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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 MILD H Y D R O T H E R M A L T R E A T M E N T O N NUTRITIVE V A L U E OF B A R L E Y GRAIN AND C A N O L A MEAL FOR CATTLE  by  C A T H E R I N E S U Z A N N E GILL  B . S c . (Agr.), T h e U n i v e r s i t y of British C o l u m b i a ,  A THESIS SUBMITTED  1994  IN P A R T I A L F U L F I L L M E N T  THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  in  THE FACULTY OF GRADUATE  D e p a r t m e n t of A n i m a l  STUDIES  Science  W e a c c e p t this thesis 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  THE UNIVERSITY  O F BRITISH  February,  COLUMBIA  1997  © Catherine S u z a n n e Gill,  1997  OF  THE  DAIRY  In  presenting this  degree at the  thesis  in  partial  University of  fulfilment  of  of  department  this thesis for or  by  his  or  requirements  British Columbia, I agree that the  freely available for reference and study. I further copying  the  representatives.  an advanced  Library shall make it  agree that permission for extensive  scholarly purposes may be her  for  It  is  granted  by the  understood  that  head of copying  my 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  DE-6 (2788)  J^A^O^'&k/  / ^  ?  ABSTRACT High ingredient costs and a competitive market h a v e promoted support for research  by the feed  industry t o i m p r o v e t h e utilization  of protein  a n d energy  feedstuffs s u c h a s barley a n d c a n o l a m e a l . T h e objective o f this r e s e a r c h w a s to determine  t h e feasibility  o f u s i n g e n z y m e a n d mild  hydrothermal  treatment  to  i m p r o v e the nutritional v a l u e o f barley grain a n d c a n o l a m e a l for u s e b y dairy cattle. 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 t o 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 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 , w a s e v a l u a t e d u s i n g 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 ( I V D M D ) , in vitro t r u e d i g e s t i b i l i t y ( I V T D ) , a c i d d e t e r g e n t f i b e r ( A D F ) a n d neutral detergent fiber ( N D F ) a n a l y s e s . A n u m b e r of interactions ( P < 0.05) w e r e o b s e r v e d for the digestibility p a r a m e t e r s . R e s u l t s o f fiber a n a l y s e s w e r e inconclusive. Overall, the addition of x y l a n a s e w a s found to b edetrimental and the mild h y d r o t h e r m a l t r e a t m e n t did not h a v e a n effect o n e n z y m e activity. T h e addition of p r o t e a s e a n d / o r p - g l u c a n a s e t o b a r l e y s u g g e s t potential a p p l i c a t i o n that m a y b e f u r t h e r 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 meal w a s subjected to protease,  x y l a n a s e e n z y m e s a n d mild hydrothermal treatment. using  I V D M D , IVTD, A D F a n d N D F . Significant  Treatments were  interactions  evaluated  ( P < 0.05)  were  o b s e r v e d f o r I V D M D , I V T D , ADF a n d NDF. T h e a d d i t i o n o f m o i s t u r e s i g n i f i c a n 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 elicited a n effect ( P < 0.05) o n t h e NDF c o n t e n t . F r o m t h i s 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 t o t h o s e i n 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 mild h e a t t r e a t m e n t w a s effective in i m p r o v i n g digestibility. H o w e v e r , t h e addition o f p r o t e a s e t o c a n o l a m e a l at a n intermediate  l e v e 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 i g e s t i b i l i t y s i g n i f i c a n t l y ( P < 0 . 0 5 ) . F i b e r a n a l y s e s resulted  in a  Experiment  3  large  n u m b e r of interactions which  evaluated  the  effects  of  the  produced variable responses.  