i ve to t he con t ro l . M o i s t u r e t r e a t m e n t o f c e r e a l g r a i n s h a s g e n e r a l l y not p r o d u c e d a g r e a t r e s p o n s e w h e n a p p l i e d in t he a b s e n c e o f hea t (Tai t 6 0 a n d B e a m e s , 1 9 8 8 ) . H o w e v e r , s o a k i n g c e r e a l h a s b e e n s u g g e s t e d to r e l e a s e e n d o g e n o u s e n z y m e s c o n t a i n e d w i th in t he g ra i n ( N e w m a n , 1 9 9 4 ) . A s p r e v i o u s l y m e n t i o n e d , m o i s t u r e m a y a l s o h a v e a n a f fec t o n e n z y m e act iv i ty ( B e a u c h e m i n a n d R o d e , 1 9 9 6 ) . T h e r e w a s a s i gn i f i can t t h r e e w a y in te rac t ion ( P = 0 . 0 0 0 1 ) n o t e d a m o n g the add i t i on o f t h e e n z y m e s : p - g l u c a n a s e , p r o t e a s e a n d x y l a n a s e ( F i g u r e 2 .5) . T h e s e resu l t s a r e s i m i l a r to t h o s e o b t a i n e d w i th I V D M D d e t e r m i n a t i o n s . N o n e of t he e n z y m e c o m b i n a t i o n s s ign i f i can t l y i n c r e a s e d ( P < 0 . 0 5 ) t h e I V T D o f b a r l e y g ra in f r o m t h e 8 9 . 4 % I V T D v a l u e o b t a i n e d fo r t h e con t ro l ( T a b l e 2 .3 ) . I V T D d id not ref lect a pos i t i ve r e s p o n s e o f b a r l e y g r a i n to t h e add i t i on of e i t he r x y l a n a s e o r p r o t e a s e . T r e a t m e n t o f b a r l e y g ra i n w i th x y l a n a s e e i t he r d e c r e a s e d o r h a d n o e f fec t o n I V T D re la t i ve to t h e c o n t r o l . T h e l o w e s t I V T D v a l u e ( 8 6 . 8 % ) w a s o b t a i n e d w h e n ba r l ey g ra in w a s t r e a t e d w i th t he c o m b i n a t i o n o f 0 . 1 % p - g l u c a n a s e , 0 . 0 0 2 5 % p r o t e a s e a n d 0 . 0 0 1 % x y l a n a s e . T h i s s a m e t r e a t m e n t a l s o r e s u l t e d in o n e o f t h e l o w e s t I V D M D v a l u e s . P o s i t i v e r esu l t s p r e v i o u s l y o b s e r v e d w i th t he a d d i t i o n o f x y l a n a s e to ba r l ey w e r e m a i n l y a t t r i bu tab le to it 's h i g h f i be r c o n t e n t ( B e a u c h e m i n a n d R o d e , 1 9 9 6 ) . H r i s t o v e t a l . ( u n p u b l i s h e d d a t a ; c i t ed by B e a u c h e m i n a n d R o d e , 1 9 9 6 ) c o m p a r e d t he a p p l i c a t i o n o f ce l l u lo l y t i c a n d x y l a n o l y t i c e n z y m e s to b a r l e y a n d c o r n a n d f o u n d that r e d u c i n g s u g a r s w e r e o n l y r e l e a s e d in ba r l ey . T h i s s u g g e s t s x y l a n a s e is a n e f f ec t i ve e n z y m e for u s e w i th ba r l ey . F u r t h e r i nves t i ga t i on into w h e t h e r f ib ro ly t ic e n z y m e t r e a t m e n t o f a b a r l e y o r c o r n b a s e d ra t ion i n c r e a s e d the d i g e s t i b l e e n e r g y c o n t e n t o f t he f e e d w a s c o n d u c t e d b y B e a u c h e m i n a n d R o d e 61 ( 1 9 9 6 ) . T h e s e a u t h o r s f o u n d that a g a i n o n l y b a r l e y r e s p o n d e d to t he e n z y m e s u p p l e m e n t a t i o n . T h e a p p l i c a t i o n o f h i gh l e v e l s o f x y l a n a s e i n c r e a s e d D M I , A D G a n d c o n s e q u e n t l y t h e f e e d c o n v e r s i o n rat io ( F C R ) o f s t e e r s f e d a h igh b a r l e y d iet . T h e p o s i t i v e r e s p o n s e w a s s u g g e s t e d to o c c u r a s a resu l t o f i m p r o v e d total t ract d iges t ib i l i t y o f b a r l e y . T h e l a c k o f r e s p o n s e o f c o r n w a s a t t r ibu ted to its l o w e r f iber c o n t e n t . In g e n e r a l , I V T D v a l u e s w e r e l e s s r e s p o n s i v e to e n z y m e t r e a t m e n t t h a n the I V D M D v a l u e s . T h i s is re f l ec ted b y f e w e r d i f f e r e n c e s b e t w e e n m e a n s in add i t i on to t he r e d u c e d s p r e a d o f m e a n s a n d l o w e r p o o l e d s t a n d a r d e r ro r in t h e I V T D resu l t s . T h e p o o l e d s t a n d a r d e r ro r ( S E p ) for t h e I V D M D m e a n s fo r t he in te rac t i on b e t w e e n P - g l u c a n a s e , p r o t e a s e a n d x y l a n a s e w a s 0 . 4 0 , w h e r e a s t he S E p for t rue d igest ib i l i ty w a s 0 . 2 6 . T h e s a m e th i rd o r d e r i n te rac t i on b e t w e e n e n z y m e s w a s d e t e c t e d in the e v a l u a t i o n o f I V T D , h o w e v e r , t he s p r e a d o f m e a n s w a s r e d u c e d f r o m 8 .3 p e r c e n t a g e un i ts w i th I V D M D a n d a r e d u c t i o n o f 3 .0 p e r c e n t a g e un i ts wi th I V T D . T h e l a rge s p r e a d in v a l u e s o b t a i n e d w i th I V D M D resu l t s m a y re f lec t m i c rob ia l c o n t a m i n a t i o n o f t h e s a m p l e s . D i f f e r e n c e s that a r e p r e s e n t at th is s t a g e o f the d i g e s t i o n p r o c e s s m a y no t b e d e t e c t e d w i th a s u b s e q u e n t I V T D d e t e r m i n a t i o n b e c a u s e th is la t ter p r o c e d u r e e l i m i n a t e s al l ce l l s o l u b l e s i n c l u d i n g al l u n d i g e s t e d m i c r o b i a l ce l l c o m p o n e n t s ( V a n S o e s t , 1 9 8 2 ) . T h e resu l t s o f t h e p r e s e n t e x p e r i m e n t s u g g e s t tha t I V T D m a y b e a bet ter i nd i ca t i on o f t h e e f fec t o f e n z y m e a c t i o n a s I V D M D resu l t s m a y b e m a s k e d by o the r c o m p o u n d i n g f a c t o r s . C h e n g et a l . , ( 1 9 9 5 ) i n d i c a t e d that d i e ta r y p - g l u c a n a s e 6 2 s u p p l e m e n t a t i o n w o u l d bene f i t d i g e s t i o n b y d e g r a d i n g t he ce l l w a l l c o m p o n e n t s of ba r l ey . P r e v i o u s r e s e a r c h h a s s h o w n that t he e n d o s p e r m w a l l s a r e d e g r a d e d by p-g l u c a n a s e h o w e v e r , d u e to it 's c o m p l e x n a t u r e , a m u l t i - e n z y m e p r e p a r a t i o n is m o r e e f fec t i ve ( C h e s s o n , 1 9 9 3 ) . T h i s e x p l a n a t i o n h o w e v e r , m a y b e o f f e red for I V D M D a n d I V T D v a l u e s fo r o n l y a s e l e c t i o n o f e n z y m e t r e a t m e n t s , m o s t no tab l y t h o s e in c o m b i n a t i o n w i th 0 . 0 1 % p - g l u c a n a s e . 2.3.3 ADF T h e r e w a s a s i gn i f i can t i n te rac t i on ( P < 0 .05 ) s h o w n in F i g u r e 2 .6 , no ted for P - g l u c a n a s e , p r o t e a s e a n d x y l a n a s e w h i c h w a s a l s o p r e v i o u s l y d e t e r m i n e d to ex i s t w i th I V D M D a n d I V T D d e t e r m i n a t i o n s . T a b l e 2 .5 s h o w s that n o n e o f t he t r e a t m e n t s s ign i f i can t l y d e c r e a s e d t h e A D F f rac t i on o f b a r l e y ( P < 0 .05 ) . T h e A D F c o n t e n t of t he con t ro l s a m p l e w a s 8 . 2 % o n a D M b a s i s . A s ign i f i can t i n c r e a s e ( P < 0 .05 ) in A D F w a s f o u n d w i th a n u m b e r o f t r e a t m e n t s . T h e a p p l i c a t i o n o f x y l a n a s e e i the r h a d n o e f fec t o n t h e A D F c o n t e n t o r i n c r e a s e d it 's c o n t e n t re la t i ve to t he con t ro l . T h e l a rges t i n c r e a s e in A D F c o n t e n t (to 9 .3 p e r c e n t a g e un i ts ) w a s o b t a i n e d w i th t he app l i ca t i on o f 0 . 0 1 % p - g l u c a n a s e a n d p a r a l l e l s t he resu l t s o f a l o w I V D M D v a l u e o b s e r v e d for t he s a m e t r e a t m e n t . M a n y o f t he t r e a t m e n t s w i th h igh A D F v a l u e s h a v e d e c r e a s e d I V D M D a n d I V T D . A shi f t t o w a r d s m o r e s o l u b l e c o m p o n e n t s in t he f e e d s u b j e c t e d to pa r t i cu la r t r e a t m e n t s , w o u l d resu l t in h i g h e r A D F v a l u e s . F o r e x a m p l e , a n i n c r e a s e in t he p ropo r t i on o f t h e s o l u b l e s t a r c h o r o t h e r f e e d c o m p o n e n t s w o u l d b e re f lec ted 6 3 in i n c r e a s e d A D F v a l u e s . T h e e n z y m e h y d r o l y s i s o f h i gh m o l e c u l a r w e i g h t ma te r i a l s u s u a l l y r esu l t s in a n i n c r e a s e in t he so lub i l i t y o f s u c h c o m p o n e n t s ( W i s e m a n , 1975 ) . A s i gn i f i can t i n te rac t i on o n A D F c o n t e n t ( P < 0 .05 ) w a s a l s o n o t e d for: du ra t i on o f h e a t t r e a t m e n t , p - g l u c a n a s e a n d p r o t e a s e ( F i g u r e 2 .7 ) . It is o f in te res t that th is t e n d e d to o c c u r in s a m p l e s w h i c h h a d h i g h e r A D F v a l u e s c o m p a r e d to t he con t ro l ( T a b l e 2 .6 ) . T h e u n t r e a t e d s a m p l e w a s not s i gn i f i can t l y d i f ferent f r o m the s a m p l e w i th t h e l o w e s t A D F c o n t e n t ( 8 . 1 % o f D M ) o b t a i n e d b y t rea t ing b a r l e y wi th h e a t fo r 3 0 m i n a n d a p p l y i n g 0 . 0 0 1 % p - g l u c a n a s e . T h e l a r g e s t i n c r e a s e in A D F c o n t e n t w a s f o u n d w h e n b a r l e y w a s t r e a t e d w i th h e a t f o r 1 5 m i n , 0 . 1 % p - g l u c a n a s e a n d 0 . 0 0 2 5 % p r o t e a s e . It is o f pa r t i cu l a r in te res t to n o t e that t he in te rac t ion b e t w e e n t he 3 e n z y m e s w a s d e t e c t e d in I V D M D , I V T D a n d A D F m e a s u r e m e n t s . In g e n e r a l , t he A D F v a l u e s i n c r e a s e d a s d iges t ib i l i t y m e a s u r e m e n t s d e c r e a s e d . T h i s is in a g r e e m e n t wi th t he n e g a t i v e a s s o c i a t i o n o f f i be r w i th d iges t ib i l i t y ( V a n S o e s t , 1 9 8 2 ) . It h a s a l s o b e e n r e p o r t e d that e n z y m e s m a y not a f fec t t h e A D F f rac t ion but m a y el ic i t the i r e f fec ts by a l te r ing t h e h e m i c e l l u l o s e f r ac t i on , w h i c h w o u l d b e re f l ec ted in t he N D F r e s i d u e ( G w a y u m b a et a l . , 1 9 9 5 ) . 2.4.4 NDF A s i gn i f i can t i n te rac t i on w a s f o u n d to ex i s t ( P < 0 .01 ) b e t w e e n h e e n z y m e s p-g l u c a n a s e a n d p r o t e a s e ( F i g u r e 2 .8 ) . W i t h i n th is i n te rac t i on , N D F c o n t e n t of the con t ro l w a s 2 1 . 8 % o f D M , w h i c h w a s t he l o w e s t o f al l t r e a t m e n t m e a n s . T h e f o l l ow ing t h r e e t r e a t m e n t c o m b i n a t i o n s s ign i f i can t l y i n c r e a s e d ( P < 0 . 0 5 ) N D F ( T a b l e 6 4 2.7) : 0 . 0 0 2 5 % p r o t e a s e ; 0 . 0 0 1 % p - g l u c a n a s e a n d 0 . 0 0 2 5 % p r o t e a s e ; a n d 0 . 0 1 % p-g l u c a n a s e . A n o t h e r s i gn i f i can t s e c o n d o r d e r i n te rac t i on ( P < 0 .01 ) w a s f o u n d b e t w e e n p-g l u c a n a s e a n d x y l a n a s e ( F i g u r e 2 .9 ) . T r e a t m e n t m e a n s a r e p r e s e n t e d in T a b l e 2 .8 . T h e a d d i t i o n o f 0 . 0 0 1 % P - g l u c a n a s e s ign i f i can t l y i n c r e a s e d ( P < 0 .05 ) N D F f r om 2 3 . 0 % to 2 4 . 5 % a s a p e r c e n t a g e o f D M . In con t ras t , t he a p p l i c a t i o n o f t he h i g h e s t c o n c e n t r a t i o n o f p - g l u c a n a s e s ign i f i can t l y d e c r e a s e d ( P < 0 .05 ) N D F f r o m 2 3 . 0 % to 2 1 . 8 % . T h e s e resu l t s d o not pa ra l l e l t h o s e o f I V D M D w h e r e it w a s d e t e r m i n e d that t he a d d i t i o n o f bo th t h e l o w e s t a n d h i g h e s t l e v e l s o f p - g l u c a n a s e d id not s ign i f i can t l y a f fec t I V D M D ( P < 0 .05 ) . T h e o b s e r v e d d e c r e a s e in N D F c o n t e n t wi th t he a p p l i c a t i o n o f 0 . 1 % p - g l u c a n a s e m a y b e e x p l a i n e d b y t he b r e a k d o w n o f ce l l wa l l c o m p o n e n t s w h i c h m a y con t r i bu te to t h e N D F f rac t i on . It a p p e a r s that t he add i t i on of P - g l u c a n a s e m a y h a v e h a d a n a f fec t o n t h e h e m i c e l l u l o s i c f rac t i on o f b a r l e y a n d not the A D F f rac t i on a s i n d i c a t e d b y t h e i n te rac t i ons i n v e s t i g a t e d . T h e r e w a s a l s o a s ign i f i can t i n te rac t i on ( P = 0 .01 ) f o u n d b e t w e e n p r o t e a s e a n d x y l a n a s e w h i c h is i l lus t ra ted in F i g u r e 2 . 1 0 . W i t h i n th is i n te rac t i on , it w a s d e t e r m i n e d that t h e t r e a t m e n t o f b a r l e y w i th 0 . 0 0 1 % x y l a n a s e s ign i f i can t l y d e c r e a s e d ( P < 0 . 0 5 ) t h e N D F c o n t e n t f r o m 2 3 . 1 % to 2 1 . 9 % o f t he D M con ten t of ba r l ey , w h e r e a s a l l o t h e r t r e a t m e n t m e a n s w e r e s i m i l a r to t h e con t ro l ( T a b l e 2 .9) . T h i s s p e c i f i c resu l t w a s re f l ec ted in a s l igh t i n c r e a s e in I V T D but not w i th a h i g h e r I V D M D v a l u e . 6 5 R e s u l t s f r o m f i be r a n a l y s e s a r e di f f icul t to in terpret , h o w e v e r , t h e y ref lect t he l a c k o f r e s p o n s e o b t a i n e d w i th I V D M D a n d I V T D d e t e r m i n a t i o n s . T h e va r ia t i on in N D F w a s m u c h g r e a t e r t h a n t he va r i a t i on o b s e r v e d w i th A D F . C o n s e q u e n t l y , f e w e r d i f f e r e n c e s w e r e o b s e r v e d b e t w e e n N D F d e t e r m i n a t i o n s . It h a s b e e n s h o w n that the h igh a m o u n t o f s t a r c h in c e r e a l g r a i n s i n te r fe res w i th t he N D F f i l t rat ion p r o c e s s a n d m a y c o n t a m i n a t e t h e s a m p l e ( V a n S o e s t et a l . , 1 9 9 1 ) . T h e p r e s e n t s t u d y i n c o r p o r a t e d t h e u s e o f a l p h a - a m y l a s e in t he N D F p r o c e d u r e in o r d e r to r e d u c e t he i n t e r f e r e n c e f r o m s t a r c h h o w e v e r , it is p o s s i b l e tha t t he e l i m i n a t i o n o f t he i n t e r f e r e n c e f r o m s t a r c h w a s not a c h i e v e d . 2.5 CONCLUSION I n c o n c l u s i v e r esu l t s f r o m t h e p r e s e n t s t u d y a n d i n c o n s i s t e n c i e s f r o m o the r s t u d i e s e x e m p l i f y t h e d i f f icu l t ies in i m p l e m e n t i n g e x o g e n o u s e n z y m e a p p l i c a t i o n s in t he r u m i n a n t f e e d indus t ry . T h e p r e s e n t s t u d y r e v e a l e d that fu r the r i nves t i ga t i on is n e c e s s a r y s h o u l d t h e r e b e a n in te res t p u r s u i n g t he a p p l i c a t i o n o f e n z y m e s to ba r l ey . A n u m b e r o f t r e a t m e n t s s u g g e s t e d a r e a s o f fu r ther r e s e a r c h . In t e r m s o f d igest ib i l i ty , r esu l t s o f t he p r e s e n t s t u d y i n d i c a t e d t h e a p p l i c a t i o n o f x y l a n a s e to b a r l e y w a s d e t r i m e n t a l . T h e a p p l i c a t i o n o f p r o t e a s e , P - g l u c a n a s e o r a c o m b i n a t i o n of t h e s e two e n z y m e s a p p e a r e d to h a v e t h e g r e a t e s t pos i t i ve r e s p o n s e . F i b e r a n a l y s e s re f lec ted s im i l a r r esu l t s tha t w e r e o b t a i n e d w i th t h e d iges t ib i l i t y m e a s u r e m e n t s . T h e e c o n o m i c s o f i m p l e m e n t i n g a n y o f t h e s e t r e a t m e n t s w a s not a t t e m p t e d in th is s tudy . P r o d u c t i o n t r ia ls a r e n e c e s s a r y to e s t a b l i s h a p r o d u c t i o n r e s p o n s e . T h e d e g r e e of 6 6 r e s p o n s e in p r o d u c t i o n wi l l d i c t a te fu r ther r e s e a r c h a n d a n y po ten t ia l a p p l i c a t i o n s o f s e l e c t e d e n z y m e s a n d the i r c o n c e n t r a t i o n s to b a r l e y . 6 7 2.6 REFERENCES A l b e r t a B a r l e y C o m m i s s i o n . 1 9 9 5 . A l b e r t a A g r i c u l t u r e , F o o d a n d R u r a l D e v e l o p m e n t . r A r o n e n , I., V a h a t a l o , A . a n d T . V a r v i k k o . 1 9 9 1 . R u m i n a l a n d in tes t ina l d e g r a d a t i o n o f ce l l w a l l c o n s t i t u e n t s o f b a r l e y , b a r l e y f i be r a n d w h e a t mil l run m e a s u r e d by t h e n y l o n b a g t e c h n i q u e . A n i m . F e e d S c i . T e c h n o l . , 3 2 : 2 0 1 - 2 0 5 . B e a u c h e m i n , K. A . a n d L. M . R o d e . 1 9 9 6 . U s e o f f e e d e n z y m e s in rum inan t nut r i t ion. P a g e s 1 0 3 - 1 3 0 in L . M . R o d e e d . , A n i m a l R e s e a r c h a n d D e v e l o p m e n t : M e e t i n g F u t u r e C h a l l e n g e s . P r o c . 1 9 9 6 C a n . S o c . A n i m . S c i . A n n u a l M e e t i n g , L e t h b r i d g e , A l b e r t a , C a n a d a . B e a u c h e m i n , K. A . , R o d e , L. M . , J o n e s , S . D. M . a n d V . J . H . S e w a l t . 1 9 9 6 . E f f ec t s o f f ib ro ly t ic e n z y m e s in c o r n a n d b a r l e y b a s e d d ie t s o n p e r f o r m a n c e a n d c a r c a s s c h a r a c t e r i s t i c s o f f e e d l o t ca t t le . C a n . J . A n i m . S c i . (Abstract in press). B e d f o r d , M . R . 1 9 9 3 . M o d e o f a c t i o n o f f e e d e n z y m e s . J . A p p l . P o u l t r y S c i . , 2 : 8 5 - 9 2 . B o y l e s , D. W . , R i c h a r d s o n , C . R. , R o b i n s o n , K. D. a n d C . W . C o b b . 1 9 9 2 . F e e d l o t p e r f o r m a n c e o f s t e e r s f e d s t e a m - f l a k e d g ra i n s o r g h u m wi th a d d e d e n z y m e s . J . A n i m . S c i . , 4 3 : 5 0 2 - 5 0 5 . B u r r o u g h s , W . , W o o d s , W . , E w i n g , S . A . G r e i g , J . a n d B . T h e u r e r . 1 9 6 0 . E n z y m e a d d i t i o n s to fa t t en ing ca t t le ra t i ons . J . A n i m . S c i . , 1 9 : 4 5 8 - 4 6 4 . C a m p b e l l , G . L. a n d M . R. B e d f o r d . 1 9 9 2 . E n z y m e a p p l i c a t i o n s fo r m o n o g a s t r i c f e e d s : a r e v i e w . C a n . J . A n i m . S c i . , 7 2 : 4 4 9 - 4 6 6 . C a n a d i a n C o u n c i l o n A n i m a l C a r e . 1 9 9 3 . G u i d e to c a r e a n d u s e o f e x p e r i m e n t a l a n i m a l s . V o l u m e 1. E . D. Ol fer t , C r o s s B . M . a n d A . A . M c W i l l i a m ( E d s . ) . 2 n d E d . , O t t a w a , O n t a r i o . C h e n g , K . - J . , M c A l l i s t e r , T . A . , S e l i n g e r , L. B . , Y a n k e , L. J . , B a e . H . D., F o r s b e r g , C . W . a n d J . A . S h e l f o r d . 1 9 9 5 . B i o t e c h n o l o g y in t he feed lo t . J . A n i m . S c i . , 4 6 : 6 0 0 - 6 0 5 . C h e s s o n , A . 1 9 9 3 . F e e d e n z y m e s . A n i m . F e e d S c i . T e c h n o l . , 4 5 : 6 5 - 7 9 . C l a r k , J . D. , D y e r , I. A . , a n d T e m p l e t o n , J . A . 1 9 6 1 . S o m e nutr i t ional a n d p h y s i o l o g i c a l e f f ec t s o f e n z y m e s fo r fa t ten ing ca t t le . J . A n i m . S c i . , 2 0 : 9 2 8 . (Abs t rac t ) . 6 8 C o c h r a n , W . G . a n d G . M . C o x . 1 9 5 0 . E x p e r i m e n t a l d e s i g n s . P a g e s 1 5 4 - 1 5 6 ; 2 4 1 -2 4 6 . J o h n W i l e y a n d S o n s Inc., N e w Y o r k , N Y . E n g s t r o m , D. F. , M a t h i s o n , L. A . a n d G o o n e w a r d e n e . 1 9 9 2 . E f fec t o f g l u c a n , s t a r c h a n d f ib re c o n t e n t o f b a r l e y g r a i n o n its d i g e s t i o n a n d ut i l i za t ion by s t e e r s . A n i m . F e e d S c i . T e c h n o l . , 3 7 : 3 3 - 4 6 . F a y , J . P . , C h e n g , K . - J . , C o s t e r t o n , J . W . , H a n n a , M . R. a n d R . E . H o w a r t h . 1 9 8 0 . In vitro d i g e s t i o n o f b loa t s a f e a n d b loa t c a u s i n g m i c r o o r g a n i s m s : g a s a n d f o a m p r o d u c t i o n . J . D a i r y S c i . , 6 3 : 1 2 7 3 - 1 2 8 1 . G o e r i n g , H . K. a n d P . J . V a n S o e s t . 1 9 7 0 . F o r a g e f ibe r a n a l y s e s , ( a p p a r a t u s , r e a g e n t s , p r o c e d u r e s , a n d s o m e a p p l i c a t i o n s ) . A g r i c . H a n d b o o k N o . 3 7 9 . , A R S - U S D A , W a s h i n g t o n , D C . G w a y u m b a , W . , F e h r , M . a n d D. A . C h r i s t e n s e n . 1 9 9 5 . T h e ef fec t o f e n z y m e t r e a t m e n t s o n t h e e n s i l i n g a n d f e e d i n g qua l i t y o f b a r l e y s i l a g e . P a g e s 1-3 In P r o c . W e s t . S e c t . , A m . S o c . A n i m . S c i . P o r t l a n d , O R . H e r r e r a - S a l d a n a , H u b e r , J . T . a n d M . H . P o o r e . 1 9 9 0 . D r y mat te r , c r u d e p ro te in , a n d s t a r c h d e g r a d a b i l i t y o f f i ve c e r e a l g r a i n s . J . D a i r y S c i . , 7 3 : 2 3 8 6 - 2 3 9 3 . Hun t , C . W . , F e n g , P . , T r e a c h e r , R. a n d G . T . P r i t c h a r d . 1 9 9 5 . E f fec t o f f ibro lyt ic e n z y m e a d d i t i v e s o n in vi t ro d e g r a d a b i l i t y o f a l fa l fa a n d tal l f e s c u e . P r o c . W e s t . S e c t . , A m . S o c . A n i m . S c i . , P o r t l a n d , O R . H u n t i n g t o n , J . A . 1 9 9 5 . R u m e n f e r m e n t a t i o n d y n a m i c s . P a g e s 1 7 - 1 9 in F e e d C o m p o u n d e r May. K e r c h e r , C . J . 1 9 6 6 . V a l u e o f d ia l l ys t i l bes t ro l a n d Z y m o - p a b s t e n z y m e p r e p a r a t i o n fo r f a t t en ing y e a r l i n g s t e e r s . J . A n i m . S c i . 1 9 : 9 6 6 (Abs t rac t ) . K o m a r e k , A . R. , R o b e r t s o n , J . B . a n d P . J . V a n S o e s t . 1 9 9 4 . A c o m p a r i s o n o f m e t h o d s fo r d e t e r m i n i n g A D F u s i n g t he F i l te r B a g T e c h n i q u e v e r s u s c o n v e n t i o n a l f i l t rat ion. J . D a i r y S c i . ( s u p p l . 1), 7 7 : 1 1 4 . (Abs t rac t ) K r a u s e , M . , F a r r , B . I., R o d e , L. M . , B e a u c h e m i n , K. A . a n d P . N o r g a a r d . 1 9 9 6 . T h e e f f ec t s o f f ib ro ly t ic e n z y m e s o n r u m i n a l d i g e s t i o n k i ne t i c s o f w h o l e ba r l ey g r a i n . C a n . S o c . A n i m . S c i . A n n u a l M e e t i n g , L e t h b r i d g e A B (Abstract in press). K r a u s e , O . G . , R i c h a r d s o n , C . R. a n d C . 0 . C o b b . 1 9 8 9 . B i o l o g i c a l r e s p o n s e s o f s t e e r s a n d ra ts f e d g r a i n s w i th a n a d d e d m i c r o b i a l e n z y m e m ix tu re . J . A n i m . S c i . ( s u p p l . 1), 6 7 : 2 8 0 (Abs t rac t ) . 6 9 K r i s h n a m o o r t h y , U . , S o l l e r , H . , S t e i n g a s s , H . a n d K. H . M e n k e . 1 9 9 1 . A c o m p a r a t i v e s t u d y o n r u m e n f e r m e n t a t i o n o f e n e r g y s u p p l e m e n t s in vitro. J . A n i m . P h y s i o l . A n i m . Nut r . , 6 5 : 2 8 - 3 5 . L e n v e n t i n i M . W . , H u n t , C . W . , Ro f f le r , R . E . a n d D. G . C a s e b o l t . 1 9 9 0 . E f fec t o f d i e t a r y l eve l o f b a r l e y b a s e d s u p p l e m e n t s a n d r u m i n a l bu f fe r o n d i g e s t i o n a n d g r o w t h b y b e e f ca t t le . J . A n i m . S c i . , 6 8 : 4 3 3 4 - 4 3 4 4 . M c A l l i s t e r , T . A . P h i l l i p p e , R. C . , R o d e , L. M . a n d K . - J . C h e n g . 1 9 9 3 . E f fec t o f the p ro te in ma t r i x o n t h e d i g e s t i o n o f c e r e a l g r a i n s b y r u m i n a l m i c r o o r g a n i s m s . J . A n i m . S c i . , 7 1 : 2 0 5 - 2 1 2 . M c A l l i s t e r , T . A . , C h e n g , K . - J . , R o d e , L. M . a n d J . G . B u c h a n a n - S m i t h . 1 9 9 0 a . U s e o f f o r m a l d e h y d e to r e g u l a t e d i g e s t i o n o f b a r l e y s t a r c h . C a n . J . A n i m . S c i . , 7 0 : 5 8 1 - 5 8 9 . M c A l l i s t e r , T . A . , R o d e , L. M . , M a j o r , D . J . , C h e n g , K . - J . a n d J . G . B u c h a n a n - S m i t h . 