treatments  on  the  fermentation  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 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 . R a t e o f gas  production  length  of  the  w a s significantly (P < 0.05) lag  phase was  significantly  affected affected  by protease, w h e r e a s , by  the  interaction  p r o t e a s e a n d x y l a n a s e ( P < 0 . 0 5 ) . In g e n e r a l , t h e t r e a t m e n t s decreased  lag  time.  Volatile  fatty  acid  (VFA) analyses  the  between  i n c r e a s e d rate a n d  revealed  a  significant  interaction (P < 0.05) b e t w e e n protease a n d x y l a n a s e . T h e r e w a s a positive linear/ r e l a t i o n s h i p ( P < 0 . 0 5 ) b e t w e e n t h e 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 ratio o f a c e t a t e t o 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 f e c t ( P < 0 . 0 5 ) o n t h e ratio o f b u t y r a t e to p r o p i o n a t e . X y l a n a s e a l s o significantly ( P < 0 . 0 5 ) production  relative  to  propionate.  Results  from  both  acetate and  experiment  3  butyrate  indicated  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  n u t r i t i o n a l v a l u e o f c a n o l a m e a l . F r o m t h e w o r k c o n d u c t e d in t h i s t h e s i s , it m a y b e concluded,  in  detrimental  to digestibility a n d that mild h y d r o t h e r m a l  enzyme  the  activities.  case  Many  of  both  enzyme  barley  and  treatments  canola  m e a l , that treatment  increased the  xylanase  did not  proportion  s u g g e s t i n g a shift in f e e d c o m p o s i t i o n . T h e r e s u l t s o f t h i s s t u d y s u g g e s t  was  improve of  fiber,  potential  applications of the treatment of barley with p r o t e a s e or a c o m b i n a t i o n of 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 of c a n o l a m e a l with p r o t e a s e b e t w e e n  the  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  nutritive  value  for  dairy  cattle.  The  in  vitro  work  presented  in  this  d e m o n s t r a t e s t h e p o t e n t i a l i m p r o v e m e n t s w i t h e n z y m e t e c h n o l o g y in t h e quality of 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 cattle.  iv  thesis feeding  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  3  of Enzymes  4  1.1.2 Mode of Action 1.1.3 Monogastric  5  Success  1.1.3.1 A n t i - N u t r i t i o n a 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  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 1.1.4.2 Fibrolytic 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 1.1.4.3.1) F o r a g e s  13  and Enzyme  Applications  1.2 IN VITRO T E C H N I Q U E S 1.2.1 Apparent  13 13  1.1.4.3.ii) C o n c e n t r a t e s 1.1.5 Feed Processing  9 10  14 16  Digestibility:  Tilley and Terry In Vitro Technique  18  1.2.2 True Digestibility  19  1.2.3 Enzymatic  19  1.2.4 Rumen  Method  20  Fluid Inoculum  1.2.4.1 A n i m a l D i e t  20  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 Inoculum P r e p a r a t i o n 1.2.5 Advantages 1.2.6 Disadvantages  21  of In Vitro Techniques of In Vitro Techniques  1.2.7 Kinetic Descriptions  of Feed Degradation  1.2.7.1 In Situ T e c h n i q u e 1.2.8 The Gas Production  22 23 23 23 25  Technique  1.2.8.1 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 v  27  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  28  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 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  28 30  1 . 2 . 8 . 5 U s e 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 in t h e E v a l u a t i o n o f Concentrates  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  32  1.3 O B J E C T I V E S O F T H E T H E S I S  33  1.4 R E F E R E N C E S  35  2.0 THE EFFECT OF ENZYME AND MILD HYDROTHERMAL TREATMENT ON THE IN VITRO DIGESTIBILITY OF B A R L E Y GRAIN  46  2.1 A B S T R A C T  46  2.2 I N T R O D U C T I O N  48  2.3 M A T E R I A L S A N D M E T H O D S 2.3.1 Feed  51 51  Treatments  2 . 3 . 2 Processing  52  Samples  2.3.3 Chemical Analyses  53  2 . 3 . 4 Animals and Feeding  54  2.3.5 Rumen Fluid Inoculum 2 . 3 . 6 In Vitro Procedure  54 55  2.3.7 Statistical  55  Analysis  2.4 R E S U L T S A N D D I S C U S S I O N :  56  2.4.7 IVDMD 2 . 4 . 2 IVTD 2.3.3 ADF  56 60 63  2.4.4 NDF  64  2.5 C O N C L U S I O N  66  2.6 R E F E R E N C E S  68  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  92  3.2 I N T R O D U C T I O N  94  3.3 M A T E R I A L S A N D M E T H O D S 3 . 3 . 7 Feed  95 95  Treatments  3 . 3 . 2 Processing 3 . 3 . 3 Chemical  of Feed  95  Treatments  96  Analyses  3.3.4 Animals and Feeding  96  3.3.5 Rumen  96  Fluid Inoculum  3 . 3 . 6 In vitro Procedure  96  3 . 3 . 7 Statistical  96  Analysis  3.4 R E S U L T S A N D D I S C U S S I O N  97  3 . 4 . 7 IVDMD  97  3.4.2 IVTD  100  3.4.3 ADF  102  3.4.4 NDF  104 vi  3.5  C O N C L U S I O N S  105  3.6  R E F E R E N C E S  107  4.0 THE EFFECT OF ENZYME AND MILD HYDROTHERMAL TREATMENT ON THE IN VITRO G A S PRODUCTION IN CANOLA MEAL  124  4.1  124  A B S T R A C T  4.2  INTRODUCTION  4.3  MATERIALS A N D  4.3.1 Feed  126 M E T H O D S  128  128  Treatments  4.3.2 Processing of Feed 4.3.3 Chemical Analyses 4.3.4 Animals 4.3.5 Rumen  128 129  Treatments  129  and Feeding  129  Fluid Collection  4.3.6 Gas Production  129  System  130  4.3.7 Gas Measurements 4.3.8 Procedure for the Gas Production 4.3.9 Data Processing 4.3.10 Kinetic Analysis of Gas Data 4.3.11 VFA Sampling 4.3.12 Statistical 4.4  R E S U L T S  A N D  131 132 132  Technique  133  and Analyses:  134  Analysis DISCUSSION  134  4.4.1 Validation and Comparison of the Gas Production 4.4.2 Rate of In Vitro Gas Production 4.4.3 Lag Period  System  135 138 141 143  4.4.4  Volatile Fatty Acids  4.4.5  VFA Ratios: acetate to propionate;  butyrate to propionate  145  4.5  C O N C L U S I O N S  147  4.6  R E F E R E N C E S  149  5.0 G E N E R A L CONCLUSIONS .'  165  5.1  B A R L E Y  5.2  C A N O L A  5.3  S U M M A R Y  167  5.4  R E F E R E N C E S  169  6.0 APPENDICES  170  APPENDIX  165 M E A L  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  PROTEASE AND APPENDIX  166  1  OF THE ENZYMES:P-GLUCANASE,  X Y L A N A S E S U P P L I E D B YFFI (WILTSHIRE,  U . K.)  171  6.2 COMPOSITION O F T H E CONCENTRATE RATION F E D T O DONOR  COWS  172  APPENDIX  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  173  APPENDIX  6.4. S A S P R O G R A M U S E D TO EVALUATE CANOLA MEAL DATA  175  APPENDIX  6.5 CALIBRATION O F T H E G A S M E A S U R E M E N TS Y S T E M (SCHOFIELD A N D  PELL, 1995)  176  vii  A P P E N D I X 6 . 6 . R E C O R D E D G A S PRODUCTION V S . STOICHIOMETRICALLY CALCULATED G A S PRODUCTION F R O M V F A COMPOSITION A T 6 H O F A RANDOM SELECTION O F CANOLA MEAL S A M P L E S A P P E N D I X 6.7. S A S P R O G R A M U S E D T O DETERMINE G A S PRODUCTION PARAMETERS F O R C A N O L A M E A L  180  A P P E N D I X 6 . 8 . R A W DATA F O R T H E ESTIMATED G A S PRODUCTION PARAMETERS F R O M CANOLA M E A L : RATE O F G A S PRODUCTION, LAG PERIOD A N D CUMULATIVE 1 8 H G A S PRODUCTION  181  viii  179  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  TRUE  DIGESTIBILITY O F B A R L E Y G R A I N T A B L E 2.2.  