1 9 9 0 b . E f f ec t o f r u m i n a l c o l o n i z a t i o n o n c e r e a l g ra i n d i g e s t i o n . C a n . J . A n i m . S c i . , 7 0 : 5 7 1 - 5 7 9 . M c A l l i s t e r , T . A . , R o d e , L. M . , C h e n g , K . - J . , S c h a e f e r , D. M . a n d J . W . C o s t e r t o n . 1 9 9 0 c . M o r p h o l o g i c a l s t u d y o f t he d i g e s t i o n o f b a r l e y a n d m a i z e g ra in by r u m e n m i c r o o r g a n i s m s . A n i m . F e e d S c i . T e c h n o l . , 3 1 : 9 1 - 1 0 5 . N e w m a n , C . W . 1 9 9 4 . T h e U n i t e d S t a t e s m a r k e t fo r f e e d e n z y m e s : W h a t o p p o r t u n i t i e s e x i s t ? P a g e 9 9 in B i o t e c h n o l o g y in t h e F e e d Indust ry . P r o c . o f A l l t e c h ' s T e n t h A n n u a l S y m p o s i u m . N o t t i n g h a m U n i v e r s i t y P r e s s . N e w m a n , C . W . a n d C . F. M c G u i r e . 1 9 8 5 . Nut r i t iona l qua l i t y o f ba r l ey . P a g e s 4 0 3 -4 3 9 in B a r l e y . R a s m u s s o n , D. C . ( E d . ) , A m . S o c . A g r o n o m y a n d A c a d e m i c P r e s s . N o . 2 6 . M a d i s o n , W i s c o n s i n . N o r d i n , M . a n d R. C . C a m p l i n g . 1 9 7 6 . D iges t ib i l i t y s t u d i e s w i th c o w s g i v e n w h o l e a n d ro l led c e r e a l g r a i n s . A n i m . P r o d . , 2 3 : 3 0 5 - 3 1 5 . N R C ( N a t i o n a l R e s e a r c h C o u n c i l ) . 1 9 8 9 . Nu t r i en t R e q u i r e m e n t s o f D a i r y C a t t l e . S e v e n t h R e v i s e d E d i t i o n . N a t i o n a l A c a d e m y P r e s s . W a s h i n g t o n , D. C . P e r r y , T . W . , P u r k h i s e r , E . R . a n d W . M . B e e s o n . 1 9 6 6 . E f f e c t s o f s u p p l e m e n t a l e n z y m e s o n n i t r ogen b a l a n c e , d iges t ib i l i t y o f e n e r g y a n d nu t r ien ts a n d o n g r o w t h a n d f e e d e f f i c i e n c y o f ca t t le . J . A n i m . S c i . , 2 5 : 7 6 0 - 7 6 4 . R i c h a r d s o n , C . R . , K r a u s e , O . G . , L o m a x , D. A . a n d C . W . C o b b . 1 9 9 0 . Ut i l i za t ion of s t e a m - f l a k e d g ra i n s o r g h u m wi th a d d e d "g ra in s o r g h u m s p e c i f i c " e n z y m e m ix tu re b y g r o w i n g s t e e r s . J . A n i m . S c i . ( s u p p l . 1), 6 8 : 5 3 8 (Abs t rac t ) . 7 0 R o o n e y , L. W . a n d R. L. P f i u g f e l d e r , 1 9 8 6 . F a c t o r s a f fec t i ng s t a r c h d iges t ib i l i t y wi th s p e c i a l e m p h a s i s o n s o r g h u m a n d c o r n . J . A n i m . S c i . , 6 3 : 1 6 0 7 . R o v i c s , J . J . a n d C . M . E l y . 1 9 6 2 . R e s p o n s e o f b e e f ca t t le to e n z y m e s u p p l e m e n t s . J . A n i m . S c i . , 2 1 : 1 0 1 2 (Abs t rac t ) . S ta t i s t i ca l A n a l y s i s S y s t e m ( S A S ) , 1 9 9 0 . S A S U s e r ' s G u i d e : S t a t i s t i c s . S A S Institute Inc., C a r y , N C . S t o k e s , S . R . , H o o v e r , W . H . , M i l le r , T . K. a n d R. B l a u w e i k e l . 1 9 9 1 . R u m i n a l d i g e s t i o n a n d m i c r o b i a l u t i l i za t ion o f d i e t s v a r y i n g in t y p e o f c a r b o h y d r a t e a n d p ro te in . J . D a i r y S c i . , 7 5 : 3 4 5 - 3 5 7 . Ta i t , R . M . a n d R. M . B e a m e s . 1 9 8 8 . P r o c e s s i n g a n d p r e s e r v a t i o n o f c e r e a l s a n d p ro te in c o n c e n t r a t e s . P a g e s 1 5 1 - 1 7 5 in F e e d S c i e n c e W o r l d A n i m a l S c i e n c e ( B 4 ) , E . R. O r s k o v , ( E d . ) , E l s e v i e r S c i . P u b l . B . V . A m s t e r d a m , N e t h e r l a n d s . T i l l ey , J . M . A . a n d R. A . T e r r y . 1 9 6 3 . A t w o - s t a g e t e c h n i q u e fo r t he in vitro d i g e s t i o n o f f o r a g e c r o p s . J . Br i t . G r a s s l . S o c , 18 : 1 0 4 - 1 1 1 . V a n S o e s t , P . J . 1 9 8 2 . Nut r i t i ona l e c o l o g y o f t he rum inan t . O & B B o o k s Inc., C o r v a l l i s , O r e g o n . V a n S o e s t , P . J . , R o b e r t s o n , J . B . a n d B . A . L e w i s . 1 9 9 1 . M e t h o d s o f d ie ta ry f iber , neu t ra l d e t e r g e n t f i be r a n d n o n s t a r c h p o l y s a c c h a r i d e s in re la t ion to a n i m a l nut r i t ion. J . D a i r y S c i . , 7 4 : 3 5 8 3 - 3 5 9 7 . v o n K e y s e r l i n g k , M . A . G . ; Sw i f t , M . L.; P u c h a l a , R. a n d J . A . S h e l f o r d . 1 9 9 6 . D e g r a d a b i l i t y c h a r a c t e r i s t i c s o f d ry ma t te r a n d c r u d e p ro te in o f f o r a g e s in r u m i n a n t s . A n i m . F e e d S c i . T e c h n o l . , 5 7 : 2 9 1 - 3 1 1 . W a l d o , D. R . 1 9 7 3 . E x t e n t a n d par t i t ion o f c e r e a l g r a i n d i g e s t i o n in r u m i n a n t s . J . A n i m . S c i . , 3 7 : 1 0 6 2 - 1 0 7 4 . W a r d , J . K. , R i c h a r d s o n , D. a n d W . S . T s i e n . 1 9 6 0 . V a l u e o f a d d e d e n z y m e p r e p a r a t i o n s in b e e f ca t t le ra t i ons . J . A n i m . S c i . , 1 9 : 1 2 9 8 (Abs t rac t ) . W e i c h e n t h a l , B . , R u s h , I. a n d B . V a n Pe l t . 1 9 9 6 . A n e n z y m e - m i c r o b i a l f e e d p r o d u c t fo r f i n i s h i n g s t e e r s . P a g e s 6 8 - 6 9 in B e e f ca t t le repor t , U n i v . N e b r a s k a -L i n c o l n , L i n c o l n , N E , U S . W i s e m a n , A . 1 9 7 5 . E n z y m e ut i l i za t ion in indus t r ia l p r o c e s s e s . P a g e s 1 1 1 - 1 2 4 in H a n d b o o k o f e n z y m e b i o t e c h n o l o g y . A . W i s e m a n , E d . E l l i s H o r w o o d L td . , C h i c h e s t e r . 71 W i n g , J . M . a n d W i l c o x , C . J . 1 9 6 0 . E f fec t o f s u p p l e m e n t a r y d i g e s t i v e e n z y m e s o n g r o w t h o f d a i r y he i f e r s . J . D a i r y S c i . , 4 3 : 1 6 5 5 - 1 6 7 5 . 7 2 T a b l e 2 . 1 . T r e a t m e n t s i n v e s t i g a t e d in o r d e r to d e t e r m i n e t he e f f ec t s o f e x o g e n o u s e n z y m e a p p l i c a t i o n w i th a n d w i thou t a d d e d m o i s t u r e a n d h e a t o n r u m e n in vitro d ry ma t te r d i s a p p e a r a n c e a n d in vitro t rue d iges t ib i l i t y o f b a r l e y g r a i n . F a c t o r L e v e l o f a p p l i c a t i o n H e a t 1 con t ro l (0), 1 5 , 3 0 , 6 0 m i n M o i s t u r e 2 0 % , 1 0 % , 2 0 % , 3 0 % P - g l u c a n a s e 3 , 4 0 % , 0 . 0 0 1 % , 0 . 0 1 % , 0 . 1 % P r o t e a s e 3 , 5 0 % , 0 . 0 0 2 5 % X y l a n a s e 3 , 6 0 % , 0 . 0 0 1 % 1 A p p l i e d at 5 5 ° C . 2 A p p l i e d o n a % v o l / w t b a s i s . 3 S u p p l i e d b y F i n n F e e d s In te rna t iona l L t d . , W i l t s h i r e , U . K . 4 A v i z y m e 1 1 1 0 5 H e p t e x s b - p r o t e a s e 6 A v i z y m e 1 3 1 0 7 3 T a b l e 2 . 2 . T h e e f fec t o f t he d u r a t i o n o f h e a t t r e a t m e n t a n d t he a p p l i c a t i o n o f m o i s t u r e a n d x y l a n a s e o n t h e 4 8 h in vitro d r y ma t te r d i s a p p e a r a n c e o f b a r l e y g ra in ( D M b a s i s ) . F e e d Treatment Durat ion of heat Mo is tu re 2 (%) X y l a n a s e 2 (%) I V D M D 3 (%) t reatment 1 (min) 0 " 0 0 8 1 . 8 c d e f g 0 0 0.001 7 9 . 3 a b 0 10 0 8 2 . 2 e f 9 0 10 0.001 g-| gcdefg 0 20 0 8 2 . 2 e f g 0 20 0.001 8 0 . 0 a b 0 30 0 8 2 . 5 f g 0 30 0.001 8 0 . 5 a b c d 15 0 0 8 2 . 1 d e f g 15 0 0.001 7 9 . 5 a b 15 10 0 8 2 gcdefg 15 10 0.001 7 9 . 6 a b 15 20 0 8 2 . 1 e f g 15 20 0.001 8 0 . 0 a b 15 30 0 8 2 3 e f g 15 30 0.001 8 0 . 4 a b c 30 0 0 g l gcdefg 30 0 0.001 7 9 . 2 a 30 10 0 g-| gcdefg 30 10 0.001 7 9 . 7 a b 30 20 0 g 0 gbcdef 30 20 0.001 8 0 . 3 a b c 30 30 0 8 2 . 2 e f g 30 30 0.001 g 0 gabcde 60 0 0 7 9 . 9 a b 60 0 0.001 8 0 g b c d e f 60 10 0 8 1 gcdefg 60 10 0.001 g 0 4 a b c 60 20 0 8 2 . 6 9 60 20 0.001 7 9 . 3 a b 60 30 0 8 2 . 8 9 60 30 0.001 8 0 . 5 a b c S . E . M . 0.56 1 Heat was applied at 55°C. 2 Applied on a % vol / wt basis. 3 IVDMD, in vitro dry matter disappearance. 4 S.E.M., pooled standard error of the mean. a ' 9 Means in a column with different superscripts are significantly different (P < 0.05). 7 4 CD O rz co Q r— > c CD E CD E N C L U Q > CD CO CD c _C0 CD co CO CD 2 IQ-CD CO CO c CO o l l a E . 0 ro <u "8 "8 TO "8 0 . 0 TO J3 CD "D E a) 03 T * CD CM O O CD C O C M 0 X — O co 0 0 CD o i 0 0 CT> CT> 1 ^ 0 0 co C O co CD CD CN C O 0 0 0 0 O O 0 0 0 0 0 0 0 0 0 0 0 0 C O C O O O 0 0 0 0 O a> TO •0 L O T i -C O 0 0 C O 0 0 C D r-C N co co ed C D cn L O o >«t cd 0 0 s o o c o c f l co co s 0 0 0 0 0 0 0 0 p o o o p o p o 000000000000000 L O L O L O L O L O L O L O C N C N C N C N C N C N C N O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O T ~ T - T ~ p O p p p p p - r - ; T - T - T -000000000000000 CO 'co C0 CD O c ca ca CD Q . Q . ca co L — CD o > T3 CO s C C o •== •a r f f 2 < > L O O V a. c CD I •4—' C CO o 4= 'c cp CO CD 1— CO CO -*-» Q . • i— c o ca co g CD E a. D CO CD O CD 5 CD ^ += -n -c to C .Ii .CO "D ^ -a c c CD -2 § -fc T3 O O ® ° 5 O TO S 8.-E . £ . co Q ^ CD 7 5 T a b l e 2 . 4 . T h e e f fec t o f m o i s t u r e l eve l o n t he 4 8 h in vitro t rue d iges t ib i l i t y o f b a r l e y g r a i n ( D M b a s i s ) . M o i s t u r e 1 (%) I V T D (%) 0 8 7 . 8 a 10 8 8 . 3 a b 2 0 8 8 . 6 b 3 0 8 8 . 6 b S . E . M . 2 0 . 14 1 A p p l i e d o n a % v o l / w t b a s i s . 2 I V T D , in vitro t r ue d iges t ib i l i t y . 3 S . E . M . , p o o l e d s t a n d a r d e r ro r o f t he m e a n . a b M e a n s in a c o l u m n w i th d i f fe rent s u p e r s c r i p t s a r e s ign i f i can t l y d i f ferent (P < 0.05). 7 6 T a b l e 2 . 5 . T h e e f fec t o f p - g l u c a n a s e , p r o t e a s e a n d x y l a n a s e a p p l i c a t i o n o n t he A D F f rac t ion o f b a r l e y g r a i n ( D M b a s i s ) . E n z y m e t r e a t m e n t 1 p - G l u c a n a s e (%) P r o t e a s e (%) X y l a n a s e (%) A D F 2 ( % ) 0 0 0 8 . 2 a 0 0 0 .001 8 . 4 a b 0 0 . 0 0 2 5 0 8 . 4 a b 0 0 . 0 0 2 5 0 .001 9 . 1 d e 0.001 0 0 g 5abc 0.001 0 0 .001 8 . 2 a 0.001 0 . 0 0 2 5 0 8 . 4 a b 0.001 0 . 0 0 2 5 0 .001 9.0°*° 0.01 0 0 9 . 3 e 0.01 0 0 .001 9 . 1 d e 0.01 0 . 0 0 2 5 0 8 . 6 a b c 0.01 0 . 0 0 2 5 0 .001 8 . 5 a b 0.1 0 0 8 . 3 a 0.1 0 0 .001 g gbcd 0.1 0 . 0 0 2 5 0 9 . 1 d e 0.1 0 . 0 0 2 5 0 .001 9 . 0 d e S . E . M . 3 0 . 1 5 1 A p p l i e d o n a % v o l / w t b a s i s . 2 A D F , a c i d d e t e r g e n t f iber . 3 S . E . M . , p o o l e d s t a n d a r d e r ro r o f t h e m e a n . a _ e M e a n s in a c o l u m n w i th d i f fe rent s u p e r s c r i p t s a r e s ign i f i can t l y d i f fe rent (P < 0.05). 7 7 T a b l e 2 . 6 . T h e e f fec t o f h e a t t r e a t m e n t a n d t he a p p l i c a t i o n o f p - g l u c a n a s e a n d x y l a n a s e o n t h e A D F f rac t i on o f b a r l e y g ra i n ( D M b a s i s ) . E n z y m e treatment 1 Durat ion of heat p -G lucanase (%) P ro tease (%) A D F 3 ( % ) t reatment 2 (min) 0 0 0 g gabcd 0 0 0.0025 g ybcdefghij 0 0.001 0 g -| abc 0 0.001 0 .0025 g 2abc 0 0.01 0 g gefghijk 0 0.01 0 .0025 g ^abcdef 0 0.1 0 g ycdefghij 0 0.1 0 .0025 g yabcdefghij 15 0 0 g gabcd 15 0 0.0025 g gabcdefghi 15 0.001 0 g ^abcdefgh 15 0.001 0.0025 g gabcdefgh 15 0.01 0 9 .2 j k l 15 0.01 0.0025 g ycdefghij 15 0.1 0 8 . 1 a b 15 0.1 0.0025 9.6' 30 0 0 g gabcde 30 0 0.0025 g gdefghijk 30 0.001 0 8 .0 a 30 0.001 0.0025 9 .1 j k l 30 0.01 0 9.4 k l 30 0.01 0 .0025 g gabcd 30 0.1 0 g gdefghijk 30 0.1 0 .0025 9 .1 i j k l 60 0 0 g gabcd 60 0 0.0025 g_0hijkl 60 0.001 0 g gdefghijk 60 0.001 0.0025 g fj9 h ii k l 60 0.01 0 9.2 i k l 60 0.01 0.0025 g gcdefghij 60 0.1 0 g ^abcdefg 60 0.1 0.0025 g o f9 h ' i k l S . E . M . 4 0.21 1 App l ied on a % vol / wt bas is . 2 .Hea t w a s appl ied at 55°C. 3 A D F , ac id detergent fiber. 4 S . E . M . , poo led s tandard error of the mean . a"' M e a n s in a co lumn with different superscr ip ts are signif icantly different (P < 0.05). 7 8 T a b l e 2 . 7 . T h e e f fec t o f p - g l u c a n a s e a n d p r o t e a s e a p p l i c a t i o n o n t he N D F c o n t e n t o f b a r l e y g r a i n ( D M b a s i s ) . E n z y m e t r e a t m e n t 1 P - G l u c a n a s e (%) P r o t e a s e (%) N D F 2 ( % ) 0 0 2 1 . 8 a 0 0 . 0 0 2 5 2 3 . 2 b c 0.001 0 2 2 . 8 a b c 0.001 0 . 0 0 2 5 2 3 . 7 C 0.01 0 2 3 . 4 C 0.01 0 . 0 0 2 5 2 2 . 1 a b 0.1 0 2 2 . 0 a 0.1 0 . 0 0 2 5 2 2 . 1 a S . E . M . 3 0 . 3 7 1 A p p l i e d o n a % v o l / w t b a s i s . 2 N D F , neu t ra l d e t e r g e n t f iber . 3 S . E . M . , p o o l e d s t a n d a r d e r ro r o f t he m e a n . a"° M e a n s in a c o l u m n w i th d i f fe rent s u p e r s c r i p t s a r e s ign i f i can t l y d i f ferent (P < 0.05). 7 9 T a b l e 2 . 8 . T h e e f fec t o f p - g l u c a n a s e a n d x y l a n a s e a p p l i c a t i o n o n t h e N D F f rac t ion of b a r l e y g r a i n ( D M b a s i s ) . E n z y m e t r e a t m e n t 1 p - G l u c a n a s e (%) X y l a n a s e (%) N D F 2 ( % ) 0 0 2 3 . 0 b c 0 0 .001 2 2 . 1 a b 0.001 0 2 4 . 5 d 0.001 0 .001 2 2 . 0 a b 0.01 0 2 2 . 4 a b c 0.01 0 .001 2 3 . 2 ° 0.1 0 2 1 . 8 a 0.1 0 .001 2 2 2a ' 3 C S . E . M . 3 0 . 3 7 1 A p p l i e d o n a % v o l / w t b a s i s . 2 N D F , neu t ra l d e t e r g e n t f iber . 3 S . E . M . , p o o l e d s t a n d a r d e r ro r o f t h e m e a n . a~° M e a n s in a c o l u m n w i th d i f fe rent s u p e r s c r i p t s a r e s ign i f i can t l y d i f fe rent (P < 0.05). 8 0 T a b l e 2 . 9 . T h e e f fec t o f p r o t e a s e a n d x y l a n a s e a p p l i c a t i o n o n t he N D F c o n t e n t o f b a r l e y g r a i n ( D M b a s i s ) . E n z y m e t r e a t m e n t 1 P r o t e a s e (%) X y l a n a s e (%) N D F 2 ( % ) 0 0 2 3 . 1 b 0 0 .001 2 1 . 9 a 0 . 0 0 2 5 0 2 2 . 7 b 0 . 0 0 2 5 0 .001 2 2 . 9 b S . E . M . 3 0 . 2 6 1 A p p l i e d o n a % v o l / w t b a s i s . 2 N D F , neu t ra l d e t e r g e n t f iber . 3 S . E . M . , p o o l e d s t a n d a r d e r ro r o f t he m e a n . a b M e a n s in a c o l u m n wi th d i f ferent s u p e r s c r i p t s a r e s ign i f i can t l y d i f ferent (P < 0.05). 81 Figure 2.1. Laboratory scale hydrothermal cooker. 82 8 4 - • - 0% X, no heat - • - 0% X, 15 min heat -A— 0% X, 30 min heat - * - 0% X, 60 min heat - * - 0.001% X, no heat - • - 0.001% X, 15 min heat -t— 0.001% X, 30 min heat - e - 0.001% X, 60 min heat 0 10 2 0 M o i s t u r e l eve l (% vo l / wt ) 3 0 F i g u r e 2 . 2 . T h e e f fec t o f t he add i t i on of m o i s t u r e , x y l a n a s e (X ) (% v o l / wt ) a n d du ra t i on o f t he h e a t t r e a t m e n t ( 5 5 ° C ) o n t he in vitro d r y ma t t e r d i s a p p e a r a n c e o f b a r l e y g r a i n . 8 3 F i g u r e 2 . 3 . T h e e f fec t o f b e t a - g l u c a n a s e , x y l a n a s e (X ) a n d p r o t e a s e ( P ) a p p l i c a t i o n (% vo l / wt) o n t he in vitro d r y ma t t e r d i s a p p e a r a n c e o f b a r l e y g r a i n . 8 4 0 10 2 0 3 0 M o i s t u r e l eve l (% vo l / wt) F i g u r e 2 .4 . Q u a d r a t i c r e l a t i onsh ip b e t w e e n l eve l o f m o i s t u r e a n d in vitro t rue d iges t ib i l i t y o f b a r l e y g ra i n (Y = 8 7 . 7 5 + 0 .071 * m o i s t u r e l eve l - 0 . 0 0 1 4 * m o i s t u r e l e v e l 2 : n = 4 , R 2 = 0 . 9 9 8 , S . E . = 0 . 0 1 8 ) . 8 5 F i g u r e 2 . 5 . T h e e f fec t o f b e t a - g l u c a n a s e , p r o t e a s e ( P ) a n d x y l a n a s e (X ) a p p l i c a t i o n (% vo l / wt ) o n t he in vitro t rue d iges t ib i l i t y o f b a r l e y g r a i n . 8 6 9 . 5 x -K-0.001% X, 0.0025% P 0 0 .001 0.01 0.1 B e t a - g l u c a n a s e l eve l (% v o l / wt ) F i g u r e 2 . 6 . T h e e f fec t o f b e t a - g l u c a n a s e , p r o t e a s e ( P ) a n d x y l a n a s e (X ) a p p l i c a t i o n (% v o l / wt ) o n t h e c o n t e n t o f a c i d d e t e r g e n t f i be r in b a r l e y g r a i n . 8 7 0% P, no heat 0% P, 15 min heat 0% P, 30 min heat * - 0% P, 60 min heat * - 0.0025% P, no heat •-0.0025% P, 15 heat H—0.0025% P, 30 heat — 0.0025% P, 60 heat —I 0.1 0 0 .001 0.01 B e t a - g l u c a n a s e l eve l (% v o l / wt) F i g u r e 2 . 7 . T h e e f fec t o f b e t a - g l u c a n a s e a n d p r o t e a s e ( P ) a p p l i c a t i o n (% vo l / wt ) a n d du ra t i on o f t h e h e a t t r e a t m e n t ( 5 5 ° C ) o n t he c o n t e n t o f a c i d d e t e r g e n t f i be r in b a r l e y g r a i n . 8 8 0 0 % P r o t e a s e 0 . 0 0 2 5 % P r o t e a s e 0 . 0 2 0 .04 0 . 0 6 0 . 0 8 B e t a - g l u c a n a s e l eve l (% vo l / wt) 0.1 F i g u r e 2 . 8 . T h e e f fec t o f t h e add i t i on o f b e t a - g l u c a n a s e a n d p r o t e a s e (% vo l / wt ) o n t he c o n t e n t o f neu t ra l d e t e r g e n t f i be r in b a r l e y g r a i n . 8 9 0 0 . 0 2 0 .04 0 . 0 6 0 . 0 8 0.1 B e t a - g l u c a n a s e l eve l (% v o l / wt ) F i g u r e 2 . 9 . T h e e f fec t o f t he a d d i t i o n o f b e t a - g l u c a n a s e a n d x y l a n a s e ( % v o l / w t ) o n t he c o n t e n t o f neu t ra l d e t e r g e n t f i be r in b a r l e y g r a i n . 9 0 2 5 2 4 . 5 v P 2 4 L _ CD 2 3 . 5 - Q M — -4—' 2 3 c CD E> 2 2 . 5 Q) "CD " O 2 2 2 1 . 5 CD z 21 2 0 . 5 2 0 0 0 . 0 0 0 5 0 .001 0 . 0 0 1 5 0 . 0 0 2 P r o t e a s e l eve l (% v o l / wt ) 0 . 0 0 2 5 F i g u r e 2 . 1 0 . T h e e f fec t o f t he add i t i on o f p r o t e a s e a n d x y l a n a s e (% v o l / wt ) o n t he c o n t e n t o f neu t ra l d e t e r g e n t f i be r in b a r l e y g r a i n . 91 CHAPTER 3 3.0 THE EFFECT OF ENZYME AND MILD HYDROTHERMAL TREATMENT ON THE IN VITRO DIGESTIBILITY OF CANOLA MEAL. 3.1 ABSTRACT C a n o l a m e a l is t he p r i m a r y a l t e rna t i ve p ro te in s u p p l e m e n t to s o y b e a n m e a l . C a n o l a m e a l is res t r i c ted in lac ta t i ng da i r y ca t t le d i e t s b e c a u s e o f it 's l o w e r e n e r g y ava i lab i l i t y par t l y a t t r i bu tab le to it 's h igh f i be r con ten t . T h e p r e s e n t s t u d y i n v e s t i g a t e d t he feas ib i l i t y o f u s i n g e n z y m e t e c h n o l o g y in c o n j u n c t i o n w i th m i ld h y d r o t h e r m a l t r e a t m e n t in o r d e r to i m p r o v e t h e nut r i t iona l v a l u e o f c a n o l a m e a l f o r da i r y ca t t le . T h e e x p e r i m e n t c o n s i s t e d o f a 4 x 4 x 4 x 2 mul t i - fac tor ia l d e s i g n in w h i c h a p p a r e n t a n d t rue in vitro d iges t ib i l i t i es a n d f ibe r a n a l y s e s w e r e u s e d to e v a l u a t e t r ea tmen t e f fec t s . T r e a t m e n t s i n c l u d e d t h e a p p l i c a t i o n o f h e a t at 5 5 ° C fo r 1 5 , 3 0 o r 6 0 m in p e r i o d s o r not a p p l i e d at a l l ; m o i s t u r e a p p l i e d at 0 % , 1 0 % , 2 0 % o r 3 0 % ; p r o t e a s e (Bacillus sp.) a p p l i e d at 0 % , 0 . 0 0 5 % , 0 . 0 1 % o r 0 . 0 5 % a n d x y l a n a s e (Trichoderma sp.) a p p l i e d at 0 % o r 0 . 0 0 1 % . Nu t r ien t c o m p o s i t i o n o f t he u n t r e a t e d c a n o l a m e a l s a m p l e w a s 8 9 . 3 % D M ; 3 6 . 6 % C P , 3 . 2 5 % E E , 1 7 . 6 7 % A D F a n d 2 1 . 8 3 % N D F o n a D M b a s i s . T h e r e w a s a s ign i f i can t i n te rac t ion ( P = 0 . 0 0 0 1 ) b e t w e e n p r o t e a s e a n d x y l a n a s e o n I V D M D . T h e a d d i t i o n o f p r o t e a s e w a s t he m a j o r f a c t o r r e s p o n s i b l e for i n c r e a s e d I V D M D . A h igh l y s i gn i f i can t q u a d r a t i c r e l a t i onsh ip ( P = 0 . 0 0 0 1 ) b e t w e e n m o i s t u r e a n d I V D M D w a s d e s c r i b e d by : Y = 7 8 . 6 2 + 0 . 1 6 5 * m o i s t u r e -9 2 0 . 0 0 3 * m o i s t u r e 2 . I V T D w a s a f f e c t e d b y the s ign i f i can t i n te rac t i on ( P < 0 .01 ) b e t w e e n p r o t e a s e a n d x y l a n a s e , w h e r e resu l t s pa ra l l e l t h o s e o b t a i n e d w i th I V D M D . A q u a d r a t i c r e l a t i o n s h i p ( P = 0 . 0 0 0 1 ) w a s a l s o f o u n d b e t w e e n m o i s t u r e a n d I V T D d e s c r i b e d b y Y = 8 7 . 2 5 + 0 . 0 7 3 * m o i s t u r e - 0 . 0 0 2 * m o i s t u r e 2 . T h e add i t i on o f 1 0 % m o i s t u r e w a s su f f i c ien t to s ign i f i can t l y i n c r e a s e bo th I V D M D a n d I V T D ( P < 0 .05 ) . H o w e v e r , w i th I V T D n o fu r the r i n c r e a s e in d iges t ib i l i t y w a s o b s e r v e d w i th a fu r ther i n c r e a s e in m o i s t u r e l e v e l . T h e r e w e r e s e v e r a l th ird o r d e r i n t e rac t i ons w h i c h a f f ec ted f ibe r c o n t e n t : d u r a t i o n o f t h e h e a t t r ea tmen t , a n d t he a d d i t i o n o f p r o t e a s e a n d x y l a n a s e ( P < 0 .05 ) a n d t he a d d i t i o n o f m o i s t u r e , p r o t e a s e a n d x y l a n a s e ( P < 0 .01) . N D F w a s s ign i f i can t l y a f f e c t e d ( P < 0 .05 ) by the a d d i t i o n o f p r o t e a s e ( P < 0 .05 ) a n d m o i s t u r e ( P = 0 . 0 0 0 1 ) . T h e mi ld h e a t t r e a t m e n t u s e d in t he p r e s e n t s t u d y d id not e n h a n c e e n z y m e act iv i ty in m o s t i n s t a n c e s . E v a l u a t i o n o f c a n o l a m e a l t r e a t m e n t s , u s i n g t h e s e l e c t e d i n d i c e s , p r o v i d e d resu l t s f r o m w h i c h s e v e r a l c o n c l u s i o n s m a y b e d r a w n . T h e a p p l i c a t i o n o f x y l a n a s e w a s not e f fec t i ve , a l t h o u g h t h e add i t i on o f p r o t e a s e in m a n y i n s t a n c e s , e l i c i ted a pos i t i ve r e s p o n s e . T h i s r e s p o n s e w a s s im i l a r to that o b t a i n e d w i th t he c o m b i n a t i o n o f p r o t e a s e a n d x y l a n a s e . T r e a t m e n t s w h i c h i n d i c a t e d a p o s s i b l e i m p r o v e m e n t in t he d igest ib i l i t y o f c a n o l a m e a l i n c l u d e d the a p p l i c a t i o n o f p r o t e a s e o r m o i s t u r e a l o n e o r in c o m b i n a t i o n w i th o t h e r f ac to r s . F u r t h e r i n v e s t i g a t i o n u s i n g o t h e r in vitro t e c h n i q u e s , s u c h a s in vitro g a s p roduc t i on m a y r e v e a l t he e f fec t s o f t he t r e a t m e n t s o n f e r m e n t a t i o n c h a r a c t e r i s t i c s w h i c h w a s u n a v a i l a b l e in t he p r e s e n t s t u d y a n d is ce r ta i n l y jus t i f ied in o r d e r to fac i l i ta te t he s e l e c t i o n o f t r e a t m e n t s fo r c a n o l a m e a l . 9 3 3.2 INTRODUCTION C a n o l a m e a l is t h e p r i m a r y a l t e rna t i ve to s o y b e a n m e a l fo r u s e a s a p ro te in s u p p l e m e n t in r a t i ons fo r l ac ta t i ng da i r y c o w s . It m a y c o m p r i s e 1 0 - 1 5 % o r u p w a r d s to 2 5 % o f t he c o n c e n t r a t e ra t ion d e p e n d i n g o n t h e c o s t o f s o y b e a n m e a l a n d the p ro te in c o n t e n t o f t h e f o r a g e ( P r o F o r m F e e d s Inc., p e r s o n a l c o m m u n i c a t i o n ) . C a n o l a m e a l is a b y - p r o d u c t f r o m c a n o l a s e e d f o l l ow ing t h e oi l ex t r ac t i on p r o c e s s . T h e hu l l s f r o m c a n o l a s e e d a r e p r e s e n t in t h e m e a l up to 3 0 % b y w e i g h t a n d a r e r ich in f i be r a n d l ign in (Be l l a n d S h i r e s , 1 9 8 2 ) . T h e h igh f i be r c o n t e n t o f c a n o l a m e a l is t h e m a j o r con t r i bu t i ng f a c t o r to it 's re la t i ve ly l o w e r e n e r g y ava i lab i l i t y w h e n c o m p a r e d to s o y b e a n m e a l ( E m a n u e l s o n , 1 9 9 4 ) . R e s e a r c h to i m p r o v e t he e n e r g y c o n t e n t o f c a n o l a m e a l is t h e r e f o r e , o f m a j o r in te res t to t he f e e d a n d da i r y i ndus t r i es . I n c r e a s i n g t h e nutr i t ive v a l u e o f f e e d s t u f f s fo r r u m i n a n t s t h r o u g h t he u s e o f e n z y m e t e c h n o l o g y h a s r e c e i v e d r e n e w e d in teres t . T h i s is p r imar i l y a resu l t o f the r e c e n t a d v a n c e s in m o n o g a s t r i c f e e d i n g r e g i m e s u s i n g e x o g e n o u s e n z y m e s ( C h e s s o n , 1 9 9 3 ; B e a u c h e m i n a n d R o d e , 1 9 9 6 ) . H o w e v e r , r e s e a r c h into the u s e of e n z y m e s a s f e e d a d d i t i v e s for r u m i n a n t s h a s y i e l d e d v a r i a b l e resu l t s ( B e a u c h e m i n a n d R o d e , 1 9 9 6 ) . In a r e v i e w o f t he l i te ra ture, it is e v i d e n t that little w o r k h a s b e e n c o n d u c t e d o n t h e u s e o f e n z y m e s in t he t r e a t m e n t o f c a n o l a m e a l a s a m e a n s of i m p r o v i n g it 's d iges t ib i l i t y fo r r u m i n a n t s . F o r fu r ther de ta i l r e g a r d i n g t h e resu l t s of e n z y m e r e s e a r c h w i th r u m i n a n t s , t h e r e a d e r m a y b e re fe r red to C h a p t e r 1, s e c t i o n 1.1. T h e u s e o f in vitro f e r m e n t a t i o n t e c h n i q u e s h a s b e e n s u c c e s s f u l l y a p p l i e d for d e c a d e s in t h e e v a l u a t i o n o f f o r a g e s fo r r u m i n a n t s (T i l ley a n d T e r r y , 1 9 6 3 ; G o e r i n g 9 4 a n d V a n S o e s t , 1 9 7 0 ) . T h e m a j o r a d v a n t a g e o f in vitro t e c h n i q u e s is that a l a rge n u m b e r o f s a m p l e s m a y b e e v a l u a t e d at o n e t i m e . T h e o b j e c t i v e o f t h e cu r ren t s t u d y w a s to ident i fy t r e a t m e n t s that w o u l d i m p r o v e t he d iges t ib i l i t y o f c a n o l a m e a l , pa r t i cu la r l y o f t h e f i be r c o m p o n e n t , in o r d e r to i m p r o v e it 's nut r i t iona l v a l u e fo r da i r y ca t t le . 3 .3 MATERIALS AND METHODS 3.3.1 Feed Treatments T h e e x p e r i m e n t c o n s i s t e d o f a c o m p l e t e l y r a n d o m i z e d 4 x 4 x 4 x 2 mul t i -f ac to r ia l d e s i g n to e v a l u a t e t he e f fec t s o f e n z y m e s a n d h y d r o t h e r m a l t r e a t m e n t o n c a n o l a m e a l u s i n g r u m e n in vitro d r y ma t te r d i s a p p e a r a n c e ( I V D M D ) a n d r u m e n in vitro t rue d iges t ib i l i t y ( I V T D ) . C a n o l a m e a l f e e d t r e a t m e n t s u n d e r i nves t i ga t i on i n c l u d e d t h e a p p l i c a t i o n o f p r o t e a s e at 0 % , 0 . 0 0 5 % , 0 . 0 1 % o r 0 . 0 5 % (Bacillus sp . ) ; x y l a n a s e at 0 % o r 0 . 0 0 1 % (Trichoderma sp.); m o i s t u r e at 0 % , 1 0 % , 2 0 % o r 3 0 % a n d h e a t a p p l i e d a t 5 5 ° C fo r 0 , 1 5 , 3 0 o r 6 0 m in p e r i o d s ( T a b l e 3 .1) . E n z y m e s w e r e not p u r e c u l t u r e s a n d s u b s t r a t e act iv i ty o f t he e n z y m e s m a y b e f o u n d in A p p e n d i x 6 . 1 . 3 .3 .2 Processing Samples C a n o l a m e a l , g r o u n d t h r o u g h a 3 m m s c r e e n , w a s o b t a i n e d f r o m P r o F o r m F e e d s Inc. F e e d s a m p l e s w e r e p r o c e s s e d a c c o r d i n g to t h e p r o c e d u r e ou t l i ned in C h a p t e r 2 , s e c t i o n 2 . 3 . 2 . 9 5 3.3.3 Chemical Analyses C h e m i c a l a n a l y s e s w e r e c o n d u c t e d o n t h e s a m p l e s a c c o r d i n g to the p r o c e d u r e s o u t l i n e d in C h a p t e r 2 , s e c t i o n 2 . 3 . 3 . 3.3.4 Animals and Feeding D o n o r a n i m a l s w e r e m a i n t a i n e d a s d e s c r i b e d in C h a p t e r 2 , s e c t i o n 2 . 3 . 4 . 3.3.5 Rumen Fluid Inoculum R u m e n f lu id w a s c o l l e c t e d a n d p r e p a r e d a c c o r d i n g to t h e p r o c e d u r e ou t l i ned in C h a p t e r 2 , s e c t i o n 2 . 3 . 5 . 3.3.6 In vitro Procedure I V D M D a n d I V T D a n a l y s e s w e r e d e t e r m i n e d o n t he f e e d s a m p l e s a c c o r d i n g to t he p r o c e d u r e s ou t l i ned in C h a p t e r 2 , s e c t i o n 2 . 3 . 6 . 3.3.7 Statistical Analysis Sta t i s t i ca l a n a l y s e s w e r e c o n d u c t e d u s i n g t he G e n e r a l L i n e a r M o d e l ( G L M ) p r o c e d u r e o f S ta t i s t i ca l A n a l y s i s S y s t e m ( S A S Inst i tute Inc., 1 9 9 0 ) u s i n g the p r o g r a m l i s ted in A p p e n d i x 6 .4 , w h i c h a l s o i n c l u d e s t he m o d e l s t a t e m e n t . In the 9 6 p r e s e n t s t u d y , it w a s a s s u m e d that t h e h i g h e s t o r d e r i n te rac t i on d id not ex is t , but it 's m e a n s q u a r e e r ro r w a s r e p r e s e n t a t i v e o f t he e x p e r i m e n t a l e r ro r ( C o c h r a n a n d C o x , 1 9 5 0 ) . S ta t i s t i ca l l y s i gn i f i can t d i f f e r e n c e s b e t w e e n m e a n s ( P < 0 .05 ) w e r e d e t e r m i n e d u s i n g t h e p r o g r a m w h i c h e m p l o y e d t he u s e o f l e a s t s q u a r e m e a n s for I V D M D , I V T D , N D F a n d A D F m e a s u r e m e n t s . F u r t h e r i n ves t i ga t i on o f t h e r e l a t i onsh ip b e t w e e n a s i gn i f i can t m a i n e f fec t a n d a m e a s u r e d p a r a m e t e r wi th o r t h o g o n a l p o l y n o m i a l s w a s c o n d u c t e d u s i n g t he C O N T R A S T s t a t e m e n t o f S A S P R O C G L M ( S A S , 1 9 9 0 ) a n d r e p o r t e d w h e n t he r e l a t i o n s h i p c o u l d b e d e f i n e d . 3.4 RESULTS AND DISCUSSION D r y ma t t e r ( D M ) c o n t e n t o f t he u n t r e a t e d c a n o l a m e a l s a m p l e w a s 8 9 . 3 % . C r u d e p ro te in ( C P ) c o n t e n t o f t h e s a m p l e w a s 3 6 . 6 % a n d c r u d e fat ( E E ) w a s 3.2 % , bo th p r e s e n t e d a s a po r t i on o f D M . A c i d d e t e r g e n t f i be r ( A D F ) w a s 1 7 . 7 % o f D M a n d t h e neu t ra l d e t e r g e n t f i be r ( N D F ) w a s 2 1 . 8 % o f t he D M f rac t i on . T h e s e resu l t s a r e c o m p a r a b l e w i th t h e v a l u e s repo r t ed b y B e l l a n d K e i t h ( 1 9 9 1 ) f r o m a s u r v e y o f c a n o l a m e a l f r o m 7 c r u s h i n g p l a n t s in W e s t e r n C a n a d a o v e r a 4 w e e k p e r i o d . T h o s e a u t h o r s r e p o r t e d m e a n v a l u e s ( D M b a s i s ) of: 4 1 . 9 % C P , 3 . 9 % E E , 1 9 . 1 % A D F a n d 2 3 . 5 % N D F . 9 7 3.4.1 IVDMD T h e I V D M D o f c a n o l a m e a l w a s s ign i f i can t l y a f f e c t e d ( P = 0 . 0 0 0 1 ) by the in te rac t i on b e t w e e n p r o t e a s e a n d x y l a n a s e ( F i g u r e 3 .1) . F o l l o w i n g a 4 8 h in vitro i n c u b a t i o n , 7 9 . 8 % o f t he u n t r e a t e d c a n o l a m e a l s a m p l e h a d d i s a p p e a r e d ( T a b l e 3 .2) . T h i s is no t m u c h h i g h e r t h a n a v a l u e r e p o r t e d for in situ d r y ma t te r d i s a p p e a r a n c e ( I S D M D ) o f 72 .1 % for a r u m i n a l i n c u b a t i o n o f 2 4 h ( M o s h t a g h i a n d Inga l ls , 1 9 9 5 ) . T h e con t ro l s a m p l e w a s i n t e r m e d i a t e l y r a n k e d w i th r e s p e c t to t h e s a m p l e s t r ea ted w i th p r o t e a s e a n d x y l a n a s e e n z y m e s . P r o t e a s e a p p l i e d a l o n e at 0 . 0 1 % d e c r e a s e d I V D M D f r o m 7 9 . 8 % to 7 7 . 8 % . T h e c o m b i n a t i o n o f 0 . 0 0 5 % p r o t e a s e a n d 0 . 0 0 1 % x y l a n a s e d e c r e a s e d I V D M D f r o m 7 9 . 8 % to 7 8 . 7 % . P r o t e a s e a p p l i e d at 0 . 0 5 % h o w e v e r , i n c r e a s e d I V D M D f r o m 7 9 . 8 % to 8 2 . 4 % . F r o m T a b l e 3 .2 , it is e v i d e n t tha t t h e r e is a s y n e r g i s t i c e f fec t b e t w e e n t h e t w o e n z y m e s w h e n p r o t e a s e is a p p l i e d at 0 . 0 1 % o r 0 . 0 5 % in c o n j u n c t i o n w i th x y l a n a s e . H o w e v e r , p r o t e a s e at ce r ta in l e v e l s h a d a pos i t i ve e f fec t w h e n a p p l i e d w i th x y l a n a s e . T h e s y n e r g y b e t w e e n t h e t w o e n z y m e s is s h o w n wi th t he a p p l i c a t i o n o f e i t he r 0 . 0 1 % o r 0 . 0 5 % p r o t e a s e in c o m b i n a t i o n w i th 0 . 0 0 1 % x y l a n a s e ( T a b l e 3 .2) . T h e c o m b i n a t i o n o f t he t w o h i g h e r l e v e l s o f p r o t e a s e ( 0 . 0 1 % a n d 0 . 0 5 % ) a p p l i e d w i th x y l a n a s e s ign i f i can t l y i n c r e a s e d ( P < 0 . 0 5 ) t h e I V D M D f r o m 7 9 . 8 % to g r e a t e r t h a n 8 1 % . T h i s s y n e r g i s t i c e f fec t w o u l d b e e x p e c t e d to el ic i t a n e f fec t b y d e g r a d i n g t he p ro te in mat r i x a n d o the r res i s tan t p lan t ce l l w a l l c o m p o u n d s . T h e p ro te in mat r i x in c e r e a l s h a s b e e n s h o w n to b e r e s p o n s i b l e fo r d i c ta t i ng t h e ra te a n d ex ten t o f d i g e s t i o n ( M c A l l i s t e r , 1 9 9 0 a , b, c ) . 9 8 T h e c o m b i n a t i o n o f t h e l o w e s t l e ve l o f p r o t e a s e w i th x y l a n a s e s ign i f i can t l y r e d u c e d ( P < 0 .05 ) I V D M D re la t i ve to t he con t ro l . X y l a n a s e a p p l i c a t i o n d i d not h a v e a n e f fec t w h e n a p p l i e d a l o n e . P r o t e a s e a p p l i e d a l o n e at a n i n t e r m e d i a t e l eve l ( 0 . 0 1 % ) p r o d u c e d a s i gn i f i can t l y l o w e r ( P < 0 .05 ) I V D M D v a l u e t h a n t he I V D M D v a l u e s o b t a i n e d w i th t he con t ro l s a m p l e a n d t he l o w e s t a n d h i g h e s t l e v e l s o f p r o t e a s e a p p l i c a t i o n . T h e e f fec t o f m o i s t u r e a p p l i e d in t he a b s e n c e o f h e a t o n t he d iges t ib i l i t y c h a r a c t e r i s t i c s o f c e r e a l g r a i n s , w i th t h e e x c e p t i o n o f s o r g h u m , h a s b e e n f o u n d to b e m i n i m a l (Tai t a n d B e a m e s , 1 9 8 8 ) . In teres t ing ly , in t h e p r e s e n t s t u d y , it w a s d e t e r m i n e d tha t m o i s t u r e h a d a h igh l y s i gn i f i can t q u a d r a t i c r e l a t i onsh ip ( P < 0 .01) w i th t he I V D M D o f c a n o l a m e a l d e s c r i b e d by Y = 7 8 . 6 2 + 0 . 1 6 5 * m o i s t u r e leve l -0 . 0 0 3 * m o i s t u r e l e v e l 2 ( F i g u r e 3 .2) . F r o m the r e g r e s s i o n a n a l y s i s , it c o u l d b e d e t e r m i n e d tha t a n o p t i m a l l e ve l o f 2 6 % m o i s t u r e w o u l d resu l t in a D M d e g r a d a b i l i t y o f 8 1 % . E v e n t h e a p p l i c a t i o n o f t he l o w e s t l eve l o f m o i s t u r e ( 1 0 % ) w a s su f f i c ien t to el ic i t a s i gn i f i can t i n c r e a s e ( P < 0 .05 ) in I V D M D f r o m 7 8 . 6 % to 7 9 . 9 % ( T a b l e 3 .3) . T h e a p p l i c a t i o n o f 2 0 % o r 3 0 % m o i s t u r e bo th p r o d u c e d I V D M D v a l u e s o f 8 0 . 7 % , w h i c h w a s s ign i f i can t l y g r e a t e r t h a n t he con t ro l a n d t h a n that o b t a i n e d w i th t he 1 0 % m o i s t u r e t r e a t m e n t . It h a s b e e n s u g g e s t e d t h e b e n e f i c i a l e f f ec t s o f m o i s t u r e add i t i on m a y b e a t t r ibu ted to t he r e l e a s e o f e n d o g e n o u s e n z y m e s w i th in t h e c e r e a l g ra in ( N e w m a n , 1 9 9 4 ) . T h i s w o u l d p r o v i d e t he oppo r tun i t y fo r t h e c e r e a l to b e par t ly d e g r a d e d pr ior to b e i n g e x p o s e d to m i c r o b i a l a t t ack in t h e r u m e n . A r e c e n t s t u d y repo r t ed t h e 12 h r u m i n a l in situ d r y ma t te r d e g r a d a b i l i t y o f c a n o l a m e a l i n c r e a s e d 9 9 f r o m 4 8 . 9 % to 5 5 . 4 % w i th t h e a d d i t i o n o f 1 5 % m o i s t u r e ( W e u r d i n g , 1 9 9 6 ) . T h e l ack o f r e s p o n s e to t h e m i ld h y d r o t h e r m a l c o n d i t i o n s u s e d in th is s t u d y , m a y b e a t t r ibuted to t he l o w t e m p e r a t u r e ( 5 5 ° C ) . P r e v i o u s s t u d i e s w h i c h h a v e r e p o r t e d h e a t e f fec ts o r e f fec ts f r o m h y d r o t h e r m a l t r e a t m e n t u s e d p r o c e s s i n g t e c h n o l o g i e s i n c l u d i n g s t e a m ro l l ing , s t e a m f l a k i n g , p r e s s u r e c o o k i n g , e x p l o d i n g , e x t r u d i n g o r pe l l e t i ng w h i c h e m p l o y t e m p e r a t u r e in e x c e s s o f 6 0 ° C (Tai t a n d B e a m e s , 1 9 8 8 ) . 3.4.2 IVTD T h e resu l t s o f I V T D d e t e r m i n a t i o n s w e r e s i m i l a r to t he resu l t s o b t a i n e d for I V D M D . T h e i n te rac t i on b e t w e e n p r o t e a s e a n d x y l a n a s e o n I V T D o f c a n o l a m e a l w a s h igh l y s i gn i f i can t ( P < 0 .01 ) ( F i g u r e 3 .3) . H o w e v e r , n o n e o f t h e t r e a t m e n t s s ign i f i can t l y i n c r e a s e d I V T D ( P < 0 .05 ) f r o m the v a l u e o f 8 7 . 9 % o b t a i n e d for the con t ro l ( T a b l e 3 .4) . T h e a p p l i c a t i o n o f 0 . 0 1 % p r o t e a s e s ign i f i can t l y r e d u c e d ( P < 0 .05 ) I V T D f r o m 8 7 . 9 % to 8 7 . 4 % . R e s u l t s f r o m I V D M D fo r th is t r e a t m e n t ref lect a s i m i l a r r e s p o n s e : t h e a d d i t i o n o f x y l a n a s e d id not h a v e a n e f fec t o n I V T D w h i c h is a l s o re f l ec ted in t h e resu l t s o f t h e I V D M D d e t e r m i n a t i o n s . D i f f e r e n c e s in t he sens i t i v i t y o f t he r esu l t s b e t w e e n I V D M D a n d I V T D m a y b e a a t t r ibu ted to the c o m p o n e n t s e a c h o n e e s t i m a t e s . T h e m a j o r d i f f e r e n c e b e t w e e n t he I V T D a n d I V D M D p r o c e d u r e s is in t h e la t ter in vitro tes t , al l ce l l s o l u b l e s a r e e l i m i n a t e d , i n c l u d i n g u n d i g e s t e d m i c r o b i a l ma t t e r ( V a n S o e s t , 1 9 8 2 ) . T h e r e w a s a q u a d r a t i c r e l a t i onsh ip b e t w e e n m o i s t u r e l eve l a n d I V T D ( P = 0 . 0 0 0 1 ) d e s c r i b e d by : Y = 8 7 . 4 5 + 0 . 7 3 * m o i s t u r e l eve l - 0 . 0 0 2 * m o i s t u r e l e v e l 2 1 0 0 ( F i g u r e 3 .4 ) . T h e o p t i m a l l e ve l o f m o i s t u r e , d e t e r m i n e d f r o m t h e r e g r e s s i o n e q u a t i o n , w a s 2 4 % w i th a c o r r e s p o n d i n g I V T D v a l u e o f 8 8 % . T h e a p p l i c a t i o n o f t he l o w e s t m o i s t u r e l eve l ( 1 0 % ) w a s su f f i c ien t to s ign i f i can t l y i n c r e a s e ( P < 0 .05 ) t he I V T D o f c a n o l a m e a l f r o m 8 7 . 3 % to 8 7 . 8 % ( T a b l e 3 .5) . In teres t ing ly , fu r ther i n c r e a s e s in m o i s t u r e l eve l d i d not fu r the r i m p r o v e d e g r a d a b i l i t y . T h i s is in c o n t r a s t to t he f i nd i ngs o f t h e e f fec t o f m o i s t u r e o n I V D M D w h e r e t he a p p l i c a t i o n o f 2 0 % a n d 3 0 % m o i s t u r e bo th r e s u l t e d in a n I V T D v a l u e o f 8 8 . 1 % . A s p r e v i o u s l y m e n t i o n e d , m o i s t u r e m a y p r o m o t e t h e r e l e a s e o f e n d o g e n o u s e n z y m e s w i th in t he g ra i n a l l o w i n g fu r ther d i g e s t i o n o f t h e f e e d b y r u m e n m i c r o b e s ( N e w m a n , 1 9 9 4 ) . In a d d i t i o n , m i c r o b i a l a t t a c h m e n t m a y b e e n h a n c e d in t h e m o i s t e n v i r o n m e n t t h r o u g h s o m e m e c h a n i s m ( C h e n g et a l . , 1 9 9 5 ) . In th is s t u d y , t h e r e w a s a l a c k o f r e s p o n s e in bo th I V D M D a n d I V T D to mi ld h e a t a p p l i e d at 5 5 ° C . R e c o m m e n d a t i o n s f r o m the e n z y m e s u p p l i e r ( F F I , W i l t sh i r e , U K ) s u g g e s t e d a p o s s i b l e i n c r e a s e in e n z y m e act iv i ty u n d e r h igh m o i s t u r e a n d o p t i m a l h e a t c o n d i t i o n s . H o w e v e r , t he resu l t s o f t he p r e s e n t e x p e r i m e n t s u g g e s t that no fu r the r i n c r e a s e in e n z y m e act iv i ty w a s a t t a i ned w i th t h e a p p l i c a t i o n of h e a t o r m o i s t u r e . A p o s s i b l e e x p l a n a t i o n m a y b e that e n z y m e act iv i ty is l im i ted by o the r f a c t o r s w h i c h c o u l d not b e o v e r c o m e w i th th is mi ld h y d r o t h e r m a l t r ea tmen t . T h e s e resu l t s m a y a l s o b e a t t r ibu ted to t he l a c k o f a n y i n c r e a s e in e n z y m e act iv i ty o n t he h y d r o t h e r m a l l y t r e a t e d i ng red ien t a n d w i th t he c o n c e n t r a t i o n s a n d / o r c o m b i n a t i o n s o f e n z y m e s u s e d in t h e p r e s e n t s t u d y . 101 T h e a p p l i c a t i o n o f h e a t at 5 5 ° C fo r a n y o f t he s e l e c t e d t i m e p e r i o d s d id not a f fec t d iges t ib i l i t y , h o w e v e r , m o i s t u r e i n c r e a s e d t he d iges t ib i l i t y o f c a n o l a m e a l . In g e n e r a l , t h e r e s p o n s e to e n z y m e add i t i on w a s i n c o n s i s t e n t , h o w e v e r , t h e pos i t i ve e f f ec t s tha t w e r e o b s e r v e d w e r e p r imar i l y a s a resu l t o f t he add i t i on o f p r o t e a s e . 3.4.3 A D F A l a r g e n u m b e r o f i n t e rac t i ons w e r e f o u n d in t he in te rp re ta t ion o f f i be r resu l t s w h i c h re f l ec ted t r e a t m e n t d i f f e r e n c e s . A s ign i f i can t th i rd o r d e r i n te rac t i on ( P < 0 .05) w a s n o t e d fo r d u r a t i o n o f t h e h e a t t r ea tmen t , a n d t he a p p l i c a t i o n o f p r o t e a s e a n d x y l a n a s e o n t h e c o n t e n t o f A D F ( F i g u r e 3 .5) . F r o m T a b l e 3 .6 , it is e v i d e n t that o n l y 3 t r e a t m e n t s r e d u c e d t h e c o n t e n t o f A D F in c a n o l a m e a l , a l t h o u g h n o n e o f t h e s e t r e a t m e n t m e a n s w e r e s ign i f i can t l y d i f ferent f r o m o n e a n o t h e r ( P < 0 .05 ) . T h e a p p l i c a t i o n o f h e a t fo r 1 5 m in r e d u c e d A D F f r o m 2 0 . 1 % to 1 8 . 9 % o f D M . T h e a p p l i c a t i o n o f p r o t e a s e at 0 . 0 0 5 % r e d u c e d A D F f r o m 2 0 . 1 % to 1 9 . 3 % o f t he D M a n d t he c o m b i n a t i o n o f 6 0 m i n o f h e a t w i th 0 . 0 0 5 % p r o t e a s e r e d u c e d A D F to 1 9 . 1 % o n a D M b a s i s . A n i n c r e a s e to g r e a t e r t h a n 2 1 % o f t he D M in t he A D F c o n t e n t w a s o b s e r v e d w i th t w o t r e a t m e n t s w h e r e h e a t w a s a p p l i e d for 3 0 o r 6 0 m i n p e r i o d s a n d t he a d d i t i o n o f 0 . 0 0 5 % p r o t e a s e a n d 0 . 0 0 1 % x y l a n a s e . T h e c o m b i n a t i o n o f e i the r 0 . 0 1 % o r 0 . 0 5 % p r o t e a s e , x y l a n a s e ( 0 . 0 0 1 % ) , in add i t i on to a n y l eve l o f hea t , p r o d u c e d A D F v a l u e s that w e r e not s ign i f i can t l y d i f fe rent ( P < 0 .05 ) f r o m the con t ro l ( T a b l e 3 .6) . 1 0 2 p r o d u c e d A D F v a l u e s that w e r e not s ign i f i can t l y d i f ferent ( P < 0 .05 ) f r o m the con t ro l ( T a b l e 3 .6) . A s i gn i f i can t i n te rac t i on ( P < 0 .01 ) w a s a l s o n o t e d fo r t he a d d i t i o n o f m o i s t u r e , p r o t e a s e a n d x y l a n a s e ( F i g u r e 3 .6) . T h e A D F c o n t e n t o f t he u n t r e a t e d s a m p l e w a s 1 8 . 3 % o f D M , w h i c h w a s s ign i f i can t l y l o w e r ( P < 0 .05 ) t h a n a n y o t h e r t r e a t m e n t ( T a b l e 3 .7 ) . A l l t r e a t m e n t s r e s u l t e d in A D F m e a n s that w e r e s ign i f i can t l y d i f ferent t h a n t h e u n t r e a t e d s a m p l e w i th t he e x c e p t i o n o f t h e a p p l i c a t i o n o f 0 . 0 1 % p r o t e a s e . I n c r e a s i n g t h e l eve l o f m o i s t u r e a p p l i e d to c a n o l a m e a l g e n e r a l l y i n c r e a s e d the A D F c o n t e n t w i th a f e w e x c e p t i o n s ( T a b l e 3 .7) . T h e add i t i on o f 2 0 % o r 3 0 % m o i s t u r e , i r r e g a r d l e s s o f e n z y m e t r ea tmen t , s ign i f i can t l y i n c r e a s e d A D F ( P < 0 .05 ) re la t i ve to t he con t ro l . A n i n c r e a s e in t h e A D F c o n t e n t m a y b e a t t r ibu ted to ar t i fact f i be r f o r m e d d u r i n g t h e h e a t i n g p r o c e s s . H o w e v e r , th i s e x p l a n a t i o n is un l i ke l y a s p re l im ina ry e x p e r i m e n t s e v a l u a t i n g A D I N c o n t e n t , w h i c h h a s b e e n s h o w n to b e a n i nd i ca to r of ar t i fact f iber , f o u n d n o e f fec t o f t r e a t m e n t s o n th is p a r a m e t e r . A n o t h e r m o r e l ike ly e x p l a n a t i o n is tha t t he p ropo r t i on of s o l u b l e f e e d c o m p o n e n t s d e c r e a s e d w i thou t a c h a n g e in f i be r c o m p o s i t i o n . T r e a t m e n t s m a y h a v e a f f e c t e d t he n o n - f i b r o u s c o m p o n e n t s o f c a n o l a m e a l . A s f i be r r esu l t s a r e p r e s e n t e d a s a por t ion o f the D M , th is w o u l d resu l t in p ropo r t i ona l l y g r e a t e r a m o u n t s o f f i be r b e i n g d e t e c t e d , t h u s a n i n c r e a s e in A D F v a l u e s . T h i s e x p l a n a t i o n w o u l d a l s o e x p l a i n t h e s i m i l a r t r end o b s e r v e d in d iges t ib i l i t y r esu l t s w h i c h p a r a l l e l e d t h o s e o f t he f i be r r esu l t s . 1 0 3 3.4.4 NDF T h e N D F f rac t i on o f c a n o l a m e a l w a s s ign i f i can t l y a f f e c t e d ( P < 0 .05 ) by p r o t e a s e t r e a t m e n t . T r e a t m e n t o f c a n o l a m e a l w i th 0 . 0 0 5 % p r o t e a s e s ign i f i can t l y ( P < 0 .05 ) i n c r e a s e d t h e N D F c o n t e n t f r o m 1 9 . 6 % to 2 0 . 5 % o f D M ( T a b l e 3 .8) . H o w e v e r , t r e a t m e n t w i th h i g h e r l e v e l s o f th is e n z y m e d id not s i gn i f i can t l y a l te r ( P < 0 .05 ) t h e N D F c o n t e n t re la t i ve to t h e con t ro l . It is o f i n te res t to n o t e t h e r e s p o n s e o f N D F to t he l o w e s t l e v e l o f e n z y m e w a s s ign i f i can t , w h e r e a s h i g h e r l e v e l s d id not e l ic i t a r e s p o n s e . A s i m i l a r e x p l a n a t i o n to t he o n e o f f e red fo r t h e i n c r e a s e in A D F c o n t e n t m a y b e a p p l i e d fo r t h e i n c r e a s e in t he N D F con ten t : that t h e r e w a s a n i n c r e a s e in s o l u b l e f e e d c o m p o n e n t s w i thou t a c h a n g e in t he f i be r c o n t e n t . E n z y m e a c t i o n o n f e e d c o m p o n e n t s o t h e r t h a n t h e f i b r o u s por t ion w o u l d b e re f l ec ted in h i g h e r f i be r v a l u e s c o m p a r e d to t he con t ro l . It is un l i ke l y t h e r e w a s c o n t a m i n a t i o n o f t he s a m p l e f r o m f e e d e n t e r i n g t h e b a g d u e to t he s m a l l p o r e s i z e . N D F c o n t e n t w a s a l s o a f f e c t e d ( P = 0 . 0 0 0 1 ) by t he l eve l o f m o i s t u r e a p p l i e d to c a n o l a m e a l . T h e a p p l i c a t i o n o f m o i s t u r e at a n y l e v e l , s i gn i f i can t l y r e d u c e d ( P < 0 .05 ) t h e N D F c o n t e n t o f c a n o l a m e a l ( T a b l e 3 .9) . T h e a d d i t i o n of 2 0 % m o i s t u r e r e d u c e d N D F f r o m 2 2 . 0 % to 2 0 . 3 % of t h e D M . T h e a p p l i c a t i o n o f e i t he r 1 0 % o r 3 0 % m o i s t u r e p r o d u c e d N D F v a l u e s o f 1 8 . 6 % a n d 1 8 . 2 % o f D M , r e s p e c t i v e l y w h i c h w e r e s ign i f i can t l y l o w e r t h a n t h e con t ro l a s we l l a s t he 2 0 % m o i s t u r e t r ea tmen t . T h e m i n o r d i f f e r e n c e s o b s e r v e d in t he A D F a n d N D F d e t e r m i n a t i o n s a m o n g t he v a r i o u s t r e a t m e n t s a r e un l i ke l y to h a v e a n y p rac t i ca l s i g n i f i c a n c e . T h e d i f f e r e n c e s b e t w e e n m e a n s a r e s ign i f i can t d u e to t h e s m a l l s t a n d a r d e r ro r o f the 1 0 4 m e a n ( S . E . M . ) . B e l l a n d K e i t h ( 1 9 9 1 ) o b t a i n e d S . E . M . v a l u e s for A D F a n d N D F of 0 . 24 a n d 0 . 1 6 fo r c a n o l a m e a l o b t a i n e d f r o m s e v e r a l c r u s h i n g p l a n t s w h i c h a r e c o m p a r a b l e to t h o s e o b t a i n e d in t h e p r e s e n t s t u d y . 3.5 CONCLUSIONS F r o m the r esu l t s o f th is s t u d y , it w a s d e t e r m i n e d that x y l a n a s e w a s not e f fec t i ve in t e r m s o f i m p r o v i n g t h e d iges t ib i l i t y o f c a n o l a m e a l . P r o t e a s e , h o w e v e r , s h o w e d po ten t i a l i m p r o v e m e n t s w h e n a p p l i e d at t he h i g h e s t l eve l ( 0 . 0 5 % ) . A l t h o u g h t he c o m b i n a t i o n o f p r o t e a s e a p p l i e d a t 0 . 0 1 % o r 0 . 0 5 % l e v e l s w i th x y l a n a s e o f ten g e n e r a t e d p o s i t i v e r e s p o n s e s , t he m a g n i t u d e o f t he r e s p o n s e w a s n o g r e a t e r t h a n w h e n p r o t e a s e w a s a p p l i e d a l o n e at t he s a m e l e v e l s . H e a t t r e a t m e n t fo r a n y leng th of t i m e w a s not e f f ec t i ve in i m p r o v i n g e n z y m e act iv i ty , n o r d i d it i n d e p e n d e n t l y el ic i t a n y r e s p o n s e f r o m a n y o f t he m e a s u r e d p a r a m e t e r s . M o i s t u r e , o n t he o t h e r h a n d , h a d a n e f fec t o n bo th t h e d iges t ib i l i t y a n d t he f i b r o u s c o m p o n e n t s o f c a n o l a m e a l . R e s u l t s w h i c h i n c r e a s e d t h e in vitro d iges t ib i l i t y o r t he f i be r c o n t e n t m a y b e a t t r ibu tab le to a shi f t in t he s o l u b l e c o m p o n e n t s w h i c h n e e d s to b e fu r ther i n v e s t i g a t e d . T h e h igh n u m b e r o f i n t e rac t i ons o b s e r v e d in t h e p r e s e n t s t u d y m a d e in te rp re ta t ion o f t h e r esu l t s dif f icult . It is o b v i o u s t he e f f ec t s o f e n z y m e t r ea tmen t c o m b i n a t i o n s p r o d u c e d bo th pos i t i ve a n d n e g a t i v e r e s p o n s e s . It is th i s i n c o n s i s t e n c y in t h e resu l t s f r o m the r e s e a r c h w i th e n z y m e s w i th r u m i n a n t d ie t s , c o m b i n e d w i th t he h igh c o s t o f e n z y m e s that wi l l c o n t i n u e to rest r ic t e n z y m e 1 0 5 a p p l i c a t i o n s in th is a r e a . T h e p r e s e n t s t u d y is not c o n c l u s i v e but t h e d a t a m a y b e a d d e d to a g r o w i n g d a t a b a s e to n a r r o w the f ie ld o f r e s e a r c h w i th r u m i n a n t e n z y m e r e s e a r c h a p p l i c a t i o n s . F u r t h e r e v a l u a t i o n o f t he e f f ec t s o f t he t r e a t m e n t s u s i n g o the r in vitro t e c h n i q u e s , s u c h a s t h e g a s p r o d u c t i o n t e c h n i q u e , wi l l a l l o w e x a m i n a t i o n of t he f e r m e n t a t i o n p ro f i l es o f t h e c a n o l a m e a l s a m p l e s a n d wi l l p r o v i d e m o r e i n fo rma t i on o n t h e e f f ec t s o f e n z y m e add i t i on a n d mi ld h y d r o t h e r m a l t r e a t m e n t o f c a n o l a m e a l . 1 0 6 3.6 REFERENCES B e a u c h e m i n , K. A . a n d L. M . R o d e . 1 9 9 6 . U s e o f f e e d e n z y m e s in r um inan t nut r i t ion. P a g e s 1 0 3 - 1 3 0 in L. M . R o d e (ed. ) , A n i m a l R e s e a r c h a n d D e v e l o p m e n t : M e e t i n g F u t u r e C h a l l e n g e s . P r o c . 1 9 9 6 C a n . S o c . A n i m . S c i . A n n . M t g . , L e t h b r i d g e , A l b e r t a , C a n a d a . B e l l , J . M . 1 9 9 3 . F a c t o r s a f fec t ing t he nut r i t ional v a l u e o f c a n o l a m e a l : A rev i ew . C a n . J . A n i m . S c i . , 7 3 : 6 7 9 - 6 9 7 . B e l l , J . M . a n d M . O . K e i t h . 1 9 9 1 . A s u r v e y o f va r i a t i on in t h e c h e m i c a l c o m p o s i t i o n o f c o m m e r c i a l c a n o l a m e a l p r o d u c e d in W e s t e r n C a n a d i a n c r u s h i n g p lan ts . C a n . J . A n i m . S c i . , 7 1 : 4 6 9 - 4 8 0 . B e l l , J . M . a n d A . S h i r e s , 1 9 8 2 . C o m p o s i t i o n a n d d iges t ib i l i t y by p i g s o f hul l f r ac t i ons f r o m r a p e s e e d cu l t i va rs w i th y e l l o w o r b r o w n s e e d c o a t s . C a n . J . A n i m . S c i . , 6 2 : 5 5 7 - 5 6 5 . C h e n g , K . - J . , M c A l l i s t e r , T . A . , S e l i n g e r , L. B . , Y a n k e , L. J . , B a e . H . D., F o r s b e r g , C . W . a n d J . A . S h e l f o r d . 1 9 9 5 . B i o t e c h n o l o g y in t h e feed lo t . J . A n i m . S c i . , 4 6 : 6 0 0 - 6 0 5 . C h e s s o n , A . 1 9 9 3 . F e e d e n z y m e s . A n i m . F e e d S c i . T e c h n o l . , 4 5 : 6 5 - 7 9 . C o c h r a n , W . G . a n d G . M . C o x . 1 9 5 0 . E x p e r i m e n t a l d e s i g n s . P a g e s 1 5 4 - 1 5 6 ; 2 4 1 -2 4 6 . J o h n W i l e y a n d S o n s Inc., N e w Y o r k , N Y . E m a n u e l s o n , M . ( 1 9 9 4 ) . P r o b l e m s a s s o c i a t e d w i th f e e d i n g r a p e s e e d m e a l to da i r y c o w s . P a g e s 1 8 9 - 2 1 4 in R e c e n t A d v a n c e s in A n i m a l Nut r i t i on . G a r n s w o r t h , P . C . a n d D. J . A . C o l e . L o u g h b o r o u g h , L e i c e s t e r s h i r e , N o t t i n g h a m U n i v . P r e s s . G o e r i n g , H . K. a n d P . J . V a n S o e s t . 1 9 7 0 . F o r a g e F i b e r A n a l y s e s . ( A p p a r a t u s , R e a g e n t s , P r o c e d u r e s , a n d s o m e A p p l i c a t i o n s ) . A g r i c . H a n d b o o k N o . 3 7 9 . , A R S - U S D A , W a s h i n g t o n , D C . M c A l l i s t e r , T . A . , C h e n g , K . - J . , R o d e , L. M . a n d J . G . B u c h a n a n - S m i t h . 1 9 9 0 a . U s e o f f o r m a l d e h y d e to r e g u l a t e d i g e s t i o n o f b a r l e y s t a r c h . C a n . J . A n i m . S c i . , 7 0 : 5 8 1 - 5 8 9 . M c A l l i s t e r , T . A . , R o d e , L. M . , M a j o r , D. J . , C h e n g , K . - J . a n d J . G . B u c h a n a n - S m i t h . 1 9 9 0 b . E f f ec t o f r u m i n a l c o l o n i z a t i o n o n c e r e a l g r a i n d i g e s t i o n . C a n . J . A n i m . S c i . , 7 0 : 5 7 1 - 5 7 9 . 1 0 7 M c A l l i s t e r , T . A . , R o d e , L. M . , C h e n g , K . - J . , S c h a e f e r , D. M . a n d J . W . C o s t e r t o n . 1 9 9 0 c . M o r p h o l o g i c a l s t u d y o f t he d i g e s t i o n o f b a r l e y a n d m a i z e g ra in by r u m e n m i c r o o r g a n i s m s . A n i m . F e e d S c i . T e c h n o l . , 3 1 : 9 1 - 1 0 5 . M o s h t a g h i N i a , S . A . a n d J . R . Inga l ls . 1 9 9 5 . E v a l u a t i o n o f m o i s t h e a t t r e a t m e n t of c a n o l a m e a l o n d i g e s t i o n in t he r u m e n , s m a l l i n tes t i ne , l a rge in tes t i ne a n d to ta l d i g e s t i v e t rac t o f s t e e r s . C a n . J . A n i m . S c i . , 7 5 : 2 7 9 - 2 8 3 . N e w m a n , C . W . 1 9 9 4 . T h e U n i t e d S t a t e s m a r k e t fo r f e e d e n z y m e s : W h a t o p p o r t u n i t i e s e x i s t ? In: B i o t e c h n o l o g y in t he F e e d Indust ry . P r o c . o f A l l t e c h ' s T e n t h A n n u a l S y m p o s i u m . N o t t i n g h a m U n i v e r s i t y P r e s s , p p . 9 9 . S ta t i s t i ca l A n a l y s i s S y s t e m ( S A S ) , 1 9 9 0 . S A S U s e r ' s G u i d e : S t a t i s t i c s . S A S Insti tute Inc., C a r y , N C . Ta i t , R. M . a n d R. M . B e a m e s . 1 9 8 8 . P r o c e s s i n g a n d p r e s e r v a t i o n o f c e r e a l s a n d p ro te in c o n c e n t r a t e s . P a g e s 1 5 1 - 1 7 5 in F e e d S c i e n c e W o r l d A n i m a l S c i e n c e ( B 4 ) , E . R . O r s k o v , (ed . ) , E l s e v i e r S c i . P u b l . B . V . A m s t e r d a m , N e t h e r l a n d s . T i l l ey , J . M . A . a n d R. A . T e r r y . 1 9 6 3 . A t w o - s t a g e t e c h n i q u e fo r t h e in vitro d i g e s t i o n o f f o r a g e c r o p s . Br i t . J . G r a s s l . S o c , 18 : 1 0 4 - 1 1 1 . V a n S o e s t , P . J . 1 9 8 2 . Nut r i t i ona l e c o l o g y o f t he rum inan t . O & B B o o k s Inc., C o r v a l l i s , O r e g o n . V a n S o e s t , P . J . , R o b e r t s o n , J . B . a n d B . A . L e w i s . 1 9 9 1 . M e t h o d s o f d ie ta ry f iber , neu t ra l d e t e r g e n t f i be r a n d n o n s t a r c h p o l y s a c c h a r i d e s in re la t ion to a n i m a l nut r i t ion. J . D a i r y S c i . , 7 4 : 3 5 8 3 - 3 5 9 7 . W e u r d i n g , R . E . 1 9 9 6 . T h e e f fec t o f h e a t a n d m o i s t u r e o n t h e d iges t ib i l i t y o f dry ma t t e r a n d c r u d e p ro te in o f b a r l e y a n d c a n o l a in t h e r u m e n a n d l o w e r g a s t r o i n t e s t i n a l t ract o f r u m i n a n t s . M . S c . T h e s i s . D e p t . A n i m . S c i . W a g e n i n g e n Inst i tute o f A n i m a l S c i e n c e A g r i c u l t u r a l Un i ve r s i t y , T h e N e t h e r l a n d s . 1 0 8 T a b l e 3 . 1 . T r e a t m e n t s i n v e s t i g a t e d in o r d e r to d e t e r m i n e t h e e f f ec t s o f e x o g e n o u s e n z y m e a p p l i c a t i o n w i th a n d w i thou t a d d e d m o i s t u r e a n d h e a t o n in vitro d ry mat te r d i s a p p e a r a n c e a n d in vitro t rue d iges t ib i l i t y o f c a n o l a m e a l . F a c t o r L e v e l o f a p p l i c a t i o n H e a t 1 con t ro l (0), 1 5 , 3 0 , 6 0 m in M o i s t u r e 2 0 % , 1 0 % , 2 0 % , 3 0 % P r o t e a s e 1 ,2, 3, 4 0 % , 0 . 0 0 5 % , 0 . 0 1 % , 0 . 0 5 % X y l a n a s e : ,2, 3, 5 0 % , 0 . 0 0 1 % 1 A p p l i e d a t 5 5 ° C . 2 A p p l i e d o n a % v o l * w t 1 b a s i s . 3 S u p p l i e d b y F i n n F e e d s In te rna t iona l L t d . , W i l t s h i r e , U . K . 4 H e p t e x s b - p r o t e a s e . 5 A v i z y m e 1 3 1 0 . 1 0 9 T a b l e 3 .2 . T h e e f fec t o f t he a d d i t i o n o f p r o t e a s e a n d x y l a n a s e to c a n o l a m e a l o n t he 4 8 h r u m e n in vitro dry ma t t e r d i s a p p e a r a n c e o f c a n o l a m e a l ( D M b a s i s ) . E n z y m e T r e a t m e n t 1 P r o t e a s e (%) X y l a n a s e (%) I V D M D 2 (%) 0 0 7 9 . 8 C 0 . 0 0 5 0 7 9 . 1 b c 0.01 0 7 7 . 8 a 0 . 0 5 0 8 2 . 4 e 0 0 .001 7 9 . 4 b c 0 . 0 0 5 0 .001 7 8 . 7 a b 0.01 0 .001 8 1 . 5 d 0 . 0 5 0 .001 8 1 . 1 d S . E . M . 3 0.31 1 A p p l i e d o n a % v o l * w t "1 b a s i s . 2 I V D M D , in vitro d r y ma t t e r d i s a p p e a r a n c e . 3 S . E . M . , p o o l e d s t a n d a r d e r ro r o f t he m e a n . a d M e a n s in a c o l u m n w i th d i f fe rent s u p e r s c r i p t s a r e s ign i f i can t l y d i f fe rent (P < 0 . 0 5 ) . 1 1 0 T a b l e 3 . 3 . T h e e f fec t o f m o i s t u r e a d d e d to c a n o l a m e a l at t he t i m e o f p r o c e s s i n g o n the 4 8 h r u m e n in vitro d r y ma t te r d iges t ib i l i t y o f c a n o l a m e a l ( D M b a s i s ) . M o i s t u r e 1 (%) I V D M D 2 (%) 0 7 8 . 6 a 10 7 9 . 9 b 2 0 8 0 . 7 C 3 0 8 0 . 7 C S . E . M . 3 0 . 2 2 1 A p p l i e d o n a % v o l * w t "1 b a s i s . 2 I V D M D , in vitro d r y ma t t e r d i s a p p e a r a n c e . 3 S . E . M . , p o o l e d s t a n d a r d e r ro r o f t h e m e a n . a _ c M e a n s in a c o l u m n wi th d i f fe rent s u p e r s c r i p t s a r e s ign i f i can t l y d i f ferent (P < 0.05). 111 T a b l e 3 .4 . T h e e f fec t o f t h e a d d i t i o n o f p r o t e a s e a n d x y l a n a s e to c a n o l a m e a l o n the r u m e n in vitro t rue d iges t ib i l i t y o f c a n o l a m e a l ( D M b a s i s ) . E n z y m e T r e a t m e n t 1 P r o t e a s e (%) X y l a n a s e (%) I V T D 2 (%) 0 0 8 7 . 9 b c 0 . 0 0 5 0 8 7 . 6 a b 0.01 0 8 7 . 4 a 0 . 0 5 0 8 8 . 2 C 0 0 .001 8 7 . 8 a b c 0 . 0 0 5 0 .001 8 7 . 5 a b 0.01 0 .001 8 8 . 2 C 0 . 0 5 0 .001 8 7 . 9 b c S . E . M . 0 . 1 8 1 A p p l i e d o n a % v o l * w t 1 b a s i s . 2 I V T D , in vitro t r ue d iges t ib i l i t y . 3 S . E . M . , p o o l e d s t a n d a r d e r ro r o f t he m e a n . ^ M e a n s in a c o l u m n w i th d i f fe rent s u p e r s c r i p t s a r e s ign i f i can t l y d i f fe rent (P < 0.05). 1 1 2 T a b l e 3 . 5 . T h e e f fec t o f m o i s t u r e a d d e d to c a n o l a m e a l at t he t i m e of p r o c e s s i n g o n the r u m e n in vitro t r ue d iges t ib i l i t y o f c a n o l a m e a l ( D M b a s i s ) . M o i s t u r e 1 (%) I V T D 2 (%) 0 8 7 . 3 a 10 8 7 . 8 b 2 0 8 8 . 1 b 3 0 8 8 . 1 b S . E . M . 3 0 . 1 3 1 A p p l i e d o n a % vo l * wt 1 b a s i s . 2 I V T D , in vitro t r ue d iges t ib i l i t y . 3 S . E . M . , p o o l e d s t a n d a r d e r ro r o f t h e m e a n . a b M e a n s in a c o l u m n w i th d i f fe rent s u p e r s c r i p t s a r e s ign i f i can t l y d i f fe rent (P < 0.05). 1 1 3 T a b l e 3 .6 . T h e e f fec t o f t he d u r a t i o n o f h e a t t r e a t m e n t a n d t he a p p l i c a t i o n o f p r o t e a s e a n d x y l a n a s e to c a n o l a m e a l o n t h e A D F f rac t i on o f c a n o l a m e a l ( D M b a s i s ) . F e e d Treatment Durat ion of heat t reatment 1 (min) P r o t e a s e 2 (%) X y l a n a s e 2 (%) A D F 3 (% 0 0 0 2 0 1 def 15 0 0 18 .9 a 30 0 0 2 0 . 0 c d e f 60 0 0 •j g a^bcd 0 0.005 0 1 9 . 3 a b c 15 0.005 0 2 0 . 3 e f 9 30 0.005 0 19 6 a b c d e 60 0.005 0 1 9 . 1 a b 0 0.01 0 19 8 b c d e f 15 0.01 0 20 o c d e f 30 0.01 0 2 0 . 0 c d e f 60 0.01 0 19 8 b c d e f 0 0.05 0 2 0 . 2 e f g 15 0.05 0 2 0 1 def 30 0.05 0 19 6a ' ) c d e 60 0.05 0 20.4ef9 0 0 0.001 2 0 . 3 e f g 15 0 0.001 2 0 . 2 d e f g 30 0 0.001 2 0 . 3 e f g 60 0 0.001 2 0 . 5 f g 0 0.005 0.001 .j g yabcdef 15 0.005 0.001 2 0 . 0 c d e f 30 0.005 0.001 2 1 . 0 9 60 0.005 0.001 2 1 . 2 g 0 0.01 0.001 2 0 . 0 c d e f 15 0.01 0.001 •| g yabcde 30 0.01 0.001 -| g ybcdef 60 0.01 0.001 2 0 . 0 c d e f 0 0.05 0.001 1 g yabcdef 15 0.05 0.001 •j g gbcdef 30 0.05 0.001 2 0 . 3 e f g 60 0.05 0.001 2 0 . 5 f g S . E . M . 4 0.27 1 Applied at 55°C. 2 Applied on a % vol * wt ' 1 basis. 3 ADF, acid detergent fiber. 4 S.E.M., pooled standard error of the mean. 3 9 Means in a column with different superscripts are significantly different (P < 0.05). 114 T a b l e 3 .7 . T h e e f fec t o f m o i s t u r e , p r o t e a s e a n d x y l a n a s e a p p l i c a t i o n to c a n o l a m e a l o n t he A D F f rac t i on o f c a n o l a m e a l ( D M b a s i s ) . F e e d Treatment 1 Moisture (%) P ro tease (%) X y l a n a s e (%) A D F 2 (%) 0 0 0 1 8 . 3 a b 10 0 0 1 9 . 3 c d 20 0 0 2 0 . 5 f g h i j 30 0 0 2 0 . 5 f g h i j 0 0.005 0 1 9 . 3 c d e 10 0.005 0 1 9 . 4 c d e 20 0.005 0 1 9 . 7 c d e 30 0.005 0 -| g gdefghi 0 0.01 0 18 .0 a 10 0.01 0 1 9 . 5 c d e 20 0.01 0 20 .9 i k l 30 0.01 0 21 .2 i k l 0 0.05 0 19 .0 b o 10 0.05 0 -|g gcdefg 20 0.05 0 20 .6 h i f k 30 0.05 0 21 .1 i k l 0 0 0.001 1 9 . 2 c d 10 0 0.001 1 9 . 6 c d e 20 0 0.001 20 .6 i j k 30 0 0.001 21.8' 0 0 .005 0.001 1 9 . 4 c d e 10 0.005 0.001 -|g gdefghi 20 0.005 0.001 21 .2 i k l 30 0.005 0.001 21 .4 k l 0 0.01 0.001 1 9 4 c d e 10 0.01 0.001 •j g gdefgh 20 0.01 0.001 1 9 . 7 c d e f 30 0.01 0.001 2 0 . 5 9 h i j 0 0.05 0.001 19 .2 c d 10 0.05 0.001 20 i e f 9 h i 20 0.05 0.001 2 0 . 5 9 h i i 30 0.05 0.001 20 .6 h i j k S . E . M . 3 0.27 1 Applied on a % vol * wt "1 basis. 2 ADF, acid detergent fiber. 3 S .E .M. , pooled standard error of the mean. a"' Means in a column with different superscripts are significantly different (P < 0.05). 1 1 5 T a b l e 3 .8 . T h e e f fec t o f t h e l eve l o f p r o t e a s e a p p l i c a t i o n to c a n o l a m e a l at t he t ime o f p r o c e s s i n g o n t h e N D F f rac t i on in c a n o l a m e a l ( D M b a s i s ) . P r o t e a s e 1 (%) 0 0 . 0 0 5 0.01 0 . 0 5 S . E . M . 3 N D F 2 (%) 1 9 . 6 a 2 0 . 5 b 1 9 . 4 a 1 9 . 4 a 0 . 2 5 1 A p p l i e d o n a % v o l * wt "1 b a s i s . 2 N D F , neu t ra l d e t e r g e n t f iber . 3 S . E . M . , p o o l e d s t a n d a r d e r ro r o f t h e m e a n . a b M e a n s in a c o l u m n w i th d i f fe rent s u p e r s c r i p t s a r e s ign i f i can t l y d i f fe rent (P < 0.05). 1 1 6 T a b l e 3 .9 . T h e e f fec t o f m o i s t u r e a d d i t i o n to c a n o l a m e a l at t he t i m e o f p r o c e s s i n g o n t h e N D F f rac t i on in c a n o l a m e a l ( D M b a s i s ) . M o i s t u r e 1 (%) N D F 2 (%) 0 2 2 . 1 ° 10 1 8 . 6 a 2 0 2 0 . 3 b 3 0 1 8 . 2 a S . E . M . 3 0 . 2 5 1 A p p l i e d o n a % v o l * wt 1 b a s i s . 2 N D F , neu t ra l d e t e r g e n t f iber . 3 S . E . M . , p o o l e d s t a n d a r d e r ro r o f t h e m e a n . a c M e a n s in a c o l u m n w i th d i f fe rent s u p e r s c r i p t s a r e s ign i f i can t l y d i f fe rent (P < 0.05). 1 1 7 0 0.01 0 . 0 2 0 . 0 3 0 . 0 4 0 . 0 5 P r o t e a s e l eve l (% vo l / wt) F i g u r e 3 . 1 . T h e e f fec t o f t he add i t i on o f x y l a n a s e a n d p r o t e a s e (% v o l / wt ) o n t he 4 8 h r u m e n in vitro d r y ma t te r d i s a p p e a r a n c e o f c a n o l a m e a l . 1 1 8 81 -r 7 7 . 5 J 1 1 h-0 10 2 0 3 0 M o i s t u r e l eve l (% v o l / wt) F i g u r e 3 .2 . Q u a d r a t i c r e l a t i onsh ip b e t w e e n m o i s t u r e l eve l a n d t h e 4 8 h in vitro d r y ma t te r d i s a p p e a r a n c e of c a n o l a m e a l (Y = 7 8 . 6 2 + 0 . 1 6 5 * m o i s t u r e l eve l - 0 . 0 0 3 * m o i s t u r e l e v e l 2 , n = 4 , R 2 = 0 . 9 8 8 , S . E . = 0 . 1 0 5 ) . 1 1 9 F i g u r e 3 . 3 . T h e e f fec t o f t he add i t i on o f p r o t e a s e a n d x y l a n a s e (% v o l / wt ) o n t he r u m e n in vitro t rue d iges t ib i l i t y o f c a n o l a m e a l . 1 2 0 8 8 . 2 -r 8 6 . 8 -\ 1 1 ; : h 0 10 2 0 3 0 M o i s t u r e l eve l (% vo l / wt ) F i g u r e 3 .4 . Q u a d r a t i c r e l a t i onsh ip b e t w e e n m o i s t u r e l eve l (% v o l / wt ) a n d t he r u m e n in vitro t rue d iges t ib i l i t y o f c a n o l a m e a l ( Y = 8 7 . 2 5 + 0 . 0 7 3 * m o i s t u r e l eve l - 0 . 0 0 2 * m o i s t u r e l e v e l 2 : n = 4 , R 2 = 0 . 9 9 1 , S . E . = 0 . 0 3 5 5 ) . 121 F i g u r e 3 . 5 . T h e e f fec t o f t he a p p l i c a t i o n o f p r o t e a s e ( P ) , x y l a n a s e (X ) (% vo l / wt) a n d du ra t i on o f h e a t t r e a t m e n t o n t h e c o n t e n t o f a c i d d e t e r g e n t f i be r in c a n o l a m e a l . 1 2 2 2 3 2 2 M o i s t u r e l eve l (% vo l / wt ) F i g u r e 3 .6 . T h e e f fec t o f t h e a p p l i c a t i o n o f m o i s t u r e , p r o t e a s e ( P ) a n d x y l a n a s e (X ) (% vo l / wt ) o n t he c o n t e n t o f a c i d d e t e r g e n t f i be r in c a n o l a m e a l . 1 2 3 CHAPTER 4 4.0 THE EFFECT OF ENZYME AND MILD HYDROTHERMAL TREATMENT ON THE IN VITRO GAS PRODUCTION IN CANOLA MEAL. 4.1 ABSTRACT T h e h i g h f i be r c o n t e n t o f c a n o l a m e a l l imi ts i t 's i n c l u s i o n in da i r y ca t t le d ie ts . A b u n d a n t in C a n a d a a n d g r o w n w o r l d w i d e , c a n o l a m e a l is a c o s t e f fec t i ve a l t e rna t i ve p ro te in s u p p l e m e n t to s o y b e a n m e a l . T h e o b j e c t i v e o f th is r e s e a r c h w a s to i n v e s t i g a t e t h e feas ib i l i t y o f u s i n g e n z y m e t e c h n o l o g y a n d mi ld h y d r o t h e r m a l t r e a t m e n t in o r d e r to i m p r o v e t he nutr i t ional v a l u e o f c a n o l a m e a l . T h e e x p e r i m e n t c o n s i s t e d o f a 4 x 4 x 4 x 2 mul t i - fac tor ia l d e s i g n . T h e in vitro g a s p r o d u c t i o n t e c h n i q u e a n d V F A a n a l y s e s w e r e u s e d to a s s e s s t he r e s p o n s e o f c a n o l a m e a l s u b j e c t e d to t h e f o l l o w i n g t r e a t m e n t s : h e a t a p p l i e d a t 5 5 ° C fo r 1 5 , 3 0 o r 6 0 m in p e r i o d s o r not a p p l i e d at a l l ; m o i s t u r e a p p l i e d at 0 % , 1 0 % , 2 0 % o r 3 0 % ; p r o t e a s e {Bacillus sp.) a p p l i e d at 0 % , 0 . 0 0 5 % , 0 . 0 1 % o r 0 . 0 5 % ; a n d x y l a n a s e a p p l i e d at 0 % o r 0 . 0 0 1 % (Trichoderma sp.). G a s p a r a m e t e r s w e r e e s t i m a t e d b y fitt ing t he g a s p r o d u c t i o n d a t a a c q u i r e d o v e r 18 h, to t he s u b s t r a t e l im i ted e x p o n e n t i a l g row th m o d e l w i th l a g (Y = A ( 1 - e'b<t'l)). T o t a l V F A p r o d u c t i o n a n d V F A ra t ios w e r e d e t e r m i n e d f r o m o n e o f t h e i n c u b a t i n g s a m p l e s fo r e a c h t r ea tmen t . T h e a p p l i c a t i o n of h e a t ( 5 5 ° C ) d i d not i m p r o v e e n z y m e act iv i ty a s d e t e r m i n e d b y t he in vitro g a s p r o d u c t i o n t e c h n i q u e a n d c o r r e s p o n d i n g V F A d a t a . T h e rate o f g a s p r o d u c t i o n w a s 1 2 4 not a f f e c t e d b y t h e a d d i t i o n o f x y l a n a s e . T h e a p p l i c a t i o n o f 0 . 0 1 % p r o t e a s e s ign i f i can t l y i n c r e a s e d ( P = 0 . 0 0 0 1 ) t h e ra te o f g a s p r o d u c t i o n f r o m 0 . 0 7 7 m L / h 1 to 0 . 1 0 8 m L / h 1 . T h e l eng th o f t he l ag p h a s e w a s a f f e c t e d by t he in te rac t i on b e t w e e n p r o t e a s e a n d x y l a n a s e ( P < 0 .05 ) . T h e r e w a s a l s o a s i gn i f i can t i n te rac t i on ( P < 0 .01 ) b e t w e e n p r o t e a s e a n d x y l a n a s e w h i c h i n c r e a s e d t h e p r o d u c t i o n V F A s . T h e rat io of a c e t a t e to p r o p i o n a t e w a s a f f e c t e d b y p r o t e a s e ( P < 0 .01 ) a n d x y l a n a s e ( P = 0 . 0 0 0 1 ) a p p l i c a t i o n . T h e rat io o f bu ty ra te to p r o p i o n a t e w a s a l s o a f f ec ted by p r o t e a s e ( P < 0 . 0 1 ) a n d x y l a n a s e a p p l i c a t i o n ( P = 0 . 0 0 0 1 ) , i n d e p e n d e n t l y . T h e 0 . 0 5 % p r o t e a s e t r e a t m e n t s ign i f i can t l y i n c r e a s e d ( P < 0 .05 ) t he p ropor t i on of a c e t a t e to p r o p i o n a t e o r bu ty ra te b e i n g p r o d u c e d . X y l a n a s e a p p l i e d to c a n o l a m e a l at 0 . 0 0 1 % a l s o r e s u l t e d in a n i n c r e a s e in t he p ropo r t i on o f a c e t a t e re la t i ve to p r o p i o n a t e o r bu ty ra te . In vitro r esu l t s o b t a i n e d in t he p r e s e n t e x p e r i m e n t s u g g e s t tha t e n z y m e s u p p l e m e n t a t i o n o f c a n o l a m e a l m a y b e a n a l t e rna t i ve p r o c e s s in i m p r o v i n g t h e ra te o f g a s a n d V F A p r o d u c t i o n , in a d d i t i o n to d e c r e a s i n g t h e leng th o f t he l ag p h a s e . In pa r t i cu la r , t h e resu l t s o f th is s t u d y i n d i c a t e that t he a p p l i c a t i o n of p r o t e a s e a n d x y l a n a s e , at o p t i m a l l e v e l s , m a y b e u s e d to i m p r o v e t he nutr i t ional v a l u e o f c a n o l a m e a l fo r da i r y ca t t le . 1 2 5 4.2 INTRODUCTION C a n o l a m e a l a s a f eeds tu f f is h i gh in p ro te in m a k i n g it t he p r i m a r y a l te rna t i ve to s o y b e a n m e a l in d i e t s for l ac ta t i ng d a i r y c o w s . It's l e v e l in r u m i n a n t d i e t s is l im i ted h o w e v e r , d u e to it 's h i g h f i be r c o n t e n t re la t i ve to tha t o f s o y b e a n m e a l . T h e l o w e r a v a i l a b l e e n e r g y c o n t e n t o f c a n o l a m e a l is re la ted to f i be r c o n t e n t , c o m p o s i t i o n o f ce l l wa l l p o l y s a c c h a r i d e s a n d t h e i m p a c t o f t h e s e f r a c t i o n s o n t he u t i l i za t ion of both t h e p ro te in a n d e n e r g y c o m p o n e n t s o f t h e f eeds tu f f ( B r e n e s et a l . , 1 9 9 3 ) . S l o m i n s k i a n d C a m p b e l l ( 1 9 9 0 ) i n v e s t i g a t e d t h e c a r b o h y d r a t e c o n t e n t of c a n o l a m e a l a n d f o u n d that 8 7 % o f t h e g l u c o s e r e l e a s e d d u r i n g e n z y m a t i c h y d r o l y s i s w a s d e r i v e d f r o m c e l l u l o s e . H i g h a m o u n t s o f x y l o s e i n d i c a t e d the p r e s e n c e o f x y l a n s a n d x y l o g l u c a n s a n d l a r g e a m o u n t o f u r o n i c a c i d r e s i d u e s ( 3 0 % ) s u g g e s t e d tha t t h e p o l y s a c c h a r i d e s in t he c e l l u l o s i c f rac t i on a l s o i n c l u d e d a l a rge a m o u n t o f p e c t i n s ( S l o m i n s k i a n d C a m p b e l l , 1 9 9 0 ) . P e c t i n s a r e g e n e r a l l y r e g a r d e d a s read i l y f e r m e n t a b l e in t h e r u m e n , h o w e v e r , in a h igh p r o d u c i n g c o w w h e r e r u m e n p a s s a g e ra te i s o f t e n g r e a t e r t h a n 8 % h" 1 a n d t h e r e s i d e n c e t i m e o f t h e f e e d in t h e r u m e n is l e s s t h a n 10 h, c o m p l e t e d e g r a d a t i o n o f p e c t i c s u b s t a n c e s is un l i ke l y ( V a n S o e s t , 1 9 8 2 ) . F o r t h e s a m e r e a s o n s , x y l a n s a n d x y l o g l u c a n s a l s o m a y not b e c o m p l e t e l y d e g r a d e d w i th in t h e sho r t r e s i d e n c e p e r i o d in t h e r u m e n . In add i t i on , l i n e a r x y l a n s a r e e a s i l y d e g r a d e d b y e n z y m e s in t h e r u m e n w h e r e a s b r a n c h e d a n d h igh ly s u b s t i t u t e d x y l a n s a r e l e s s r ead i l y h y d r o l y z e d ( V a n S o e s t , 1 9 9 4 ) . T h e f i b rous c a r b o h y d r a t e f rac t i on o f c a n o l a m e a l h a s b e e n d e t e r m i n e d to b e t he m a i n 1 2 6 con t r i bu t i ng f a c t o r to it 's l o w e n e r g y ava i lab i l i t y in r u m i n a n t a n i m a l s a n d is the re fo re a n impo r tan t f a c t o r in f e e d f o r m u l a t i o n ( B e l l , 1 9 9 3 ) . M a n y s t u d i e s h a v e s h o w n t he benef i t o f a d d i n g e x o g e n o u s e n z y m e s to c o n c e n t r a t e s f e d to r u m i n a n t s , a s w e l l a s d e l e t e r i o u s e f f ec t s . S e v e r a l s t u d i e s w e r e i n c o n c l u s i v e in the i r r e s u l t s . F o r a br ie f r e v i e w o f t he l i terature t he r e a d e r is re fe r red to C h a p t e r 1, s e c t i o n s 1.1.4.2. i i a n d 1.1.4.3. i i . A r e v i e w o f t he l i te ra ture r e v e a l s t he re h a s b e e n l im i ted i nves t i ga t i on into t he p o s s i b l e a p p l i c a t i o n o f e x o g e n o u s e n z y m e s a p p l i e d in pa r t i cu l a r to c a n o l a m e a l in o r d e r to i m p r o v e it 's nut r i t iona l v a l u e w h e n fed to r u m i n a n t s . P r e l i m i n a r y a s s e s s m e n t o f t he e f fec ts o f mi ld h y d r o t h e r m a l a n d e n z y m e t r e a t m e n t o n t he nut r i t iona l v a l u e o f c a n o l a m e a l w a s c o n d u c t e d u s i n g in vitro a n a l y s e s ( re fer to C h a p t e r 3) . In vitro f e r m e n t a t i o n t e c h n i q u e s h a v e b e e n s u c c e s s f u l l y u s e d in the e v a l u a t i o n o f f o r a g e s fo r r u m i n a n t s for d e c a d e s (T i l ley a n d T e r r y , 1 9 6 3 ; G o e r i n g a n d V a n S o e s t , 1 9 7 0 ) . G a s p r o d u c t i o n in pa r t i cu la r h a s b e e n s h o w n to b e ind ica t i ve o f the d iges t ib i l i t y a n d f e r m e n t a t i o n o f a va r ie t y o f f e e d s t u f f s a s it m e a s u r e s the a p p e a r a n c e o f f e r m e n t a t i o n p r o d u c t s ra the r t h a n the i r d i s a p p e a r a n c e o v e r t ime ( M e n k e e t a l . , 1 9 7 9 ; M e n k e a n d S t e i n g a s s , 1 9 8 8 ; B l u m m e l a n d O r s k o v , 1 9 9 3 ; O p a t p a t a n a k i t e t a l . , 1 9 9 4 , K r i s h n a m o o r t h y et a l . , 1 9 9 5 ) . T h e p r o d u c t i o n o f g a s ( C 0 2 a n d C H 4 ) a r i s e s a s a resu l t o f t h e f e r m e n t a t i o n o f c a r b o h y d r a t e s by r u m e n m i c r o b e s to vo la t i l e fat ty a c i d s ( V F A ) , p r imar i l y a c e t a t e , bu ty ra te a n d p r o p i o n a t e , t he lat ter o f w h i c h d o e s not d i rec t l y con t r i bu te to the p r o d u c t i o n o f g a s . A c o m p u t e r i z e d s y s t e m w a s d e v e l o p e d b y P e l l a n d S c h o f i e l d ( 1 9 9 3 ) in w h i c h c h a n g e s in p r e s s u r e w e r e 1 2 7 i nd i ca t i ve o f t h e g a s p r o d u c e d a n d t h e r e f o r e t he f e r m e n t a t i o n prof i le o f v a r i o u s f e e d s t u f f s . T h e p r e s e n t s t u d y i n v e s t i g a t e d t he u s e o f a s i m i l a r s y s t e m in the e v a l u a t i o n o f t h e nutr i t ive v a l u e o f c a n o l a m e a l . T h e p r i m a r y o b j e c t i v e o f th i s s t u d y w a s to u s e t h e g a s p r o d u c t i o n t e c h n i q u e to i n v e s t i g a t e t h e feas ib i l i t y o f u s i n g e n z y m e s a n d mi ld h y d r o t h e r m a l t r ea tmen t a p p l i e d to c a n o l a m e a l , in o r d e r to i m p r o v e it 's nut r i t iona l v a l u e fo r d a i r y ca t t le . 4.3 MATERIALS AND METHODS 4.3.1 Feed Treatments T h e e x p e r i m e n t c o n s i s t e d o f a c o m p l e t e l y r a n d o m i z e d 4 x 4 x 4 x 2 mul t i -f ac to r i a l d e s i g n w h i c h e v a l u a t e d t h e e f f e c t s o f e n z y m e s a n d h y d r o t h e r m a l t r e a t m e n t o n c a n o l a m e a l u s i n g t he in vitro g a s p r o d u c t i o n t e c h n i q u e . T r e a t m e n t s u n d e r i n ves t i ga t i on a r e d e s c r i b e d in C h a p t e r 3 , s e c t i o n 3 . 3 . 1 . 4.3.2 Processing of Feed Treatments C a n o l a m e a l t r e a t m e n t s w e r e p r o c e s s e d a c c o r d i n g to t he p r o c e d u r e ou t l i ned in C h a p t e r 2 , s e c t i o n 2 . 3 . 2 . 1 2 8 4.3.3 Chemical Analyses C h e m i c a l a n a l y s e s w e r e c o n d u c t e d o n t he s a m p l e s a c c o r d i n g to the p r o c e d u r e s o u t l i n e d in C h a p t e r 2 , s e c t i o n 2 . 3 . 3 . 4.3.4 Animals and Feeding D o n o r a n i m a l s w e r e m a i n t a i n e d a s d e s c r i b e d in C h a p t e r 2 , s e c t i o n 2 .3 .4 . 4.3.5 Rumen Fluid Collection R u m e n f lu id w a s c o l l e c t e d pr ior to t he m o r n i n g f e e d i n g a s s u g g e s t e d by M e n k e a n d S t e i n g a s s ( 1 9 8 8 ) . F o l l o w i n g c o l l e c t i o n , r u m e n f lu id w a s f i l tered t h r o u g h 4 l a y e r s o f c h e e s e c l o t h a n d a l a y e r o f g l a s s w o o l into a p r e w a r m e d a n d air t ight c o n t a i n e r fo r t r a n s p o r t to t he l abo ra to r y . 4.3.6 Gas Production System G a s p r o d u c t i o n m e a s u r e m e n t s w e r e m a d e u s i n g a c o m p u t e r i z e d s y s t e m a d a p t e d f r o m P e l l a n d S c h o f i e l d ( 1 9 9 3 ) ( F i g u r e 4 .1 ) w i th t h e f o l l ow ing m o d i f i c a t i o n s . A l a b o r a t o r y i n c u b a t o r m a i n t a i n e d at 3 9 ± 0 . 5 ° C w a s m o d i f i e d to a c c o m m o d a t e 3 2 c u s t o m i z e d E r l e n m e y e r f l a s k s (50 m L ) . A ro ta t ing b o t t o m p la te a l l o w e d for c o n t i n u o u s m i x i n g t h r o u g h o u t t h e e x p e r i m e n t . E a c h f l a s k w a s f i t ted w i th a s m a l l port in w h i c h a v a c u t a i n e r s t o p p e r w a s p l a c e d w h i c h a l l o w e d for s y s t e m ca l i b ra t i on 1 2 9 ( A p p e n d i x 6 .5 ) , r u m e n f lu id in jec t ion a n d V F A s a m p l i n g . A n a e r o b i c c o n d i t i o n s w e r e m a i n t a i n e d b y m o i s t e n i n g t h e s e a l w i th w a t e r . P r e s s u r e t r a n s d u c e r s ( P X 1 7 0 s e r i e s , O m e g a E n g i n e e r i n g , Inc., S t a m f o r d , C T , U . S . A . ) w e r e a t t a c h e d t h r o u g h t he top of s i l i c o n e s t o p p e r s w h i c h c o n t a i n e d a 0 . 5 c m h o l e . T h e s t o p p e r s a n d t r a n s d u c e r s w e r e s e c u r e d w i th e l a s t i c b a n d s a n c h o r e d to two s m a l l " e a r s " l o c a t e d at t he n e c k of t he f l a s k t h r o u g h o u t t h e a c q u i s i t i o n o f g a s m e a s u r e m e n t s ( F i g u r e 4 .2 ) . 4.3.7 Gas Measurements G a s m e a s u r e m e n t s w e r e m a d e u s i n g 0 to 1 0 3 k P a (0 to 15 ps i ) p r e s s u r e t r a n s d u c e r s w h i c h m e a s u r e d t he p r e s s u r e w i th in a f l a s k a n d g e n e r a t e d a d i f ferent ia l ou tpu t v o l t a g e p r o p o r t i o n a l to tha t p r e s s u r e . O u t p u t v o l t a g e w a s m e a s u r e d u s i n g T e m p s c a n / 1 0 0 0 ( O m e g a E n g i n e e r i n g , Inc., S t a m f o r d , C T , U . S . A . ) , a h igh s p e e d m e a s u r e m e n t s y s t e m s e t to a c q u i r e d a t a f r o m 3 2 c h a n n e l s a n d e q u i p p e d wi th a d i rec t t r a n s f e r to a c o m p u t e r . T h e c h a n n e l s w e r e c o n f i g u r e d to a c q u i r e d a t a at 5 m in in te rva ls o v e r a n 1 8 h i n c u b a t i o n p e r i o d . T h e f o l l ow ing r e g r e s s i o n e q u a t i o n (r 2 = 0 . 9 9 4 ) w a s u s e d to d e t e r m i n e t h e g a s v o l u m e f r o m a v o l t a g e r e s p o n s e ( A p p e n d i x 6 .5) : Y = 64.60 mL*Y1 X -1.27 mL, ( E q u a t i o n 1) w h e r e Y is t h e g a s v o l u m e a n d X is v o l t a g e r e s p o n s e . 1 3 0 4 . 3 . 8 Procedure for the Gas Production Technique S a m p l e s o f 0 . 1 5 0 g w e r e w e i g h e d in t r ip l ica te into 5 0 ml_ e r l e n m e y e r f l a s k s to w h i c h 12 m L o f p r e h e a t e d p h o s p h a t e - b i c a r b o n a t e m e d i u m a n d r e d u c i n g so lu t i on ( G o e r i n g a n d V a n S o e s t , 1 9 7 0 ) w a s a d d e d . T h e f l a s k s w e r e f l u s h e d w i th C 0 2 to e s t a b l i s h a n a e r o b i c c o n d i t i o n s pr io r t o s e a l i n g . R u m e n f lu id (3 m L ) w a s t r a n s f e r r e d v i a a n 18 g a u g e n e e d l e t h r o u g h t h e port into t he f l a s k s to m a k e up a 2 0 % i n o c u l u m ( G o e r i n g a n d V a n S o e s t , 1 9 7 0 ; M e n k e a n d S t e i n g a s s , 1 9 8 8 ) . T h e s o l u t i o n s in t he f l a s k s w e r e a l l o w e d to equ i l i b ra te f o r 5 m i n a n d t h e e x c e s s g a s r e l e a s e d w i th a n e e d l e ( 2 6 . 5 g a u g e ) t h r o u g h t he port pr ior to d a t a a c q u i s i t i o n . E a c h run i n c l u d e d t h r e e b l a n k s ( r u m e n f lu id a n d buf fer ) w h i c h w e r e a v e r a g e d in o r d e r to c o r r e c t e a c h c h a n n e l fo r r u m e n f lu id act iv i ty . S t a n d a r d h a y a n d c a n o l a s a m p l e s w e r e a l s o i n c u b a t e d to m o n i t o r da i l y va r i a t i on a n d d e t e c t a b n o r m a l r u m e n f lu id act iv i ty . T h e bu f fe r c a p a c i t y o f t h e g a s p r o d u c t i o n s y s t e m w a s m o n i t o r e d u s i n g p H m e a s u r e m e n t s c o n d u c t e d o n a r a n d o m s e l e c t i o n of f l a s k s at t h e e n d of e a c h g a s run . T h e bu f fe r c a p a c i t y o f t he s y s t e m w a s m a i n t a i n e d w i th in a p H r a n g e o f 6 . 7 - 7 . 0 . 131 4.3.9 Data Processing A l l v o l t a g e m e a s u r e m e n t s a c q u i r e d w i th t he d a t a l o g g e r w e r e r e c o r d e d u s i n g T e m p W i n d o w s 3 . 1 ® s o f t w a r e ( O m e g a E n g i n e e r i n g , Inc., S t a m f o r d , C T , U . S . A . ) . D e t e r m i n a t i o n o f g a s p r o d u c t i o n fo r a s p e c i f i c c h a n n e l r e q u i r e d : 1) S u b t r a c t i o n o f t h e b a s e l i n e v o l t a g e at t i m e z e r o f r o m t h e o b s e r v e d v o l t a g e at a n y t ime . 2) C o n v e r s i o n o f t h e v o l t a g e ou tpu t to g a s v o l u m e u s i n g t h e ca l i b ra t i on co r rec t i on f ac to r ( c o r r e c t e d to 1 0 3 k P a , 3 9 ° C ) , ( A p p e n d i x 6 .5) . 3 ) N o r m a l i z a t i o n o f t h e g a s p r o d u c t i o n d a t a to 1 g d r y s a m p l e w e i g h t . 4 ) U s e o f s t e p s 1 a n d 2 to c a l c u l a t e g a s p r o d u c t i o n o f t he b lank , h a y a n d c a n o l a s t a n d a r d s . 4.3.10 Kinetic Analysis of Gas Data T h e g a s p r o d u c t i o n prof i le o f a c a n o l a m e a l s a m p l e is i l lus t ra ted in F i g u r e 4 . 3 . T h e s i m p l e l im i ted e x p o n e n t i a l g r o w t h ( S L E G ) m o d e l w i th l a g w a s u s e d to d e s c r i b e t he g a s p r o d u c t i o n d a t a . T h i s m o d e l h a s b e e n p r e v i o u s l y u s e d to d e t e r m i n e g a s p r o d u c t i o n p a r a m e t e r s in t rop i ca l f e e d s t u f f s ( K r i s h n a m o o r t h y et a l . , 1 9 9 1 ; K r i s h n a m o o r t h y e t a l . , 1 9 9 5 ) : Y = A ( 1 - e b ( t l > ) , ( E q u a t i o n 2 ) 1 3 2 w h e r e V is t h e c u m u l a t i v e g a s p r o d u c t i o n a t a g i v e n t i m e t, I is t h e init ial l ag for t he o n s e t o f f e r m e n t a t i o n a n d A is t h e po ten t ia l c u m u l a t i v e g a s p r o d u c t i o n w h i c h m a y b e p r o d u c e d at a s p e c i f i c ra te b ( m L / h 1 ) . R a t e s o f f e r m e n t a t i o n a n d l ag p e r i o d s w e r e e s t i m a t e d u s i n g n o n l i n e a r r e g r e s s i o n a n d t h e S e c a n t m e t h o d to m i n i m i z e t he r e s i d u a l s u m s o f s q u a r e s a s s o c i a t e d w i th t h e r e g r e s s i o n m o d e l (S ta t i s t i ca l A n a l y s i s S y s t e m s , ( S A S ) , 1 9 9 0 ) ( A p p e n d i x 6 .7 ) . T h e S e c a n t m e t h o d is a l i nea r i terat ive c u r v e f i t t ing p r o c e d u r e of the P R O C N L I N p r o c e d u r e s o f S A S (1990 ) . 4.3.11 VFA Sampling and Analyses: P r e l i m i n a r y e x p e r i m e n t s c o n d u c t e d in o u r l a b o r a t o r y i n d i c a t e d that 6 h total g a s p r o d u c t i o n m e a s u r e m e n t s o f c a n o l a m e a l t r e a t m e n t s w e r e re f l ec ted in a 6 h s a m p l i n g t i m e fo r V F A s ( A p p e n d i x 6 .6) . P r e l i m i n a r y i nves t i ga t i on o f t he c h a n g e s in t h e ra t i os o f V F A s p r o d u c e d a l s o i n d i c a t e d th i s w a s a n a d e q u a t e s a m p l i n g t ime . Inheren t a n a l y t i c e r ro r a s s o c i a t e d wi th V F A a n a l y s e s in th is l a b o r a t o r y is 5 % . O n e f l a s k o f e a c h f e e d t r e a t m e n t w a s s a m p l e d a n d a n a l y z e d in d u p l i c a t e for total a n d i nd i v i dua l V F A s ( a c e t a t e , p r o p i o n a t e , i s o b u t y r a t e , n -bu ty ra te , i s o - v a l e r a t e a n d n-v a l e r a t e ) a c c o r d i n g to t h e p r o c e d u r e o f E r w i n et al., ( 1961 ) . A l ue r l o c k f i t ted wi th a t u b e (I.D. = 1.19 m m , O . D . 1.70 m m ) w a s i m m e r s e d b e l o w the r u m e n f lu id i n o c u l u m a n d w a s u s e d to s a m p l e f r o m a f l ask u s i n g a s y r i n g e . A f t e r 6 h o f i n c u b a t i o n , a 0 .5 m L r e p r e s e n t a t i v e s a m p l e w a s w i t h d r a w n f r o m the f e r m e n t a t i o n f l a s k s . S a m p l e s w e r e c e n t r i f u g e d at 3 0 0 0 g fo r 1 0 m i n u t e s , f r o z e n a n d a c i d i f i e d to p H 2 w i th a 2 0 % 1 3 3 p h o s p h o r i c a c i d s o l u t i o n (v/v) . T h e s u p e r n a t a n t w a s la ter i n j ec ted into a g a s c h r o m a t o g r a p h ( M o d e l G C 1 4 A , S h i m a d z u C o r p . , K y o t o , J a p a n ) e q u i p p e d w i th a f u s e d s i l i c a c a p i l l a r y c o l u m n ( 3 0 m x 0 . 2 5 I.D. S t a b i l w a x - D A , R e s t e k C o r p . , B e l l e f o n t e , P A , U S A ) . C o l u m n t e m p e r a t u r e w a s s e t to i n c r e a s e at 1 0 ° m i n 1 f r o m 1 2 0 ° C to 1 8 0 ° C a n d i s o - c a p r o i c a c i d w a s u s e d fo r t he in te rna l s t a n d a r d . 4.3.12 Statistical Analysis Sta t i s t i ca l a n a l y s e s w e r e c o n d u c t e d u s i n g t h e G e n e r a l L i n e a r M o d e l ( G L M ) p r o c e d u r e o f S ta t i s t i ca l A n a l y s i s S y s t e m ( S A S Inst i tute Inc., 1 9 9 0 ) u s i n g the p r o g r a m l i s ted in A p p e n d i x 6 .4 . In t he p r e s e n t s t u d y , it w a s a s s u m e d that the h i g h e s t o r d e r i n te rac t i on d i d not ex i s t , but it 's m e a n s q u a r e e r ro r w a s r e p r e s e n t a t i v e o f t h e e x p e r i m e n t a l e r ro r ( C o c h r a n a n d C o x , 1 9 5 0 ) . S ta t i s t i ca l l y s ign i f i can t d i f f e r e n c e s ( P < 0 . 0 5 ) b e t w e e n m e a n s w e r e d e t e r m i n e d u s i n g t he p r o g r a m w h i c h e m p l o y e d t h e u s e o f l e a s t s q u a r e m e a n s for ra te o f g a s p r o d u c t i o n , l eng th of the l ag p h a s e , to ta l V F A p r o d u c t i o n , a c e t a t e to p r o p i o n a t e ra t ios a n d bu ty ra te to p r o p i o n a t e ra t ios . F u r t h e r i n v e s t i g a t i o n o f t h e r e l a t i onsh ip b e t w e e n a s i gn i f i can t m a i n e f fec t a n d a m e a s u r e d p a r a m e t e r w i th o r t h o g o n a l p o l y n o m i a l s w a s c o n d u c t e d u s i n g t he C O N T R A S T s t a t e m e n t o f S A S P R O C G L M ( S A S , 1 9 9 0 ) . 1 3 4 4.4 RESULTS AND DISCUSSION Nut r ien t a n a l y s i s o f t he con t ro l s a m p l e c a n b e f o u n d in C h a p t e r 3 , s e c t i o n 4.4.1 Validation and Comparison of the Gas Production System T h e r e s u l t s o f t h e p r e s e n t s t u d y w i th c a n o l a m e a l a r e c o m p a r a b l e to t h o s e o b t a i n e d f r o m o t h e r s t u d i e s u s i n g v a r i o u s g a s s y s t e m s to e v a l u a t e a va r ie ty of f e e d s t u f f s . In t h e p r e s e n t s t u d y , g a s p r o d u c t i o n p a r a m e t e r s w e r e e v a l u a t e d u s i n g t h e e x p o n e n t i a l m o d e l i n c l u d i n g l a g . T h e ra te o f g a s p r o d u c t i o n o f t he con t ro l t r e a t m e n t w a s d e t e r m i n e d to b e 0 . 0 7 7 ± 0 . 0 0 4 m L / f r 1 a n d t h e l eng th of t he l ag p h a s e w a s d e t e r m i n e d to b e 0 . 4 4 9 ± 0 . 0 4 6 h. A l t h o u g h s e l e c t i o n o f t h e a p p r o p r i a t e m o d e l h a s b e e n s h o w n to a f fec t t h e e s t i m a t i o n o f g a s p r o d u c t i o n p a r a m e t e r s ( K r i s h n a m o o r t h y et a l . , 1 9 9 1 ) , r a tes o f g a s p r o d u c t i o n f r o m s t u d i e s w i th o t h e r f e e d s h a v e p r o d u c e d c o m p a r a b l e resu l t s to t h o s e o b t a i n e d in t he p r e s e n t s t u d y i r r e s p e c t i v e o f w h e t h e r s i n g l e o r d u a l - p o o l e d l og i s t i c m o d e l s w e r e u s e d . R a t e s o f g a s p r o d u c t i o n in a s t u d y w i th r y e g r a s s w e r e d e t e r m i n e d u s i n g a d u a l p h a s e log is t i c m o d e l ( F r a n c e et a l . , 1 9 9 3 ) w h i c h y i e l d e d v a l u e s b e t w e e n 0 . 0 0 7 4 m L / h ' 1 a n d 0 . 0 1 9 2 m L / h " 1 f r o m d a t a o b t a i n e d w i th a m a n u a l s y s t e m ( T h e o r o d o u et a l . , 1 9 9 4 ) . A r e v i e w o f t h e l i terature r e v e a l s that m a n u a l m e t h o d s o f r e c o r d i n g g a s p r o d u c t i o n a r e i nhe ren t l y a s s o c i a t e d w i th g r e a t e r e x p e r i m e n t a l e r ro r t h a n a c o m p u t e r i z e d s y s t e m . T h e p r e s e n t s t u d y e m p l o y e d t he u s e o f a c o m p u t e r i z e d 1 3 5 s y s t e m w h i c h c l o s e l y r e s e m b l e d that d e v e l o p e d b y S c h o f i e l d a n d P e l l ( 1 9 9 5 ) wi th t he m i n o r m o d i f i c a t i o n s p r e v i o u s l y n o t e d . T h e c o m p u t e r i z e d s y s t e m h a s b e e n p r e v i o u s l y u s e d to e v a l u a t e a l f a l f a , t imo thy a n d c o r n s i l a g e a n d p r o d u c e d a s t a n d a r d e r ro r r a n g i n g f r o m 0 . 7 % to 2 . 6 % o f t he e s t i m a t e d p a r a m e t e r s u s i n g e i t he r s i n g l e o r d u a l p h a s e m o d e l s ( S c h o f i e l d a n d P e l l , 1 9 9 5 ) . In t he p r e s e n t s t u d y , t he p o o l e d s t a n d a r d e r ro r fo r ra te w a s ± 0 . 0 0 4 m L / h " 1 w i th v a l u e s fo r t r e a t m e n t s r a n g i n g f r o m 0 . 0 7 7 m L / h " 1 to 0 . 1 0 8 m L / h " 1 . T h e e r ro r a s s o c i a t e d w i th t h e l eng th o f t he l ag p h a s e w a s ± 0 . 0 4 6 h w i th v a l u e s for t r e a t m e n t s r a n g i n g f r o m 0 . 1 3 6 h to 0 . 4 4 9 h. T h e v a l u e s fo r l a g in t h e p r e s e n t s t u d y h a v e a g r e a t e r e r ro r a s s o c i a t e d w i th t h e m t h a n m o s t o t h e r r e p o r t e d f e e d v a l u e s w i th o t h e r s y s t e m s . T h i s m a y b e at t r ibuted to va r i a t i on w i th in a f eeds tu f f b e i n g i n v e s t i g a t e d o r t he l a rge n u m b e r o f o b s e r v a t i o n s o v e r a s p e c i f i c t i m e in te rva l w h i c h is u n i q u e to th is t y p e o f s y s t e m . T h i s va r ia t i on h o w e v e r , is not r e f l ec ted in t h e v a l u e s fo r ra te . T h e m a n u a l s y s t e m u s e d b y K r i s h n a m o o r t h y et a l . ( 1 9 9 1 ) to e v a l u a t e e n e r g y s u p p l e m e n t s , p r e s e n t e d s t a n d a r d e r ro r s r a n g i n g f r o m 1 .8% to 3 . 4 % for rate of g a s p r o d u c t i o n a n d e r r o r s r a n g i n g f r o m 8 % to 5 7 % for l ag p e r i o d . In t he p r e s e n t s tudy , t he s p r e a d o f o b s e r v a t i o n s a s a p ropo r t i on o f t he e s t i m a t e d v a l u e w a s o n a v e r a g e 5 . 2 % fo r ra te o f g a s p r o d u c t i o n a n d 1 0 . 2 % for l eng th o f t h e l a g p e r i o d . T h e l a rge r e r ro r a s s o c i a t e d w i th t h e l eng th o f t h e l ag p h a s e in bo th m e t h o d s m a y b e at t r ibuted to a n u m b e r o f p h y s i o l o g i c a l c o n d i t i o n s ( K r i s h n a m o o r t h y et a l . , 1 9 9 1 ) . S o m e fac to r s w h i c h m a y r e q u i r e c o n s i d e r a t i o n a r e : s a m p l e s i z e , r u m e n f lu id act iv i ty , m ic rob ia l p o p u l a t i o n a n d c h e m i c a l c o m p o s i t i o n o f t he feeds tu f f . 1 3 6 T h e g a s p a r a m e t e r s e s t i m a t e d in t he p r e s e n t s t u d y w i th c a n o l a m e a l , u s i n g a c o m p u t e r i z e d s y s t e m , a r e c o m p a r a b l e to t h o s e o b t a i n e d w i th a m a n u a l g a s p r o d u c t i o n s y s t e m u s e d to e v a l u a t e e n e r g y s u p p l e m e n t s ( K r i s h n a m o o r t h y et a l . , 1 9 9 1 ) . E m p l o y i n g t he e x p o n e n t i a l m o d e l wi th l a g , t he ra tes o f g a s p r o d u c t i o n for w h e a t , b a r l e y , m a i z e , t a p i o c a , oa t , r ye , s o r g h u m w e r e 0 . 1 1 9 6 , 0 . 1 1 9 3 , 0 . 0 7 6 7 , 0 . 1 3 4 9 , 0 . 0 8 6 6 , 0 . 0 8 7 5 a n d 0 .1101 m L / h " 1 . T h e l a g p e r i o d s fo r t he s a m e f e e d s w e r e 1.1, 0 . 5 , 1.2, 0 . 7 , - 0 . 3 , - 0 . 2 a n d 1.5 h, r e s p e c t i v e l y . A s u b s e q u e n t s t u d y e v a l u a t e d r a p e s e e d m e a l a n d d e t e r m i n e d that t h e ra te o f g a s p r o d u c t i o n w a s 0 . 1 2 5 m L / h 1 a n d t he l eng th o f t h e l a g p h a s e w a s - 0 . 3 7 h ( K r i s h n a m o o r t h y et a l . , 1 9 9 5 ) . T h e v a l u e for ra te o f g a s p r o d u c t i o n o b t a i n e d b y t h e s e a u t h o r s w a s f a s t e r t h a n t he v a l u e o f 0 . 0 7 7 m L / h " 1 d e t e r m i n e d in t h e p r e s e n t s t u d y . In con t ras t , t he v a l u e for l ag o b t a i n e d in the p r e s e n t s t u d y is m u c h l o n g e r t h a n t h e v a l u e o b t a i n e d b y t h e s e a u t h o r s . T h e r e a r e a n u m b e r o f p o s s i b l e e x p l a n a t i o n s for t he l a rge d i f f e r e n c e in l a g t i m e s b e t w e e n t h e s e two s t u d i e s (- 0 . 3 7 h v s . 0 . 4 5 h). T h e g r e a t e r a m o u n t s o f ant i -nut r i t iona l f ac to r s p r e s e n t in r a p e s e e d m e a l c o m p a r e d to c a n o l a m e a l , m a y h a v e a de t r imen ta l e f fect o n l ag t i m e t h r o u g h s o m e a c t i o n o n r u m e n m i c r o b e s . A n o t h e r p o s s i b l e e x p l a n a t i o n m a y b e that m a n u a l r e a d i n g s a r e s u b j e c t to g r e a t e r e r ro r t h a n a c o m p u t e r i z e d s y s t e m . In a d d i t i o n , t h e f r e q u e n c y o f g a s m e a s u r e m e n t s m a y i m p a c t o n the e s t i m a t i o n o f p a r a m e t e r s f r o m t h e m o d e l w h i c h is d e p e n d e n t t h e a c c u r a c y of a c c u m u l a t e d m e a s u r e m e n t s . A c o m p u t e r i z e d s y s t e m is m o r e l ike ly to d e t e c t the po in t o f m a x i m u m ra te o f g a s p r o d u c t i o n f r o m w h i c h , l a g is e s t i m a t e d t h a n a m a n u a l s y s t e m w i th f e w m e a s u r e m e n t s . 1 3 7 4.4.2 Rate of In Vitro Gas Production E v a l u a t i o n o f t he ra tes o f g a s p r o d u c t i o n f r o m the c a n o l a m e a l t r e a t m e n t s e s t a b l i s h e d tha t t h e a p p l i c a t i o n o f p r o t e a s e h a d a s i gn i f i can t i m p a c t ( P < 0 .05) . P r o t e a s e a p p l i e d a t 0 . 0 1 % s ign i f i can t l y i n c r e a s e d t h e ra te o f g a s p r o d u c t i o n ( P < 0 .05 ) f r o m 0 . 0 7 7 m L / h " 1 to 0 . 1 0 8 m L / h " 1 ( T a b l e 4 .2 ) . H o w e v e r , t h e l o w e s t a n d h i g h e s t l e v e l s o f p r o t e a s e p r o d u c e d ra tes o f 0 . 0 7 5 m L / h " 1 a n d 0 . 0 8 9 m L / h " 1 , r e s p e c t i v e l y w h i c h w e r e not s ign i f i can t l y d i f ferent ( P < 0 .05 ) f r o m t h e con t ro l . T h e i n c r e a s e d r a t e s o f g a s p r o d u c t i o n o b s e r v e d w i th t he a d d i t i o n o f 0 . 0 1 % p r o t e a s e m a y re f lec t t he a c c e s s i b i l i t y a n d t he e a s e o f f e r m e n t a t i o n o f t h e c a r b o h y d r a t e s in c a n o l a m e a l b y r u m e n m i c r o b e s . W i t h f o r a g e s , i n c r e a s e d bac te r i a l c o l o n i z a t i o n d e n s i t y h a s b e e n a s s o c i a t e d w i th i n c r e a s e d ra tes o f g a s p r o d u c t i o n ( H i d a y a t et a l . , 1993 ) . T h e i m p r o v e d d iges t ib i l i t y o f t he f i b r o u s c a r b o h y d r a t e f rac t ion o f c a n o l a m e a l is impor tan t in o r d e r to i m p r o v e it 's e n e r g y v a l u e a n d o p t i m i z e t he u s e o f a n e x p e n s i v e c o m m o d i t y . T h e n o n l i n e a r e f fec t o f p r o t e a s e is i l lus t ra ted in F i g u r e 4 . 4 . Inves t iga t ion into t h e n a t u r e o f th i s e f fec t w a s i n c o n c l u s i v e . F u r t h e r r e s e a r c h is w a r r a n t e d o n the e f f ec t s o f c a n o l a m e a l t r e a t e d w i th l e v e l s o f p r o t e a s e b e t w e e n 0 . 0 1 % a n d 0 . 0 5 % . A n o n l i n e a r e f fec t o f e n z y m e t r e a t m e n t o f a f eeds tu f f h a s b e e n p r e v i o u s l y r epo r ted . W h e n s t e e r s w e r e f e d c u b e d a l fa l fa h a y that h a d b e e n s p r a y e d w i th v a r i o u s l e v e l s of x y l a n a s e , t h e a n i m a l s h a d i m p r o v e d D M I , A D G a n d f e e d e f f i c i e n c i e s at 4 t i m e s the a p p l i c a t i o n d o s e t h a n at 8 o r 16 t i m e s t h e a p p l i c a t i o n d o s e ( B e a u c h e m i n et a l . , 1 3 8 1 9 9 5 ) . T h i s n o n l i n e a r r e s p o n s e to e n z y m e c o n c e n t r a t i o n m a y b e a t t r ibu ted to the s a t u r a t i o n o f e n z y m e b i n d i n g s i t e s w h e r e a n o p t i m a l r a the r t h a n m a x i m u m d o s e r e s p o n s e is f o u n d . T h e l a c k o f r e s p o n s e o f ra te f r o m x y l a n a s e t r e a t m e n t m a y b e a c o n s e q u e n c e o f it 's l o w l eve l o f a p p l i c a t i o n . A l a c k o f r e s p o n s e w a s a l s o o b s e r v e d to m i ld h e a t a n d m o i s t u r e t r e a t m e n t s w h i c h w e r e a p p l i e d in o r d e r to bene f i t e n z y m e act iv i ty a s r e c o m m e n d e d b y t h e e n z y m e s u p p l i e r ( F F I , p e r s o n a l c o m m u n i c a t i o n ) . C u r r e n t r e s e a r c h o f h y d r o t h e r m a l t r e a t m e n t o f c a n o l a m e a l n o w f o c u s e s o n m o d e r a t e hea t p r o c e s s e s w h i c h e m p l o y t e m p e r a t u r e s in e x c e s s o f 1 0 0 ° C in o r d e r to i n c r e a s e r u m i n a l e s c a p e p ro te in (M i r e t a l . , 1 9 8 4 ; M o s h t a g h i N i a a n d Inga l ls , 1992 ) . T h e r e f o r e , t h e l a c k o f r e s p o n s e o b s e r v e d w i th t h e mi ld h y d r o t h e r m a l t r e a t m e n t s w a s not u n e x p e c t e d . It m a y b e c o n c l u d e d that h y d r o t h e r m a l t r e a t m e n t d id not i m p r o v e e n z y m e act iv i ty a s t h e r e w e r e n o in te rac t i ve e f fec ts o f e n z y m e a n d h y d r o t h e r m a l t r e a t m e n t s . T h e r e a r e re la t i ve ly f e w e x p e r i m e n t s w h i c h h a v e u s e d t he g a s p roduc t i on t e c h n i q u e in o r d e r to e v a l u a t e t h e e f fec t o f e n z y m e s o n t he f e r m e n t a t i o n k ine t i cs of a feeds tu f f . O n e e x p e r i m e n t i n v e s t i g a t e d t he u s e o f ce l l wa l l d e g r a d i n g e n z y m e s to i m p r o v e t h e qua l i t y o f g r a s s s i l a g e u s i n g g a s p r o d u c t i o n p a r a m e t e r s ( B e u v i n k a n d S p l o e s t r a , 1 9 9 4 ) . R e s u l t s o b t a i n e d b y B e u v i n k a n d S p l o e s t r a ( 1 9 9 4 ) w h e r e rate of g a s p r o d u c t i o n w a s a f f e c t e d but to ta l g a s p r o d u c t i o n r e m a i n e d t h e s a m e , w e r e a t t r ibu ted to a shi f t in t h e so lub i l i t y o f f e e d c o m p o n e n t s a s a resu l t o f e n z y m e a c t i o n . T h e s e a u t h o r s f o u n d that in g r a s s s i l a g e that w a s e n z y m e t r e a t e d , t he e n z y m e s 1 3 9 a p p e a r e d to m o d i f y t h e ce l l w a l l ma te r i a l to m o r e rap id l y f e r m e n t a b l e c o m p o n e n t s l e a v i n g a m o r e s l o w l y d e g r a d a b l e f rac t i on a s i n d i c a t e d b y t he s h o r t e r init ial lag p h a s e a n d l o w e r m a x i m u m rate o f g a s p r o d u c t i o n . In tha t i n s t a n c e , t he a u t h o r s s p e c u l a t e d tha t c h a n g e s in t h e c h e m i c a l c o m p o s i t i o n ( N D F , lac t i c a c i d ) o f t he f e e d w e r e r e s p o n s i b l e fo r t he o b s e r v e d c h a n g e s in t he ra te o f g a s p r o d u c t i o n . A s im i l a r e x p l a n a t i o n m a y b e o f f e red in th is s t u d y w h e r e tota l g a s p r o d u c t i o n b e t w e e n t r e a t m e n t s w a s d e t e r m i n e d to b e s i m i l a r h o w e v e r , t h e ra tes o f f e r m e n t a t i o n a n d l ag t i m e s w e r e s i gn i f i can t l y a f f e c t e d by e n z y m e p r e t r e a t m e n t ( A p p e n d i x 6 .8) . F e e d t r e a t m e n t s h a d n o e f fec t o n tota l g a s p r o d u c t i o n , th is i n d i c a t e s t h e t r e a t m e n t s d id no t a f fec t t h e to ta l a m o u n t o f f e r m e n t a b l e m a t e r i a l bu t p e r h a p s c a u s e d a shi f t t o w a r d s m o r e rap id l y d e g r a d a b l e c o m p o n e n t s . C u m u l a t i v e 18 h g a s p r o d u c t i o n for t he con t ro l t r e a t m e n t w a s 2 0 1 . 1 2 m L / g D M a n d w a s not s i gn i f i can t l y d i f ferent ( P < 0 .05 ) f r o m a n y o t h e r o f t he 1 2 8 t r e a t m e n t m e a n s ( A p p e n d i x 6 .8) . T h e so lub i l i t y o f f e e d c o m p o n e n t s m a y h a v e a n e f fec t o n t he rate o f g a s p r o d u c t i o n h o w e v e r , a s i m i l a r c h e m i c a l c o m p o s i t i o n w o u l d e x p l a i n t h e l a c k o f r e s p o n s e f r o m total g a s p r o d u c t i o n a t 1 8 h. C u m u l a t i v e g a s p r o d u c t i o n h a s b e e n a s s o c i a t e d w i th t he ex ten t o f d i g e s t i o n o f a f e e d s t u f f ( B e u v i n k a n d S p l o e s t r a , 1 9 9 4 ) . T h e ex ten t o f d i g e s t i o n of a f o r a g e h a s b e e n s h o w n to b e d e t e r m i n e d by it 's c h e m i c a l c o m p o s i t i o n ( M e r t e n s , 1 9 7 7 ) . 1 4 0 4.4.3 Lag Period T h e l eng th o f t h e l ag p h a s e w a s s ign i f i can t l y ( P < 0 .05 ) a f f ec ted by the in te rac t i ve e f fec t o f p r o t e a s e a n d x y l a n a s e w h i c h is i l lus t ra ted in F i g u r e 4 . 5 . T h e l eng th o f t he l a g p h a s e o f t he u n t r e a t e d s a m p l e in t he p r e s e n t s t u d y w a s 0 . 4 4 9 ± 0 . 0 4 6 h. F r o m T a b l e 4 . 3 it m a y b e d e t e r m i n e d that t h e con t ro l t r e a t m e n t h a d a s ign i f i can t l y l o n g e r l a g p h a s e t h a n a n y o f t he e n z y m e t r e a t m e n t s ( P < 0 .05) . In a d d i t i o n , a n y c o m b i n a t i o n o f e n z y m e t r e a t m e n t s h o r t e n e d t he l eng th o f t he l ag p h a s e o f c a n o l a m e a l to l e s s t h a n 0 . 2 9 4 h. T h e a p p l i c a t i o n o f p r o t e a s e at 0 . 0 1 % wi th o r w i t hou t 0 . 0 0 1 % x y l a n a s e s h o w e d t h e g r e a t e s t r e d u c t i o n in t h e l eng th o f t he l ag p h a s e c o m p a r e d to the con t ro l . P r o t e a s e a p p l i e d at 0 . 0 1 % a l o n e s h o r t e n e d l ag t i m e f r o m 0 . 4 4 9 h to 0 . 1 4 0 h; w h e r e a s p r o t e a s e a p p l i e d at 0 . 0 1 % in c o n j u n c t i o n w i th 0 . 0 0 1 % x y l a n a s e d e c r e a s e d t he l a g p h a s e f r o m 0 . 4 4 9 h to 0 . 1 3 6 h. T h e a p p l i c a t i o n o f 0 . 0 0 1 % x y l a n a s e w a s a s e f fec t i ve in d e c r e a s i n g l a g p e r i o d a s a n y t r e a t m e n t w h e r e a n e n z y m e h a d b e e n a p p l i e d . P r o t e a s e a p p l i e d at 0 . 0 5 % s ign i f i can t l y i n c r e a s e d ( P < 0 .05 ) t h e l e n g t h o f t h e l a g p h a s e to 0 . 2 7 3 h f r o m a l ag p e r i o d o f 0 . 1 4 0 h for the 0 . 0 1 % p r o t e a s e t r ea tmen t . T h i s o b s e r v a t i o n is h o w e v e r , di f f icul t to e x p l a i n s i n c e m a n y f a c t o r s h a v e b e e n a s s o c i a t e d w i th l a g . L a g t i m e m a y b e i n te rp re ted a s t h e t i m e r e q u i r e d for: hyd ra t i on o f t he f e e d par t i c le , f e r m e n t a t i o n o f s o l u b l e s u g a r s , a t t a c h m e n t a n d c o l o n i z a t i o n of r u m e n m i c r o b e s to t he f e e d a s we l l a s t he l eng th o f t i m e r e q u i r e d for t he c o m m e n c e m e n t of e n z y m a t i c d e g r a d a t i o n ( C h e n g et a l . , 1 9 8 0 ) . A s h o r t e r l ag p h a s e re f lec ts t he e a s e in 141 w h i c h t h e s e p r o c e s s e s o c c u r . In a d d i t i o n , t h e s t ruc tu re a n d c o m p o s i t i o n of the f e e d , t he r u m e n m i c r o b i a l p o p u l a t i o n a n d t h e in vitro e n v i r o n m e n t m a y a l s o s ign i f i can t l y i m p a c t t he l eng th o f t h e l a g p h a s e . N e g a t i v e l a g h a s b e e n a s s o c i a t e d w i th a n u m b e r o f t r e a t m e n t m e a n s in th is e x p e r i m e n t ( A p p e n d i x 6 .8) . N e g a t i v e l ag m a y b e e x p l a i n e d by t h e p r o d u c t i o n of g a s in t h e e a r l y s t a g e s o f t h e f e r m e n t a t i o n p r o c e s s f r o m rap id l y f e r m e n t a b l e s o l u b l e s ( K r i s h n a m o o r t h y e t a l . , 1 9 9 1 ) . S m a l l f e e d pa r t i c l es a n d m i c r o b i a l d e b r i s p r e s e n t in t he r u m e n f lu id a r e f e r m e n t e d a n d con t r i bu te to t he g a s v o l u m e . It h a s b e e n s h o w n that t he ra te a n d ex ten t o f d i g e s t i o n o f c e r e a l g r a i n s m a y b e a t t r ibu ted to t h e p r e s e n c e o f a p ro te in mat r i x w h i c h r e n d e r s s t a r c h a n d pro te in l e s s read i l y d i g e s t i b l e b y r u m e n m i c r o o r g a n i s m s ( M c A l l i s t e r et a l . , 1 9 9 0 a , b, c ) . T h e resu l t s o f t h e p r e s e n t s t u d y s h o w that ra te o f g a s p r o d u c t i o n m a y b e i n c r e a s e d f r om 0 . 0 7 7 m L / h 1 to a m a x i m u m o f 0 . 1 0 8 m L / h " 1 w i th t he a d d i t i o n o f p r o t e a s e . T h e leng th o f t he l a g p h a s e w a s s h o r t e n e d f r o m 0 . 4 4 9 h to a m i n i m u m o f 0 . 1 3 6 h wi th the a d d i t i o n o f a c o m b i n a t i o n o f p r o t e a s e a n d x y l a n a s e . T h e c o m b i n a t i o n o f e n z y m e s w o u l d b e e x p e c t e d to el ic i t a s y n e r g i s t i c e f fec t by d e g r a d i n g t h e p ro te in mat r ix a n d o t h e r r e s i s t a n t p l an t c o m p o u n d s . In t h e p r e s e n t s t u d y a s i n g l e e n z y m e w a s su f f i c ien t to e l ic i t a pos i t i ve e f fec t o n ra te a n d l a g v a l u e s ( T a b l e 4 . 2 a n d 4 . 3 , r e s p e c t i v e l y ) . It a p p e a r s f r o m t h e s e resu l t s tha t t h e l a g p h a s e is m o r e s e n s i t i v e to t he e f f ec t s of e n z y m e s u p p l e m e n t a t i o n t h a n ra tes o f g a s p r o d u c t i o n . In t h e c a s e o f l a g , a l t h o u g h t rea tmen t m e a n s w e r e o f ten l o w e r w i th t he a p p l i c a t i o n o f t he c o m b i n a t i o n o f p r o t e a s e a n d 1 4 2 x y l a n a s e , t h e y w e r e no t s i gn i f i can t l y d i f ferent f r o m the m e a n s o b s e r v e d wi th e i the r x y l a n a s e o r p r o t e a s e a p p l i e d a t 0 . 0 0 5 % o r 0 . 0 1 % . E v a l u a t i o n o f t h e c h a n g e s in g a s p r o d u c t i o n k i ne t i c s h a s b e e n s h o w n to b e a u s e f u l a n d rap id m e t h o d to d e t e r m i n e t h e e f f ec t s o f e n z y m e s u p p l e m e n t a t i o n o n g r a s s s i l a g e ( B e u v i n k a n d S p l o e s t r a , 1 9 9 4 ) . T h e s e s a m e p r i n c i p l e s m a y b e a p p l i e d to t he cu r ren t s t u d y w h e r e e n z y m e s a r e e x p e c t e d to el ic i t the i r r e s p o n s e in t e r m s of i n c r e a s i n g t h e f e rmen tab i l i t y o f c a n o l a m e a l . G a s p r o d u c t i o n p a r a m e t e r s s u c h a s ra te a n d l a g p r o v i d e i n fo rma t i on a b o u t t he f e r m e n t a t i o n pa t te rn o f v a r i o u s f eeds tu f f s w h i c h m a y b e u s e f u l in f o r m u l a t i n g r a t i ons to m a x i m i z e t h e u t i l i za t ion o f nu t r ien ts in t he r u m e n . 4.4.4 Volatile Fatty Acids I n fo rmat ion a b o u t t h e c o m p o s i t i o n o f V F A s a n d the i r a m o u n t s is e s p e c i a l l y impor tan t in t h e e v a l u a t i o n o f g a s p r o d u c t i o n resu l t s f r o m c o n c e n t r a t e s . T h e f e r m e n t a t i o n o f c o n c e n t r a t e s p r o d u c e s a l a rge p ropo r t i on o f p r o p i o n i c a c i d w h i c h d o e s not con t r i bu te to t he f o r m a t i o n o f C 0 2 n o r C H 4 a n d t h u s g a s v o l u m e m a y b e u n d e r e s t i m a t e d . T h e r e f o r e , in te rp re ta t ion o f r esu l t s f r o m g a s p r o d u c t i o n s t u d i e s i nvo l v i ng c o n c e n t r a t e s r e q u i r e s c o n s i d e r a t i o n o f t he p r o d u c t i o n of tota l V F A s in a d d i t i o n to t he i r p r o p o r t i o n s . It h a s b e e n r e p o r t e d that t he a p p l i c a t i o n o f e x o g e n o u s e n z y m e s to c a n o l a m e a l in o r d e r to d e g r a d e t he ce l l wa l l r esu l t s in t he r e l e a s e o f s t a r c h a n d p ro te in that w a s p r e v i o u s l y u n a v a i l a b l e ( S l o m i n s k i a n d C a m p b e l l , 1 9 9 0 ) . T h i s m a y e x p l a i n t he 1 4 3 s ign i f i can t i n c r e a s e ( P < 0 .05 ) in t h e p r o d u c t i o n o f V F A s f r o m 2 . 5 8 m m o l / g D M - 1 for t he u n t r e a t e d s a m p l e to V F A p r o d u c t i o n v a l u e s r a n g i n g b e t w e e n 3 .24 a n d 3 .92 m m o l / g D M 1 fo r s a m p l e s t r ea ted w i th a n e f fec t i ve c o m b i n a t i o n o f p r o t e a s e a n d x y l a n a s e ( T a b l e 4 .4 ) . F i g u r e 4 . 6 i l l us t ra tes t he s i gn i f i can t i n te rac t i on b e t w e e n p r o t e a s e a n d x y l a n a s e ( P < 0 .01 ) w h i c h a f f ec ted t h e p r o d u c t i o n o f V F A s . T h e i n c r e a s e in a v a i l a b l e s u b s t r a t e s fo r r u m e n m i c r o b i a l f e r m e n t a t i o n m a y b e t he c a u s e o f t he o b s e r v e d i n c r e a s e in V F A p r o d u c t i o n . S i m i l a r to t he e f fec t o f p r o t e a s e s u p p l e m e n t a t i o n o n t he ra te o f g a s p r o d u c t i o n , t he e f fec t o f e n z y m e add i t i on is n o n l i nea r w i th t h e c o m b i n a t i o n o f e n z y m e s . A p o s s i b l e e x p l a n a t i o n for th is w a s p r e v i o u s l y e l u d e d to a s e n z y m e b i n d i n g s i t e s o n t he s u b s t r a t e b e c o m e s a t u r a t e d . In t he p r e s e n t s t u d y , t r e a t m e n t m e a n s fo r V F A p r o d u c t i o n that w e r e s ign i f i can t l y h i g h e r t h a n t h e con t ro l w e r e , (in i n c r e a s i n g o rde r ) : 1) 0 . 0 0 1 % x y l a n a s e , 2) 0 . 0 0 5 % p r o t e a s e , 3 ) 0 . 0 0 1 % x y l a n a s e a n d 0 . 0 1 % p r o t e a s e a n d 4 ) 0 . 0 1 % p r o t e a s e . O n l y t h e 0 . 0 0 1 % x y l a n a s e t r e a t m e n t w a s s ign i f i can t l y ( P < 0 .05 ) l o w e r t h a n t he o t h e r t r e a t m e n t m e a n s l i s ted . T h e i n c r e a s e in V F A p r o d u c t i o n o b s e r v e d w i th a n u m b e r o f t he e n z y m e t r e a t m e n t s m a y b e a t t r ibu ted to a n i n c r e a s e in t he ava i lab i l i t y o f s u b s t r a t e s for m i c r o b i a l f e r m e n t a t i o n resu l t i ng f r o m e n z y m a t i c h y d r o l y s i s o f v a r i o u s c o m p o n e n t s . P o l y s a c c h a r i d e s not i n c l u d e d in t he c e l l u l o s i c f rac t ion i n c l u d e d a l a r g e a m o u n t of p e c t i n s , x y l a n s a n d x y l o g l u c a n s , w h i c h a r e l a rge l y i n s o l u b l e in w a t e r ( S l o m i n s k i a n d C a m p b e l l , 1 9 9 0 ) . T h e so lub i l i t y o f f e e d c o m p o n e n t s is k n o w n to b e re la ted to the d iges t ib i l i t y o f a f eeds tu f f ( V a n S o e s t , 1 9 9 4 ) . S l o m i n s k i et a l . , ( 1 9 9 3 ) d e t e r m i n e d that 1 4 4 t he s u p p l e m e n t a t i o n o f i n c r e a s i n g l e v e l s o f c a r b o h y d r a s e - l i k e e n z y m e s to c a n o l a m e a l r e s u l t e d in m i n o r i n c r e a s e s in t he s o l u b l e c a r b o h y d r a t e f r ac t i on . It h a s a l s o b e e n s u g g e s t e d that c a r b o h y d r a s e s resu l t in t he r e l e a s e o f shor t c h a i n p o l y s a c c h a r i d e s f r o m t h e c e l l w a l l b y s y s t e m a t i c a l l y r e m o v i n g t h e s u r f a c e c o m p o n e n t s ( H o t t e n , 1 9 9 1 ) . T h e i n c r e a s e in V F A p r o d u c t i o n f r o m 2 . 5 8 m m o l / g D M 1 to 3 . 8 8 m m o l / g D M " 1 o b s e r v e d w i th t he c o m b i n a t i o n o f 0 . 0 1 % p r o t e a s e a n d 0 . 0 0 1 % x y l a n a s e t r e a t m e n t m a y b e a t t r ibu ted to i n c r e a s e d m i c r o b i a l s u b s t r a t e ava i lab i l i t y . P r o t e o l y t i c e n z y m e s h a v e b e e n s h o w n to i n c r e a s e t he tota l w a t e r s o l u b l e c a r b o h y d r a t e c o n t e n t w h e n a p p l i e d to c a n o l a m e a l ( S l o m i n s k i et a l . , 1993 ) . It s h o u l d b e n o t e d h o w e v e r , tha t x y l a n a s e a l o n e r e s u l t e d in h i g h e r V F A t h a n p r o t e a s e a p p l i e d a l o n e . T h e e f f e c t i v e n e s s o f x y l a n a s e is e v i d e n t f r o m it's con t r i bu t i on to t he i n t e r a c t i o n s a s w e l l a s it 's m a i n e f fec t s . A s p r e v i o u s l y m e n t i o n e d , m a n y o f t he c a r b o h y d r a t e s c o n t a i n e d w i th in c a n o l a m e a l s u c h a s t he h igh a m o u n t s o f x y l a n s a n d x y l o g l u c a n s ( S l o m i n s k i a n d C a m p b e l l , 1 9 9 0 ) a r e s u s c e p t i b l e to x y l a n o l y t i c a t t a c k (B ie l y , 1 9 8 5 ) . T h e r e f o r e , i m p r o v e m e n t s in t he fe rmen tab i l i t y o f c a n o l a m e a l a s re f l ec ted w i th i n c r e a s e d V F A p r o d u c t i o n w o u l d b e e x p e c t e d wi th t he a d d i t i o n o f x y l a n a s e . T h e h e a t a n d m o i s t u r e t r e a t m e n t s o f c a n o l a m e a l d i d not s ign i f i can t l y a f fec t t he p r o d u c t i o n o f V F A s n o r d id it i m p r o v e e n z y m e act iv i ty . T h e s e resu l t s a r e c o m p a r a b l e to t h o s e o b t a i n e d w i th t he e s t i m a t e d g a s p r o d u c t i o n p a r a m e t e r s . 1 4 5 4.4.5 VFA Ratios: acetate to propionate; butyrate to propionate R a t i o s w e r e e v a l u a t e d in th is e x p e r i m e n t a s t h e y w e r e m o r e l ike ly to ref lect d i f f e r e n c e s in t h e p r o p o r t i o n s o f V F A s b e i n g p r o d u c e d a n d t h e r e f o r e re f lec t the t y p e o f s u b s t r a t e b e i n g f e r m e n t e d . It w a s d e t e r m i n e d that p r o t e a s e e l i c i ted a h igh ly s i gn i f i can t l i n e a r e f fec t ( P = 0 . 0 0 0 1 ) o n t he rat io o f a c e t a t e re la t i ve to p r o p i o n a t e p r o d u c t i o n ( F i g u r e 4 .7 ) . T h e rat io o f bu ty ra te to p r o p i o n a t e w a s a l s o s ign i f i can t l y a f f e c t e d b y t h e p r o t e a s e t r e a t m e n t ( P = 0 . 0 0 0 1 ) , a l t h o u g h t he n a t u r e o f t he ef fect c o u l d not b e d e t e r m i n e d . T h e h i g h e s t l e v e l o f p r o t e a s e s ign i f i can t l y i n c r e a s e d t he a c e t a t e to p r o p i o n a t e rat io f r o m 2 . 7 9 to 3 .74 w h e r e a s t he l o w e r l e v e l s o f a p p l i c a t i o n d i d not s ign i f i can t l y a f fec t t h e rat io ( T a b l e 4 .5 ) . T h e a p p l i c a t i o n o f p r o t e a s e at 0 . 0 1 % d id not a f fect the rat io o f a c e t a t e to p r o p i o n a t e n o r t he rat io o f bu ty ra te to p r o p i o n a t e . In con t ras t , t he rat io o f bu ty ra te to p r o p i o n a t e s h o w e d t h e g r e a t e s t i n c r e a s e f r o m 0 . 5 9 to 0 . 6 8 wi th bo th t h e l o w e s t a n d h i g h e s t l eve l o f p r o t e a s e a p p l i c a t i o n ( T a b l e 4 .5 ) . T h e n o n l i near e f fec t o f p r o t e a s e w a s p r e v i o u s l y o b s e r v e d w i th ra te o f g a s p r o d u c t i o n a n d total V F A p r o d u c t i o n . T a b l e 4 . 6 s h o w s that x y l a n a s e a p p l i e d at 0 . 0 0 1 % i n c r e a s e d t he rat io of a c e t a t e to p r o p i o n a t e p r o d u c t i o n f r o m 2 . 7 8 to 3 . 5 0 , a n d t he rat io o f bu ty ra te to p r o p i o n a t e p r o d u c t i o n f r o m 0 . 5 7 to 0 . 6 9 . G r e a t e r i n c r e a s e s in t h e rat io of a c e t a t e to p r o p i o n a t e p r o d u c t i o n w e r e o b t a i n e d w i th t he a d d i t i o n o f 0 . 0 5 % p r o t e a s e t h a n wi th a n y o t h e r t r e a t m e n t . T h e g r e a t e s t i n c r e a s e in t h e rat io o f bu ty ra te w a s o b t a i n e d wi th t he s u p p l e m e n t a t i o n o f 0 . 0 0 1 % x y l a n a s e . T h e c e l l u l o s i c f rac t i on o f a feeds tu f f is o f ten a s s o c i a t e d w i th a n i n c r e a s e in t h e p r o d u c t i o n o f a c e t a t e ( V a n S o e s t , 1994 ) . 1 4 6 T h e i n c r e a s e s in a c e t a t e p r o d u c t i o n o b s e r v e d w i th t he d e s c r i b e d e n z y m e t r e a t m e n t s s u g g e s t tha t t h e e n z y m e s e l i c i t ed the i r a c t i o n s o n t h e s t ruc tu ra l c a r b o h y d r a t e c o m p o n e n t s o f c a n o l a m e a l . N o n e o f t h e t r e a t m e n t s i n c r e a s e d p r o p i o n a t e re la t i ve to a c e t a t e n o r to bu t y ra te p r o d u c t i o n . I n c r e a s e d p r o p i o n a t e p r o d u c t i o n w o u l d i nd i ca te be t te r u t i l i za t ion o f e n e r g y a s a c e t i c a n d bu ty r i c a c i d s resu l t in a h i g h e r p r o d u c t i o n of g a s e s ( C 0 2 a n d C H 4 ) , w h i c h a r e a s o u r c e o f d i e ta r y e n e r g y los t to t h e a n i m a l ( V a n S o e s t , 1 9 8 2 ) . T h i s e x p l a n a t i o n m a y b e a p p r o p r i a t e for d e s c r i b i n g t he resu l t s of the p r e s e n t s t u d y w h e r e p ro teo ly t i c a n d x y l a n o l y t i c e n z y m e s e l i c i ted the i r e f fec ts o n the f ibro ly t ic c o m p o n e n t s o f c a n o l a m e a l a s a c e t a t e p r o d u c t i o n in pa r t i cu la r i n c r e a s e d w i th s p e c i f i c t r e a t m e n t s . T h e m e c h a n i s t i c a c t i o n a n d l im i ta t ions o f p r o t e a s e a n d x y l a n a s e h e l p e x p l a i n s o m e o f t h e o b s e r v e d resu l t s o f t h e p r e s e n t s t u d y . T h e e n z y m e p r o t e a s e a t t a c k s t he p ro teo ly t i c b o n d s w h e r e a s x y l a n a s e a t t a c k s t he x y l a n c h a i n a n d bo th o f w h i c h r e n d e r m o r e c a r b o h y d r a t e s a v a i l a b l e fo r d i g e s t i o n b y r u m e n m i c r o b e s . X y l a n a s e act iv i ty h o w e v e r , m a y b e l im i ted b y t he n u m b e r o f g l u c u r o n i c a c i d r e s i d u e s a n d a r a b i n o s e ' s i d e c h a i n s (B ie l y , 1 9 8 5 ) . T h i s m a y e x p l a i n s o m e o f t h e l a c k o f r e s p o n s e f r o m x y l a n a s e s u p p l e m e n t a t i o n in t h e p r e s e n t s t u d y . T h e r e s p o n s e o b t a i n e d wi th t h e p r o t e a s e e n z y m e m a y b e a t t r ibu ted to a n i n c r e a s e in s u b s t r a t e ava i lab i l i t y for r u m e n m i c r o b i a l d i g e s t i o n w h i c h is re f l ec ted in a g r e a t e r p r o d u c t i o n o f V F A s , h i g h e r rate o f g a s p r o d u c t i o n a n d s h o r t e r l ag p h a s e s re la t i ve to t h e u n t r e a t e d c a n o l a m e a l . 1 4 7 4.5 CONCLUSIONS E v a l u a t i o n o f t h e f e r m e n t a t i o n k i ne t i c s o f c a n o l a m e a l u s i n g g a s p r o d u c t i o n w a s s u c c e s s f u l in d e t e r m i n i n g t h e e f f e c t i v e n e s s o f e n z y m e a n d mi ld h y d r o t h e r m a l t r e a t m e n t o f c a n o l a m e a l . T h e add i t i on o f 0 . 0 1 % p r o t e a s e to c a n o l a m e a l i n c r e a s e d ra te o f g a s p r o d u c t i o n , d e c r e a s e d l ag t i m e a n d i n c r e a s e d V F A p r o d u c t i o n but h a d n o e f fec t o n t h e rat io o f V F A s . X y l a n a s e w a s o n l y e f fec t i ve w h e n u s e d in c o m b i n a t i o n w i th p r o t e a s e , h o w e v e r , it 's e f fec ts w e r e n o g r e a t e r t h a n w h e n p r o t e a s e w a s a p p l i e d a l o n e fo r bo th t he l eng th o f t he l a g p h a s e a n d t h e p r o d u c t i o n of V F A s . T h e a p p l i c a t i o n o f x y l a n a s e a n d o f t he h i g h e s t l e ve l o f p r o t e a s e i n c r e a s e d the p r o d u c t i o n o f a c e t a t e re la t i ve to p r o p i o n a t e . T h e l o w e s t a n d h i g h e s t l eve l o f p r o t e a s e a p p l i c a t i o n o r t he add i t i on o f x y l a n a s e , a l s o i n c r e a s e d t h e rat io of bu ty ra te re la t i ve to p r o p i o n a t e . T h e mi ld h y d r o t h e r m a l t r e a t m e n t o f c a n o l a m e a l i m p o s e d in th is e x p e r i m e n t d id not bene f i t e n z y m e act iv i ty a s i n d i c a t e d b y t h e l a c k o f r e s p o n s e to a n y o f t h e m e a s u r e d v a r i a b l e s . T h e resu l t s o f t he p r e s e n t s t u d y s u p p o r t fur ther r e s e a r c h into t h e e f f e c t i v e n e s s o f t he a p p l i c a t i o n o f p r o t e a s e in o r d e r to i m p r o v e the nut r i t iona l v a l u e o f c a n o l a m e a l fo r da i r y ca t t le . 1 4 8 4.6 REFERENCES B e a u c h e m i n , K. A . , R o d e , L. M . a n d V . J . H . S e w a l t . 1 9 9 5 . F ib ro l y t i c e n z y m e s i n c r e a s e f i be r d iges t ib i l i t y a n d g r o w t h ra te o f s t e e r s f e d d r y f o r a g e s . C a n . J . A n i m . S c i . , 7 5 : 6 4 1 - 6 4 4 . B e l l , J . M . 1 9 9 3 . F a c t o r s a f fec t i ng t he nut r i t iona l v a l u e o f c a n o l a m e a l : A rev iew . C a n . J . A n i m . S c i . 7 3 : 6 7 9 - 6 9 7 . B i e l y , J . 1 9 8 5 . M i c r o b i a l x y l a n o l y t i c s y s t e m s . T r e n d s in B i o t e c h n o l . , 3 ( 1 1 ) : 2 8 6 - 2 9 0 . B e u v i n k , J . M . W . a n d S . F . S p l o e s t r a . 1 9 9 4 . In vitro g a s p r o d u c t i o n k i ne t i cs o f g r a s s s i l a g e s t r e a t e d w i th d i f fe rent ce l l w a l l - d e g r a d i n g e n z y m e s . G r a s s F o r a g e S c i . , 4 9 : 2 7 7 - 2 8 3 . B l u m m e l , M . a n d E . R. O r s k o v . 1 9 9 3 . C o m p a r i s o n o f in vitro g a s p r o d u c t i o n a n d n y l o n b a g d e g r a d a b i l i t y o f r o u g h a g e s in p red i c t i ng f e e d i n take in cat t le . A n i m . F e e d S c i . T e c h n o l . , 4 0 : 1 0 9 - 1 1 9 . B r e n e s , A . , G u e n t e r , W . , M a r q u a r t , R. R . a n d B . A . Ro t te r . 1 9 9 3 . E f fec t o f p-g l u c a n a s e / p e n t o s a n a s e e n z y m e s u p p l e m e n t a t i o n o n t he p e r f o r m a n c e of c h i c k e n s a n d l a y i n g h e n s f e d w h e a t , b a r l e y , n a k e d o a t s a n d rye d i e t s . C a n . J . A n i m . S c i . , 7 3 : 9 4 1 - 9 5 1 . C h e n g , K . - J . , F a y , J . P . , H o w a r t h , R . E . a n d J . W . C o s t e r t o n . 1 9 8 0 . S e q u e n c e of e v e n t s in t h e d i g e s t i o n o f f r e s h l e g u m e l e a v e s by r u m e n b a c t e r i a . A p p l . E n v i r o n . M i c r o b i o l . , 4 0 : 6 1 3 - 6 2 5 . C o c h r a n , W . G . a n d G . M . C o x . 1 9 5 0 . E x p e r i m e n t a l d e s i g n s . P a g e s 1 5 4 - 1 5 6 ; 2 4 1 -2 4 6 . J o h n W i l e y a n d S o n s Inc., N e w Y o r k , N Y . E r w i n , E . S . , M a r c o , G . J . a n d E . M . E m e r y . 1 9 6 1 . V o l a t i l e fat ty a c i d a n a l y s i s o f b l ood a n d r u m e n f lu id b y g a s c h r o m a t o g r a p h y . J . D a i r y S c i . , 4 4 : 1 7 6 8 - 1 7 7 1 . F r a n c e , J . , D h a n o a , M . S . , T h e o d o r o u , M . K. , L i s te r , S . J . , D a v i e s , D. R . a n d D. Isac . 1 9 9 3 . A m o d e l to in terpre t g a s a c c u m u l a t i o n p ro f i l es a s s o c i a t e d w i th in vitro d e g r a d a t i o n o f r u m i n a n t f e e d s t u f f s . J . T h e o r . B i o l . , 1 6 3 : 9 9 - 1 1 1 . G o e r i n g , H . K. a n d P . J . V a n S o e s t . 1 9 7 0 . F o r a g e F i b e r A n a l y s e s . ( A p p a r a t u s , R e a g e n t s , P r o c e d u r e s , a n d s o m e A p p l i c a t i o n s ) . A g r i c . H a n d b o o k N o . 3 7 9 . , A R S - U S D A , W a s h i n g t o n , D C . 1 4 9 H i d a y a t , K. ; H i l l m a n , N e w b o l d , C . J . a n d C S . S t e w a r t . 1 9 9 3 . T h e con t r i bu t i ons o f b a c t e r i a a n d p r o t o z o a to r u m i n a l f o r a g e f e r m e n t a t i o n in vitro, a s d e t e r m i n e d b y m i c r o b i a l g a s p r o d u c t i o n . A n i m . F e e d S c i . T e c h n o l . , 4 2 : 1 9 3 - 2 0 8 . H o t t e n , P . 1 9 9 1 . W h y c o n s i d e r e n z y m e s a s a f e e d a d d i t i v e ? M i s s e t - W o r l d Pou l t . , 7 : 1 3 - 1 5 . K r i s h n a m o o r t h y , U . , S o l l e r , H . , S t e i n g a s s , H . a n d K. H . M e n k e . 1 9 9 1 . A c o m p a r a t i v e s t u d y o n r u m e n f e r m e n t a t i o n o f e n e r g y s u p p l e m e n t s in vitro. J . A n i m . P h y s i o l . A n i m . Nut r . , 6 5 : 2 8 - 3 5 . K r i s h n a m o o r t h y , U . , S o l l e r , H . , S t e i n g a s s , H . a n d K. H . M e n k e . 1 9 9 5 . E n e r g y a n d p ro te in e v a l u a t i o n o f t rop i ca l f e e d s t u f f s fo r w h o l e t ract a n d r u m i n a l d i g e s t i o n b y c h e m i c a l a n a l y s e s a n d r u m e n i n o c u l u m s t u d i e s in vitro. A n i m . F e e d . S c i . T e c h n o l . , 5 2 : 1 7 7 - 1 8 8 . M c A l l i s t e r , T . A . , C h e n g , K . - J . , R o d e , L. M . a n d J . G . B u c h a n a n - S m i t h . 1 9 9 0 a . U s e o f f o r m a l d e h y d e to r e g u l a t e d i g e s t i o n o f b a r l e y s t a r c h . C a n . J . A n i m . S c i . , 7 0 : 5 8 1 - 5 8 9 . M c A l l i s t e r , T . A . , R o d e , L. M . , C h e n g , K . - J . , S c h a e f e r , D . M . a n d J . W . C o s t e r t o n . 1 9 9 0 c . M o r p h o l o g i c a l s t u d y o f t h e d i g e s t i o n o f b a r l e y a n d m a i z e g r a i n b y r u m e n m i c r o o r g a n i s m s . A n i m . F e e d S c i . T e c h n o l . , 3 1 : 9 1 - 1 0 5 . M c A l l i s t e r , T . A . , R o d e , L. M . , M a j o r , D. J . , C h e n g , K . - J . a n d J . G . B u c h a n a n - S m i t h . 1 9 9 0 b . E f f ec t o f r u m i n a l c o l o n i z a t i o n o n c e r e a l g r a i n d i g e s t i o n . C a n . J . A n i m . S c i . , 7 0 : 5 7 1 - 5 7 9 . M e n k e , K. H . a n d H . S t e i n g a s s . 1 9 8 8 . E s t i m a t i o n o f t he e n e r g e t i c f e e d v a l u e o b t a i n e d f r o m c h e m i c a l a n a l y s i s a n d in vitro g a s p r o d u c t i o n u s i n g r u m e n f lu id . A n i m . R e s . D e v . , 2 8 : 7 - 5 5 . M e n k e , K. H . , R a a b , L , S a l e w s k i , A . , S t e i n g a s s , H . , F r i t z , D . a n d W . S c h n e i d e r . 1 9 7 9 . T h e e s t i m a t i o n o f t h e d iges t ib i l i t y a n d m e t a b o l i s a b l e e n e r g y c o n t e n t o f r u m i n a n t f e e d i n g s t u f f s f r o m t h e g a s p r o d u c t i o n w h e n t h e y a r e i n c u b a t e d wi th r u m e n l i quo r in vitro. J . A g r i c . S c i . ( C a m b . ) , 9 3 : 2 1 7 - 2 2 2 . M e r t e n s , D . R . 1 9 7 7 . D i e t a r y f i be r c o m p o n e n t s ; r e l a t i onsh ip to ra te a n d ex ten t o f r u m i n a l d i g e s t i o n . F e d . P r o c . F e d . A m . S o c . E x p . B i o l . , 3 6 : 1 8 7 - 1 9 2 . M i r , Z . ; M a c L e o d , G . K. ; B u c h a n a n - S m i t h , J . G . ; G r i e v e , D. G . a n d W . L. G r o v u m . 1 9 9 2 . M e t h o d s fo r p r o t e a c t i n g s o y b e a n a n d c a n o l a p ro te i ns f r o m d e g r a d a t i o n in t he r u m e n . C a n . J . A n i m . S c i . , 6 4 : 8 5 3 - 8 6 5 . 1 5 0 M o s h t a g h i N i a , S . A . a n d J . R . Inga l ls . 1 9 9 2 . E f fec t o f h e a t i n g o n c a n o l a m e a l p ro te in d e g r a d a t i o n in t he r u m e n a n d d i g e s t i o n in t h e l o w e r gas t ro i n tes t i na l t rac t o f s t e e r s . C a n . J . A n i m . S c i . , 7 2 : 8 3 - 8 8 . O p a t p a t a n a k i t , Y . , K e l l a w a y , R . C , L e a n , I. J . , A n n i s o n , G . a n d A . K i rby . 1 9 9 4 . M i c r o b i a l f e r m e n t a t i o n o f c e r e a l g r a i n s in vitro. A u s t . J . A g r i c . R e s . , 4 5 : 1 2 4 7 -1 2 6 3 . P e l l , A . N . a n d P . S c h o f i e l d . 1 9 9 3 . C o m p u t e r i z e d m o n i t o r i n g o f g a s p r o d u c t i o n to m e a s u r e f o r a g e d i g e s t i o n in vitro. J . D a i r y S c i . , 7 6 : 1 0 6 3 - 1 0 7 3 . S c h o f i e l d , P . a n d A . N . P e l l . 1 9 9 5 . Va l i d i t y o f u s i n g a c c u m u l a t e d g a s p r e s s u r e r e a d i n g s to m e a s u r e f o r a g e d i g e s t i o n in vitro: a c o m p a r i s o n i nvo l v i ng t h ree f o r a g e s . J . D a i r y S c i . , 7 8 : 2 2 3 0 - 2 2 3 8 . S l o m i n s k i , B . A . a n d L. D. C a m p b e l l . 1 9 9 0 . N o n - s t a r c h p o l y s a c c h a r i d e s o f c a n o l a m e a l : Q u a n t i f i c a t i o n , d iges t ib i l i t y in pou l t ry a n d po ten t i a l bene f i t o f d ie ta ry e n z y m e s u p p l e m e n t a t i o n . J . S c i . F o o d A g r i c , 5 3 : 1 7 5 - 1 8 4 . S l o m i n s k i , B . A . , G u e n t e r , W . a n d L. D. C a m p b e l l . 1 9 9 3 . N e w a p p r o a c h to wa te r -s o l u b l e c a r b o h y d r a t e d e t e r m i n a t i o n a s a too l fo r e v a l u a t i o n o f p lan t ce l l wa l l d e g r a d i n g e n z y m e s . J . A g r i c . F o o d C h e m . , 4 1 : 2 3 0 4 - 2 3 0 8 . S ta t i s t i ca l A n a l y s i s S y s t e m ( S A S ) , 1 9 9 0 . S A S U s e r ' s G u i d e : S t a t i s t i c s . S A S Insti tute Inc., C a r y , N C . T h e o d o r o u , M . K. , W i l l i a m s , B . A . D h a n o a , M . S . , M c A l l a n , A . B . a n d J . F r a n c e . 1 9 9 4 . A s i m p l e g a s p r o d u c t i o n m e t h o d u s i n g a p r e s s u r e t r a n s d u c e r to d e t e r m i n e t h e f e r m e n t a t i o n k i ne t i cs o f r um inan t f e e d s . A n i m . F e e d S c i . T e c h n o l . , 4 8 : 1 8 5 - 1 9 7 . T i l l ey , J . M . A . a n d R . A . T e r r y . 1 9 6 3 . A t w o - s t a g e t e c h n i q u e for t h e in vitro d i g e s t i o n o f f o r a g e c r o p s . Br i t . J . G r a s s l . S o c , 18 : 1 0 4 - 1 1 1 . V a n S o e s t , P . J . 1 9 8 2 . Nut r i t i ona l e c o l o g y o f t h e rum inan t . O & B B o o k s Inc., C o r v a l l i s , O r e g o n . V a n S o e s t , P . J . 1 9 9 4 . Nut r i t i ona l e c o l o g y o f t he rum inan t . 2 n d e d . , O & B B o o k s Inc., C o r v a l l i s , O r e g o n . 151 T a b l e 4 . 1 . T r e a t m e n t s i n v e s t i g a t e d in o r d e r to d e t e r m i n e t he e f f ec t s o f e x o g e n o u s e n z y m e a p p l i c a t i o n w i th a n d w i thou t a d d e d m o i s t u r e a n d h e a t o n in vitro g a s p r o d u c t i o n p a r a m e t e r s a n d V F A a n a l y s e s o f c a n o l a m e a l . F a c t o r L e v e l o f a p p l i c a t i o n H e a t 1 con t ro l (0), 1 5 , 3 0 , 6 0 m in M o i s t u r e 2 0 % , 1 0 % , 2 0 % , 3 0 % P r o t e a s e : ,2, 3, 4 0 % , 0 . 0 0 5 % , 0 . 0 5 % , 0 . 0 1 % X y l a n a s e : ,2, 3, 5 0 % , 0 . 0 0 1 % 1 A p p l i e d at 5 5 ° C . 2 A p p l i e d o n a % v o l / w t b a s i s . 3 S u p p l i e d b y F i n n F e e d s In te rna t iona l L t d . , W i l t s h i r e , U . K . 4 H e p t e x s b - p r o t e a s e . 5 A v i z y m e 1 3 1 0 . 1 5 2 T a b l e 4 . 2 . T h e e f fec t o f t he p r o t e a s e a p p l i c a t i o n o n t he in vitro ra te o f g a s p r o d u c t i o n o f c a n o l a m e a l . P r o t e a s e 1 (%) R a t e (ml_*h- 1 ) 2 0 0 . 0 7 7 3 0 . 0 0 5 0 . 0 7 5 3 0.01 0 . 1 0 8 b 0 . 0 5 0 . 0 8 9 a b S . E . M . 3 0 . 0 0 4 1 A p p l i e d o n a % v o l / w t b a s i s . 2 R a t e o f g a s p r o d u c t i o n (b) w a s c a l c u l a t e d u s i n g t he e q u a t i o n Y = A (1 - e <-b('-l>). 3 S . E . M . , p o o l e d s t a n d a r d e r ro r o f t he m e a n . a b M e a n s in a c o l u m n wi th d i f fe rent s u p e r s c r i p t s a r e s ign i f i can t l y d i f ferent (P < 0.05). 1 5 3 T a b l e 4 . 3 . T h e e f fec t o f p r o t e a s e a n d x y l a n a s e a p p l i c a t i o n o n t h e l eng th o f t he lag p h a s e in vitro o f c a n o l a m e a l . E n z y m e t r e a t m e n t 1 P r o t e a s e (%) X y l a n a s e (%) L a g (h ) 2 0 0 0 . 4 4 9 c 0 . 0 0 5 0 0 . 2 6 6 a b 0.01 0 0 . 1 4 0 a 0 . 0 5 0 0 . 2 7 3 b 0 0 .001 0 . 1 8 7 a b 0 . 0 0 5 0 .001 0 . 2 9 4 b 0.01 0 .001 0 . 1 3 6 a 0 . 0 5 0 .001 0 . 1 8 7 a b S . E . M . 3 0 . 0 4 6 1 A p p l i e d o n a % v o l / w t b a s i s . 2 L e n g t h o f t h e l a g p h a s e (/) w a s c a l c u l a t e d u s i n g t he e q u a t i o n Y = A ( 1 - e ( b ( t ~ l > ) . 3 S . E . M . , p o o l e d s t a n d a r d e r ro r o f t h e m e a n . a c M e a n s in a c o l u m n w i th d i f fe rent s u p e r s c r i p t s a r e s ign i f i can t l y d i f fe rent (P < 0.05). 1 5 4 T a b l e 4 . 4 . T h e e f fec t o f p r o t e a s e a n d x y l a n a s e a p p l i c a t i o n o n t h e 6 h in vitro V F A p r o d u c t i o n o f c a n o l a m e a l . E n z y m e t r e a t m e n t 1 P r o t e a s e (%) X y l a n a s e (%) V F A ( m m o l / g ) 0 0 2 . 5 8 a 0 . 0 0 5 0 3 . 3 6 ^ 0.01 0 3 . 9 2 d 0 . 0 5 0 3 . 2 6 a b 0 0 .001 3 . 2 4 b c 0 . 0 0 5 0 .001 2 . 3 4 a 0.01 0 .001 3 . 8 8 d 0 . 0 5 0 .001 2 . 6 6 a b S . E . M . 2 0 . 2 0 8 1 A p p l i e d o n a % v o l / w t b a s i s . 2 S . E . M . , p o o l e d s t a n d a r d e r ro r o f t h e m e a n . a _ d M e a n s in a c o l u m n w i th d i f fe rent s u p e r s c r i p t s a r e s ign i f i can t l y d i f ferent (P < 0.05). 1 5 5 T a b l e 4 . 5 . T h e e f fec t o f p r o t e a s e a p p l i c a t i o n o n t h e 6 h in vitro rat io o f a c e t a t e to p r o p i o n a t e a n d bu t y ra te to p r o p i o n a t e o f c a n o l a m e a l . P r o t e a s e 1 (%) A : P 2 B : P 2 0 2 . 7 8 9 a 0 . 5 9 a 0 . 0 0 5 3 . 1 2 8 a 0 . 6 8 b 0.01 2 . 8 9 0 a 0 . 5 7 a 0 . 0 5 3 . 7 3 6 b 0 . 6 8 b S . E . M . 3 0 . 1 5 4 0 . 2 5 3 1 A p p l i e d o n a % v o l / w t b a s i s . 2 M o l a r ra t ios 3 S . E . M . , p o o l e d s t a n d a r d e r ro r of t he m e a n . a b M e a n s in a c o l u m n wi th d i f fe rent s u p e r s c r i p t s a r e s ign i f i can t l y d i f fe rent (P < 0.05). 1 5 6 T a b l e 4 . 6 . T h e e f fec t o f x y l a n a s e a p p l i c a t i o n o n t h e 6 h in vitro rat io o f a c e t a t e to p r o p i o n a t e ( A : P ) a n d bu ty ra te to p r o p i o n a t e ( B : P ) o f c a n o l a m e a l . X y l a n a s e 1 (%) A : P 2 B : P 2 0 2 . 7 7 9 0 . 5 7 0 .001 3 . 4 9 3 0 . 6 9 S . E . M . 3 0 . 1 0 8 0 . 0 1 8 1 A p p l i e d o n a % v o l / w t b a s i s . 2 M o l a r ra t ios 3 S . E . M . , p o o l e d s t a n d a r d e r ro r o f t h e m e a n . 1 5 7 F i g u r e 4 . 1 . T h e c o m p u t e r i z e d g a s p r o d u c t i o n s y s t e m ( a d a p t e d f r o m P e l l a n d S c h o f i e l d , 1993 ) . 1 5 8 Figure 4.2. Customized Erlenmeyer flasks with sampling port containing luer lock and tubing apparatus for V F A sampling. 159 091-0 . 1 2 -, 0.1 0 . 0 8 0 . 0 6 0 . 0 4 0 . 0 2 0 0 0 . 0 0 5 0.01 P r o t e a s e l eve l (% v o l / wt) a b 0 . 0 5 F i g u r e 4 . 4 . T h e e f fec t o f t he add i t i on o f p r o t e a s e o n t h e ra te o f r u m e n in vitro g a s p r o d u c t i o n o f c a n o l a m e a l i n c u b a t e d w i th r u m e n f lu id . 161 0 0.01 0 . 0 2 0 . 0 3 0 . 0 4 0 . 0 5 P r o t e a s e l eve l (% vo l / wt ) F i g u r e 4 . 5 . T h e e f fec t o f t he add i t i on o f p r o t e a s e a n d x y l a n a s e (% v o l / wt ) o n t he l eng th o f t he l ag p h a s e o f c a n o l a m e a l i n c u b a t e d in vitro w i th r u m e n f lu id . 1 6 2 F i g u r e 4 . 6 . T h e e f fec t o f t he add i t i on o f x y l a n a s e a n d p r o t e a s e (% v o l / wt ) o n t he p r o d u c t i o n o f V F A s in c a n o l a m e a l f o l l o w i n g a 6 h in vitro i n c u b a t i o n w i th r u m e n f lu id . 1 6 3 CD CD -+—' CD O < 2 . 5 + + 0 0.01 0 . 0 2 0 . 0 3 0 .04 0 . 0 5 P r o t e a s e l eve l (% vo l / wt ) F i g u r e 4 . 7 . L i n e a r r e l a t i onsh ip b e t w e e n l eve l o f p r o t e a s e a p p l i c a t i o n a n d t he rat io o f a c e t a t e to p r o p i o n a t e in c a n o l a m e a l f o l l ow ing a 6 h in vitro i n c u b a t i o n w i th r u m e n f lu id ( Y = 1 7 . 5 4 6 * p r o t e a s e l eve l + 2 .851 : n = 4 , R 2 = 0 . 8 3 9 , S . E . = 0 .17 ) . 1 6 4 CHAPTER 5 5.0 GENERAL CONCLUSIONS In t h e L o w e r F r a s e r V a l l e y r e g i o n o f Br i t i sh C o l u m b i a ( B . C . ) , m a x i m i z i n g f e e d u t i l i za t ion is a p r i m a r y g o a l a s f e e d c o s t s i n c r e a s e . G r e a t e r a w a r e n e s s o f t he e n v i r o n m e n t a l i m p a c t o f t h e i n t e n s i v e da i r y a n d a n i m a l p r o d u c t i o n in th is a r e a a l s o p r o m o t e s r e s e a r c h into m i n i m i z i n g nut r ient w a s t e s . B a r l e y g ra i n is t he m a j o r e n e r g y s o u r c e a n d c a n o l a m e a l is t h e m a j o r a l t e rna t i ve p ro te in s o u r c e to s o y b e a n m e a l u s e d in ra t i ons fo r d a i r y ca t t le . C a n o l a m e a l is res t r i c ted in da i r y ca t t le d i e t s b e c a u s e o f it 's h i g h f i be r c o n t e n t w h i c h l o w e r s it 's e n e r g y v a l u e . B a r l e y g ra i n is re la t i ve ly h igh in s t a r c h , h o w e v e r r e s e a r c h fo r i m p r o v i n g it 's nut r i t iona l v a l u e is a l s o a s a c o n s e q u e n c e o f it 's re la t i ve ly h igh f i be r con ten t . C u r r e n t p r o c e s s i n g t e c h n o l o g y fo r r um inan t f e e d s t u f f s en ta i l s c o n v e n t i o n a l m e t h o d s . T h e poss ib i l i t y o f e n z y m e s u s e d in c o n j u n c t i o n w i th p r o c e s s i n g t e c h n o l o g y h a s ye t to b e r e a l i z e d . T h e o b j e c t i v e o f t he cu r ren t s t u d y w a s to i n v e s t i g a t e t he feas ib i l i t y o f a p p l y i n g e n z y m e s a n d mi ld h y d r o t h e r m a l t r e a t m e n t to b a r l e y a n d c a n o l a m e a l in o r d e r to i m p r o v e the i r nut r i t iona l v a l u e for da i r y ca t t le . 5.1 BARLEY In vitro a n a l y s e s w e r e u s e d to e v a l u a t e t h e e f fec t s o f b a r l e y t r ea ted wi th p-g l u c a n a s e , p r o t e a s e , h e a t a n d m o i s t u r e . R e s u l t s o f in vitro d iges t ib i l i t i es o f ba r l ey a r e p r e s e n t e d in C h a p t e r 2 , s e c t i o n 2 . 3 . A s m e n t i o n e d in C h a p t e r 2 , da i r y p r o d u c e r s 165 in t he L o w e r F r a s e r V a l l e y r e g i o n o f B . C . f a c e a u n i q u e c h a l l e n g e to m a t c h the h igh s o l u b l e n i t r ogen c o n t e n t p r e s e n t in g r a s s s i l a g e s ( von K e y s e r l i n g k et a l . , 1 9 9 6 ) wi th a read i l y a v a i l a b l e c a r b o h y d r a t e s o u r c e in o r d e r to m a x i m i z e r u m e n m ic rob ia l p ro te in p r o d u c t i o n a n d o p t i m i z e p e r f o r m a n c e ( K r i s h n a m o o r t h y et a l . , 1 9 9 1 ; S t o k e s et a l . , 1 9 9 1 ) . T h e i n t e n s i v e ag r i cu l tu ra l p r a c t i c e s in th is e n v i r o n m e n t a l l y f rag i le r e g i o n a l s o r e q u i r e a n a c t i v e ro le by p r o d u c e r s to r e d u c e nut r ient w a s t e s by b a l a n c i n g t h e s u p p l y o f n i t r ogen a n d c a r b o h y d r a t e s to t h e r u m e n ( H u n t i n g t o n , 1 9 9 5 ) a n d t h e m a x i m i z i n g o f f e e d u t i l i za t ion . M i l d h e a t a p p e a r e d not to h a v e a n ef fect , w h e r e a s t he a d d i t i o n o f m o i s t u r e h a d a q u a d r a t i c r e l a t i onsh ip w i th d igest ib i l i ty . F r o m th is s t u d y , it a p p e a r s x y l a n a s e a l o n e is d e t r i m e n t a l t o w a r d s t h e d iges t ib i l i t y o f ba r l ey . H o w e v e r , po ten t i a l a p p l i c a t i o n s ex i s t fo r p r o t e a s e a p p l i e d a l o n e at a m o d e r a t e leve l ( 0 . 0 1 % ) o r a c o m b i n a t i o n o f p r o t e a s e a n d p - g l u c a n a s e to i m p r o v e t he f e e d i n g qua l i t y o f b a r l e y . 5.2 CANOLA MEAL R e s u l t s o f t h e in vitro d iges t ib i l i t i es o f t h e c a n o l a m e a l t r e a t m e n t s m a y b e f o u n d in C h a p t e r 3 , s e c t i o n 3 . 3 . A s e c o n d e x p e r i m e n t u t i l i zed t he g a s p r o d u c t i o n t e c h n i q u e to o b t a i n i n f o rma t i on o f t he e f fec ts o f t he e n z y m e a n d mi ld h y d r o t h e r m a l t r e a t m e n t s o n t h e f e r m e n t a t i o n c h a r a c t e r i s t i c s o f c a n o l a m e a l . T h e resu l t s o f t he g a s p r o d u c t i o n e x p e r i m e n t m a y b e f o u n d in C h a p t e r 4 , s e c t i o n 4 . 3 . R e s u l t s f r o m t h e s e two e x p e r i m e n t s s u g g e s t e d that s o m e t r e a t m e n t s m a y el ic i t the i r e f f ec t s o n s p e c i f i c f e e d c o m p o n e n t s . T h e g a s p r o d u c t i o n s t u d y a p p e a r e d to b e m o r e s e n s i t i v e to the 1 6 6 d i f f e r e n c e s b e t w e e n t r e a t m e n t s t h a n t he d iges t ib i l i t y e x p e r i m e n t , a l t h o u g h s im i l a r c o n c l u s i o n s w e r e r e a c h e d in s e l e c t i n g b e n e f i c i a l t r e a t m e n t s . T h e p r i nc ip l e b e h i n d a p p l y i n g h e a t a n d m o i s t u r e t r e a t m e n t s w a s to o p t i m i z e e n z y m e act iv i ty . T h e resu l t s o f t he g a s p r o d u c t i o n e x p e r i m e n t i n d i c a t e d t h e s e c o n d i t i o n s w e r e not c o n d u c i v e to o p t i m i z i n g e n z y m e a c t i o n o n t h e f e e d s u b s t r a t e . M o i s t u r e e l i c i t ed a q u a d r a t i c e f fec t o n t h e d iges t ib i l i t y o f c a n o l a m e a l that w a s not d e t e c t e d in t h e e v a l u a t i o n o f g a s p r o d u c t i o n . T r e a t m e n t o f c a n o l a w i th x y l a n a s e w a s d e t r i m e n t a l to d iges t ib i l i t y , s i m i l a r to t he resu l t s p r e v i o u s l y o b s e r v e d w i th ba r l ey , w h e r e a s , t h e a d d i t i o n o f p r o t e a s e at a m o d e r a t e l eve l a p p e a r e d to b e s u c c e s s f u l in i n c r e a s i n g t h e f e rmen tab i l i t y o f c a n o l a m e a l . 5.3 SUMMARY In c o n c l u s i o n , it a p p e a r s that t r e a t m e n t s w h i c h i m p r o v e d t h e nutr i t ive v a l u e of b a r l e y a n d c a n o l a m e a l a s d e t e r m i n e d u s i n g in vitro a n a l y s e s , m a y b e p r imar i l y e x p l a i n e d b y t h e a d d i t i o n o f p r o t e a s e . H o w e v e r , it is impo r tan t that a n i m a l p e r f o r m a n c e t r ia ls b e c o n d u c t e d o n s e l e c t e d t r e a t m e n t s to c l e a r l y d e f i n e the po ten t ia l a p p l i c a t i o n s o f p r o t e a s e a n d o t h e r e n z y m e s in r u m i n a n t f e e d s . I n c r e a s i n g f e e d c o s t s a n d a c o m p e t i t i v e m a r k e t fo r bo th da i r y p r o d u c e r s a n d t he f e e d indus t ry a l i ke , wi l l p r o m o t e r e s e a r c h into i n n o v a t i v e p r o c e s s i n g t e c h n o l o g y s u c h a s that i n v e s t i g a t e d in t he e x p e r i m e n t s d i s c u s s e d in th is t h e s i s . A l t h o u g h t h e " e c o n o m i c s " of i m p l e m e n t i n g e n z y m e s into r u m i n a n t f e e d s h a v e not b e e n d i s c u s s e d in th is t h e s i s , e n z y m e s a r e a n e x p e n s i v e c o m m o d i t y a n d the i r p rac t i ca l a p p l i c a t i o n s r e m a i n 1 6 7 d e p e n d e n t u p o n t h e re turn o n i n v e s t m e n t into m o s t l ike ly , m o r e e x p e n s i v e , " v a l u e a d d e d " p r o d u c t . 1 6 8 5.4 REFERENCES H u n t i n g t o n , J . A . 1 9 9 5 . R u m e n f e r m e n t a t i o n d y n a m i c s . P a g e s 1 7 - 1 9 in F e e d C o m p o u n d e r , M a y i s s u e . K r i s h n a m o o r t h y , U . , S o l l e r , H . , S t e i n g a s s , H . a n d K. H . M e n k e . 1 9 9 1 . A c o m p a r a t i v e s t u d y o n r u m e n f e r m e n t a t i o n o f e n e r g y s u p p l e m e n t s in v i t r o . J . A n i m . P h y s i o l . A n i m . Nut r . , 6 5 : 2 8 - 3 5 . S t o k e s , S . R. , H o o v e r , W . H . , M i l l e r , T . K. a n d R. B l a u w e i k e l . 1 9 9 1 . R u m i n a l d i g e s t i o n a n d m i c r o b i a l u t i l i za t ion o f d i e t s v a r y i n g in t y p e o f c a r b o h y d r a t e a n d p ro te in . J . D a i r y S c i . , 7 5 : 3 4 5 - 3 5 7 . v o n K e y s e r l i n g k , M . A . G . , Swi f t , M . L , P u c h a l a , R . a n d J . A . S h e l f o r d . 1 9 9 6 . D e g r a d a b i l i t y c h a r a c t e r i s t i c s o f d r y ma t te r a n d c r u d e p ro te in o f f o r a g e s in r u m i n a n t s . A n i m . F e e d S c i . T e c h n o l . , 5 7 : 2 9 1 - 3 1 1 . 1 6 9 6.0 APPENDICES 1 7 0 CO CD ' CO 1— 00 X> CO CO 3 g CD > c O T3 CD -*—< CO CD 0 CD CD 1 C LU CO J D .Q CD C 'CD CO CD o to co >, 0 _CD C 1 X I O j | >% CD "O SZ 0) CD O i : CD £ -!= < CD c CD O CQ. CD O CD D_ o CD •4-» CO c TO X o o CD co o "CD O 0- O £ CM CD E N C LU 9 1 ° ^ " LO I s -T— CO O I S -0 0 oo CD CO CD c CD -=• CD CM 0 0 O 0 0 co oo Tt-<M I S -co co O t- CD T - 1 CM CD CD CN o CO CN CO CD L O CM oo d I S - 1 -C N 05 c o CN CM I S -CD CN O CO I S -CO T— CD CM CD 3 CD _ -t—• X CQ.CL CD CD •c CD X I CD T3 Z) CD" L — x: CO CD _ l c o "-»—» CD -•—» CO x o I— CD CD CO CD or CD CD | | o TO D) a) < C CD — CT CO T3 T3 • C CD X I CD -C LL -*—» CD C —I C >.u. •° >, 0 3 -n -*-» CJ O CD ZS = T3 Q. C C L O =3 O CO CD CD ^ CO ~ ~ " E o LO CM CD E N C CD CD SZ X I T3 CD CO CD 0) CD CD CO o o CT M — O E CL Q. CO CD T3 CD CO CO CD L -C L X CD CO o CD co" CD -i—» CO CO N CM >,<6 X CD E N C CD CT E o LO C N CD CD-S' C£ to ' c =3 CD > -t—< _co CD L — to To co ro X I <D T3 3 CD to CD W r w ° x co $ 11 CO o ct* CD O CO CD CO O X CD < LU Z 2 T - CM CO CD -t—» 2 I.0" 171 APPENDIX 6.2 COMPOSITION OF THE CONCENTRATE RATION FED TO DONOR COWS. T a b l e 6 . 2 . Nu t r i en t c o m p o s i t i o n o f t h e da i r y c o n c e n t r a t e ra t ion . Ing red ien t C o n t e n t (% a s f e d b a s i s ) B a r l e y ( 1 1 . 5 % C P ) 5 8 . 6 W h e a t mi l l run 1 4 . 7 C a n o l a m e a l 11 .2 D a i r y f i n e s 3 .5 B e e t p u l p 3 .0 M o l a s s e s / c a n e / L I 2 . 7 L i m e s t o n e 1.75 C o r n ( 8 .5%) 1.0 S a l t a n d t r a c e m i n e r a l m i x 0 . 8 8 D is t i l l e rs g r a i n 0 . 8 7 S o y a oi l 0 . 7 5 R a y p e l l b i n d e r 0 .7 M e a t a n d b o n e m e a l 0 . 3 5 1 7 2 APPENDIX 6.3. SAS PROGRAM USED TO EVALUATE BARLEY DATA. o p t i o n s n o c e n t r e l i n e s i z e = 7 5 ; f i l e n a m e invi t ro ' f i l e n a m e ' ; d a t a ; inf i le inv i t ro; input f e e d r e p i v d m d ivtd a d f nd f h e a t m o i s t b e t a prot x y l ; t it le ' in v i t ro r esu l t s a n d f i be r a n a l y s e s o f ba r l ey ' ; p r o c g l m ; c l a s s h e a t m o i s t b e t a prot x y l ; m o d e l i v d m d ivtd a d f nd f = h e a t | m o i s t | b e t a | prot | xy l / n o u n i s s 3 ; r a n d o m h e a t * m o i s t * b e t a * p r o t * x y l / tes t ; I s m e a n s h e a t m o i s t b e t a prot xy l h e a t * m o i s t h e a t * b e t a h e a t * p r o t h e a t * x y l m o i s t * b e t a mo i s t *p ro t mo i s t * xy l be ta *p ro t be ta *xy l p ro t *xy l h e a t * m o i s t * b e t a h e a t * m o i s t * p r o t h e a t * m o i s t * xy l hea t *p ro t *xy l h e a t * b e t a * x y l h e a t * b e t a * p r o t m o i s t * b e t a * p r o t m o i s t * b e t a * x y l mo is t *p ro t *xy l be ta *p ro t * xy l h e a t * m o i s t * b e t a * p r o t h e a t * m o i s t * p r o t * x y l h e a t * b e t a * p r o t * x y l h e a t * m o i s t * b e t a * x y l m o i s t * b e t a * p r o t * x y l / s t d e r r e = h e a t * m o i s t * b e t a * p r o t * x y l ; run ; N o r m a l l y , o n l y r e l e v a n t i n t e rac t i ons a r e i n c l u d e d a s t e r m s in t h e L S M E A N S s t a t e m e n t . D u e to t h e l a rge n u m b e r o f f a c t o r s in th is e x p e r i m e n t , t he e r ro r t e rm i n c l u d e s t he h i g h e s t o r d e r i n te rac t i on a s a n i nd i ca t i on o f e x p e r i m e n t a l er ror . T h e 1 7 3 m o d e l s t a t e m e n t i n c l u d e s t h e s u m o f a l l o f t e r m s i n c l u d e d in t h e L S M E A N S s t a t e m e n t . 1 7 4 APPENDIX 6.4. SAS PROGRAM USED TO EVALUATE CANOLA MEAL DATA. o p t i o n s n o c e n t r e l i n e s i z e = 7 5 ; f i l e n a m e invi t ro ' f i l e n a m e ' ; d a t a ; inf i le inv i t ro; input f e e d r e p i v d m d ivtd a d f nd f h e a t m o i s t prot x y l ; t it le ' in v i t ro r e s u l t s a n d f i be r a n a l y s e s o f c a n o l a m e a l ; p r o c g l m ; c l a s s h e a t m o i s t prot x y l ; m o d e l i v d m d ivtd a d f nd f = h e a t | m o i s t | prot | xy l / n o u n i s s 3 ; r a n d o m h e a t * m o i s t *p ro t *xy l / tes t ; I s m e a n s h e a t m o i s t prot xy l h e a f m o i s t hea t *p ro t h e a t * x y l mo i s t *p ro t mo i s t * xy l p ro t *xy l h e a t * m o i s t * p r o t h e a t * m o i s t * xy l hea t *p ro t * xy l mo i s t *p ro t * xy l h e a t * m o i s t * p r o t * x y l / s t de r r e = h e a t * m o i s t * p r o t * x y l ; r un ; N o r m a l l y , o n l y r e l e v a n t i n t e rac t i ons a r e i n c l u d e d a s t e r m s in t he L S M E A N S s t a t e m e n t . D u e to t h e l a r g e n u m b e r o f f a c t o r s in th is e x p e r i m e n t , t he e r ro r t e r m i n c l u d e s t h e h i g h e s t o r d e r i n te rac t i on a s a n i nd i ca t i on o f e x p e r i m e n t a l er ror . T h e m o d e l s t a t e m e n t i n c l u d e s t he s u m o f al l o f t e r m s i n c l u d e d in t he L S M E A N S s t a t e m e n t . 1 7 5 APPENDIX 6.5 CALIBRATION OF THE GAS MEASUREMENT SYSTEM (Schofield and Pell, 1995). C a l i b r a t i o n o f t h e g a s p r o d u c t i o n s y s t e m w a s r e q u i r e d in o r d e r to e s t a b l i s h t he l i nea r r e l a t i o n s h i p b e t w e e n t h e c h a n g e in p r e s s u r e a r i s i n g f r o m g a s p r o d u c t i o n a n d t h e s u b s e q u e n t v o l t a g e r e s p o n s e . A r e g r e s s i o n e q u a t i o n w a s d e v e l o p e d to d e s c r i b e th is r e l a t i o n s h i p a n d to c o n v e r t v o l t a g e r e s p o n s e to t h e resu l t i ng g a s v o l u m e ( P e l l a n d S c h o f i e l d , 1 9 9 3 ) . In terpre ta t ion o f g a s p r o d u c t i o n resu l t s r e q u i r e s a c c o m m o d a t i o n f o r h igh l y s o l u b l e g a s e s s u c h a s C 0 2 ( P e l l a n d S c h o f i e l d , 1995 ) . G a s c o m p o s i t i o n is a l s o impor tan t b e c a u s e o f d i f fer ing so lub i l i t i es . V o l u m e c h a n g e s re la ted to c a l i b r a t i o n c o r r e c t i o n s i nvo l v i ng m e t h a n e p r o d u c t i o n w e r e a s s u m e d to b e ins ign i f i can t in th is e x p e r i m e n t ( S c h o f i e l d a n d P e l l , 1 9 9 5 ) . A s f e r m e n t a t i o n p r o c e e d s , a n i n c r e a s e in p r e s s u r e w i th in t he f l a s k resu l t s in s o m e o f t he g a s to d i s s o l v e a n d r e m a i n in s o l u t i o n . O n l y i n s o l u b l e g a s e s a r e a v a i l a b l e to i n d u c e a p r e s s u r e c h a n g e . T h e f o l l o w i n g e q u a t i o n is H e n r y ' s l a w u s e d to d e s c r i b e t he r e l a t i o n s h i p b e t w e e n the m o l a r v o l u m e o f g a s d i s s o l v e d into a m e d i u m in r e s p o n s e to it 's par t ia l p r e s s u r e w i th in a f i xed v o l u m e c o n t a i n e r ( S c h o f i e l d a n d P e l l , 1 9 9 5 ) . vol. C02 (aqueous) / vol. C02 (gas) = 0.0246 R T (VaA/g), ( E q u a t i o n 1) w h e r e R is t h e u n i v e r s a l g a s c o n s t a n t ( 0 . 0 8 2 L * a t m / m o l * 0 K ) , T is t h e t e m p e r a t u r e in d e g r e e s K e l v i n , Va is t h e l iqu id v o l u m e in t h e f l a s k a n d Vg is t h e a v a i l a b l e g a s v o l u m e w i th in t h e f l ask . C a l i b r a t i o n o f t h e g a s p r o d u c t i o n s y s t e m w a s c o n d u c t e d u n d e r t he s a m e c o n d i t i o n s a s d e s c r i b e d in C h a p t e r 4 , s e c t i o n 4 . 3 . 6 . T h e s y s t e m , s o l u t i o n s a n d 1 7 6 e q u i p m e n t w e r e p r e w a r m e d to 3 9 ° C ± 1 ° C a n d 15 m L o f bu f fe r w a s a d d e d to 12 E r l e n m e y e r f l a s k s (50 m L ) . T h e f l a s k s w e r e r e p e a t e d l y i n jec ted w i th e i t he r 5 , 10 , 15 o r 2 0 m L o f C 0 2 t h r o u g h t h e port a n d a l l o w e d to equ i l i b ra te fo r 10 m i n . T h e d a t a in T a b l e 6 . 3 i l l us t ra tes t h e e f fec t o f p r e s s u r e o n t he so lub i l i t y o f C 0 2 . S e n s o r r e s p o n s e w a s o b t a i n e d b y s u b t r a c t i n g t he d i f f e r e n c e b e t w e e n t he v o l t a g e r e c o r d e d at b a s e l i n e f r o m t h e v o l t a g e r e c o r d e d at 10 m i n . A ca l i b ra t i on c o r r e c t i o n f a c t o r w a s o b t a i n e d for C 0 2 so lub i l i t y in th is s y s t e m u s i n g t h e a b o v e e q u a t i o n ( E q u a t i o n 1) fo r C 0 2 at 3 9 ° C : [COJ aq/[C02]gas = 0.0246 R T *15 mL/41.5 mL = 0.227 T h e g a s p r o d u c t i o n d a t a c o l l e c t e d fo r t he p r e s e n t s t u d y w a s c a l c u l a t e d u s i n g t he r e g r e s s i o n b e l o w ( E q u a t i o n 2 ) (r 2 = 0 . 9 9 4 ) w h i c h d e s c r i b e s t he re l a t i onsh ip b e t w e e n g a s p r o d u c t i o n a n d s u b s e q u e n t v o l t a g e r e s p o n s e : Y = 64.60 ±3.58 (mL /V)X-1.27±0.47 mL, ( E q u a t i o n 2 ) w h e r e Y r e p r e s e n t s t h e g a s v o l u m e a n d X is t h e v o l t a g e r e s p o n s e p r o d u c e d by the s e n s o r s . T a b l e 6 . 3 . T h e e f fec t o f C 0 2 so lub i l i t y o f g a s o n it 's abi l i ty to i n d u c e a p r e s s u r e co2 In jec ted A m o u n t o f C 0 2 d i s s o l v e d in buf fe r C 0 2 a v a i l a b l e to i n d u c e p r e s s u r e c h a n g e V o l t a g e R e s p o n s e (V) 5 m L 1.14 m L 3 . 8 6 m L 0 . 0 7 4 7 10 m L 2 . 2 7 m L 7 . 7 3 m L 0 . 1 4 5 0 15 m L 3 . 4 0 m L 1 1 . 6 0 m L 0 . 2 0 3 9 2 0 m L 4 . 5 4 m L 15 . 4 6 m L 0 . 2 5 3 4 1 7 7 6.5.1 REFERENCES P e l l , A . N . a n d P . S c h o f i e l d . 1 9 9 3 . C o m p u t e r i z e d mon i t o r i ng o f g a s p r o d u c t i o n to m e a s u r e f o r a g e d i g e s t i o n in vitro. J . D a i r y S c i . , 7 6 : 1 0 6 3 - 1 0 7 3 . S c h o f i e l d , P . a n d A . N . P e l l . 1 9 9 5 . V a l i d i t y o f u s i n g a c c u m u l a t e d g a s p r e s s u r e r e a d i n g s to m e a s u r e f o r a g e d i g e s t i o n in vitro: a c o m p a r i s o n i nvo l v i ng t h ree f o r a g e s . J . D a i r y S c i . , 7 8 : 2 2 3 0 - 2 2 3 8 . 1 7 8 < LL >- O _l CO _l a: < o o X CO LU 1-< o X O o l-o co CO Od CO co" LU > O AMPL o AMPL o < 11 AMPL h- CO o _i z> < Q o LU O 0£ LL. Q. 3 CO o o < 1-CD o < O 3 o UJ Q LL Q O o o a. z O CO o LU GA 1-GA EC a _j CO UJ LU X Q i - CO 5 Z O LU O a Q. _l z 0. < < < o r2 V • • • « • • • • o o o o CN CD O) CD + X °° E ° d II II • • •• % o d o o co o CD o d o d CM o d (6/-|iu) uouonpojd se6 papjooa^j 1 7 9 APPENDIX 6.7. SAS PROGRAM USED TO DETERMINE GAS PRODUCTION PARAMETERS FOR CANOLA MEAL. o p t i o n s n o c e n t r e l i n e s i z e = 7 5 ; f i l e n a m e g a s p r o d u c t i o n ' f i l e n a m e ' ; d a t a ; inf i le g a s p r o d u c t i o n ; input t i m e f e e d r e p l f e e d r e p 2 f e e d r e p 3 ; tit le ' t es t o f f i t t ing s i m p l e e x p o n e n t i a l w i th l a g ' ; p r o c nl in m e t h o d = d u d ; p a r m s a=1 b=0.1 l ag=0 ; m o d e l y = a*(1 - e x p ( - b * ( t - l a g ) ) ) ; ou tpu t ou t=b p r e d i c t e d = y h a t r e s i d u a l = y r e s ; p r o c p r i n t ; p r o c p l o t ; p lot y*t='o' yha t * t="p" / o v e r l a y ; run ; 1 8 0 q '•4-» o T3 2 Q . CO CD CD H— o CD -f—< CO c o CO c o -4—' c o o "co E 1_ CD •o >. xz ~o c CO . CO c CD Q E o > ^ D N - a c o I * * i CO > CO 5 CO T3 _,-B CO CO T -2 CD — > n o " ca c O CO O CD - I CO o co CD 0) CO CD CD T t : : CD O _CD £ _Q CO i— a? Q CO CD > w o ^ |£« CT 3 " 2 r O O C Q CO re _ i c CO CD a co 2 0C CD CO CO c _ra ^ > ^ CD Ui (0 CD 2 ^ Q. e. CD 3 CO 5 0 s CO c 2 i £ •o CD CD Q LL. id CD co C N CM 00 CD T i - L O L O C N T — CO C D L O a> L O L O ed od CD CD cd L O 0 O 0 TI- C D 0 0 0 0 O 0 C N co 00 L O C D L O 0 L O O 0 co L O T i - L O rl- CO C N 0 T l - T l - L O T — i< d ed d T f T — C D T f d L O d O co TI- L O CD x— C N C D 00 00 CO CO C N C N C N T — C N C N T — CM T T CO T l - CO CO T f CD CO L O T t -C N C N C N C N C N CM C N C N C N C N C N O O 0 O O O O O O 0 O 0 d d d d d d d d d d d d L O 0 0 0 CD 0 C D L O O CO L O N - 0) C D L O 00 CM C D — 00 CO C N C N •*~ C N C N T — CM T f C N CM O d d d d d d d d d d d N - L O C N CM T l - T l - CO T t O 0 0 0 O 0 0 0 0 0 0 O O 0 0 0 O 0 0 0 0 0 0 O d d d d d d d d d d d d co L O CD co TI- 0 0 CO 00 O ) C D 00 T l - C D CO r-~-0 O T - 0 0 0 T — 0 C D O 0 d d d d d d d d d d d d T — 0 O 0 0 0 O 0 O 0 0 0 O 0 d 0 d d 0 d 0 0 d 0 d L O L O L O 0 T — L O 0 L O 0 L O 0 q 0 0 O O 0 O 0 0 d d d 0 d d d 0 d d d 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 CM C N co T t L O C D 00 CD T — v — 181 Q (0 O = 5 •- 2 ° « « o> o J 3 £ 3 ^ | oo o _i Q O) (0 —I c (0 0) Q CO , Q re (A re c JH o CO (0 0) 4-1 2 ^ 3 CO O) c '3 co o o CO o o co o o ZOO d d d o CD CO o LO LO CD I S -CSI o 0 0 Is-CM d CD Is-o d o o T - CD CO LO o o O CD o d LO" I S - CD o d CTJ CD CM CD o d C D C O CM ^ CD CD CD O O O O CD <fr Is- CD CD LO O O CM LO O T - LO o o o o d d o CD LO d C O CD CM O O d d CM CD CM LO CM d d o o d C D o LO co o d CM CO cd IS-' Is- CD 0 0 CD CD o CM CM CD LO LO 0 0 CD CO d CM CD LO d 0 0 CM I S -CM CM O O d d CM CO CM CM O O d d T— Is- CD oo CD CD CD LO o T— CM CM CM CO o d d d d d d LO CM d d CD CD LO LO d d CO LO CM CM LO CO CM o o o o co d CO CD r- 0 0 CM oo d oo CD CD CD o o LO LO LO CD •St-LO oo LO LO d d d LO 0 0 CM 0 0 CD CM CO CM CM CM Is- t^- «fr CM CM CM CM CM o o O O o d d d d d d L O CD CO CD CM d d o o CM CM d o o CM LO CM CM CO CM CO LO CM CM CM O O CD CO Is- LO CD co CO Is- CD "< — *~ O O o o o o o T— d d d d d d d d d d d d o o o o d o o o o o o o o CO o d o o Is-o o d CM CM CM 0 0 LO co LO CD CD CD o o o o o o o o o o o o o o o o o o o o o o o o d d d d d d d d d d d d CM Is- O 0 0 O CO O T - O o d d LO o o o LO o LO o o o LO o LO o o o LO o LO o o d d d o d d d o d d d o d o o o o o o o o o o o o o o o o o • ^ - « - t - C M C M C M C M C M C M C M C M C 0 C 0 C 0 C 0 C 0 C 0 o o o o o o o o o o o o o o o o o CO • ^ - l 0 C D I s - 0 0 C D O T - C N C 0 " « t l 0 C D I s - 0 0 C D T - T - - < - T - T - - « - C M C M C M C N C M C M C M C M C M C M O CO 182 n ti CO T t CO 5 c si CO o - J T t T t LO CN Q CO D) CO —I c CO CD Q CO CO B , O CO CD CO (0 c JO CD (A (0 CD IQ. 2^  3 CO o C o CO c "co g .S |T3 CD ,?0 T t CN O C O C N C D O O O L O o I S -co C N d o C N T t C N O d o o C N CO o o I s -o o o o d L O o o co o o C O oo CO C N oo CD T t o co I S - T t L O L O co oo CD L O CD CD od T t od cn od CD L O CT) od L O CD CD o o O O co C D o o I S - o C N CD r t L O L O L O L O L O o L O L O CD T t oo T t CT) L O L O T t T t T i - T t T t L O L O C N L O L O T t C D C D T t co C N d ed T t CD L O d cd C N od C N L O T t CN cd C D C D r— L O I S - C N C N CT) cn O cn cn C N CN I S -T — C N C N C N CN ' T — C N T — C N C N CN T t co Tt- T t T t Tf- T t I S - T t o co L O T t cn C N co CN CN CN CN C N o cn O C N co L O x— o O o O O O O O o o O O o O O O o d d d d d d d d d d d d d d d d I S - T t C N 00 I S - CD C N T — oo CD co CT) CN T t 00 C N I S - TI- C N oo C N I S - T t T t CD CD CD o I S -C N L O C O L O co C N C N T t C N C N C N O C N C N CN d d d d d d d d d d d d d d d d C D L O CO C D C D C N o L O L O CO L O T t CD T t CO o o o o o O T — O o o o o O o o o o o o o O o O o o o o o O o o d d d d d d d d d d d d d d d d co TI- T t oo T t L O CO o CD o C D co I S - C O o C D T i - CD L O TI- co I S - I S - oo cn o o 00 CT) o o cs o o o o T — o o o o X — o o T — d d d d d d d d d d d d d d d d T — T — T — o o o o o o o o o o o o o o o o o d o d d o d o o d o d d o d o L O L O L O L O o L O o T — L O o T — L O o T — L O o o o o o o o o o o o o o d d d o d d d o d d d o d d d o o o o o o o o o o o o o o o o L O L O L O L O L O L O L O L O L O L O L O L O L O L O L O L O t — C N co TT L O CD I S - oo cn o CN co T t L O CD TT T t T t T l - T t Tt- TJ- T i " T t L O L O L O L O L O L O L O 1 8 3 Q CO > w o ^ * - JO +3 Q _ 00 o - J . 3 T - i_ c O Q-3 Q (O CO (0 - J c (0 CO to Q CO CD (Q CD 10 (0 c CD CO (0 CD I d . 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