73  T H E E F F E C T O F T H E DURATION O F HEAT T R E A T M E N T A N D T H E APPLICATION  OF MOISTURE A N D XYLANASE O N T H E 4 8 O F B A R L E Y G R A I N (DM T A B L E 2.3. THE 48  H IN VITRO  75  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 APPLICATION ON F R A C T I O N O F B A R L E Y G R A I N (DM  F R A C T I O N O F B A R L E Y G R A I N (DM  77  BASIS)  78  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 APPLICATION O N T H E  NDF  BASIS)  79  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 APPLICATION O N T H E  NDF  BASIS)  80  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 APPLICATION O N T H E  C O N T E N T O F B A R L E Y G R A I N (DM T A B L E 3.1.  BASIS)  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 APPLICATION O F P - G L U C A N A S E  F R A C T I O N O F B A R L E Y G R A I N (DM T A B L E 2.9.  H IN W T R O T R U E D I G E S T I B I L I T Y 76  C O N T E N T O F B A R L E Y G R A I N (DM T A B L E 2.8.  DIGESTIBILITY  BASIS)  A N D X Y L A N A S E O N T H E ADF T A B L E 2.7.  TRUE  BASIS)  T H EE F F E C T O F MOISTURE LEVEL O N T H E 4 8  T H E ADF T A B L E 2.6.  74  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  O F B A R L E Y G R A I N (DM T A B L E 2.5.  DRY MATTER DISAPPEARANCE  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 APPLICATION O N  O F B A R L E Y G R A I N (DM T A B L E 2.4.  H IN VITRO  BASIS)  NDF  BASIS)  81  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  TRUE  DIGESTIBILITY  OF CANOLA MEAL T A B L E 3.2.  MEAL O N T H E4 8 M E A L (DM T A B L E 3.3.  109  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 H R U M E N IN VITRO  110  T H E E F F E C T O F MOISTURE A D D E D T O C A N O L A MEAL AT T H E TIME O F  PROCESSING ON THE 48 M E A L (DM T A B L E 3.4.  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  BASIS)  111  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 A B L E 3.5.  DRY MATTER DISAPPEARANCE O F CANOLA  BASIS)  T R U E D I G E S T I B I L I T Y O F C A N O L A M E A L (DM  BASIS). ... 1 1 2  T H E E F F E C T O F MOISTURE A D D E D T O C A N O L A MEAL A T T H E TIME 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  BASIS) T A B L E 3.6.  113 T H E E F F E C T O F T H E DURATION O F HEAT T R E A T M E N TA N D T H E APPLICATION  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 M E A L (DM T A B L E 3.7.  FRACTION O F CANOLA  BASIS)  114  T H E E F F E C T O F MOISTURE, P R O T E A S E A N D X Y L A N A S E APPLICATION 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  ix  BASIS)  115  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 TIME O F  P R O C E S S I N G O N T H E NDF T A B L E 4.1.  F R A C T I O N IN C A N O L A M E A L (DM  BASIS)  117  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  ANALYSES OF CANOLA  MEAL T A B L E 4.2.  152 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  RATE OF GAS  PRODUCTION OF CANOLA MEAL T A B L E 4.3.  T H E L A G P H A S E IN VITRO T A B L E 4.4. VITRO T A B L E 4.5.  153  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 APPLICATION O N T H E LENGTH OF OF CANOLA MEAL  154  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 APPLICATION O N T H E VFA  6 H / N  PRODUCTION OF CANOLA MEAL  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  155 RATIO O F  A C E T A T E TO PROPIONATE AND BUTYRATE TO PROPIONATE OF CANOLA MEAL 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  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)  MEAL  156  RATIO O F OF CANOLA 157  X  LIST OF FIGURES F I G U R E 2.1.  LABORATORY SCALE HYDROTHERMAL COOKER  82  F I G U R E 2.2.  T H E E F F E C T O F T H E ADDITION O F MOISTURE, X Y L A N A S E (X) (% V O L / WT)  A N D DURATION O F H E A T T R E A T M E N T (55°C) O N T H E IN VITRO  DRY 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 GRAIN F I G U R E 2.3.  83  T H E E F F E C T O F P - 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 )  APPLICATION (% V O L / WT) O N T H E IN VITRO DRY 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 GRAIN F I G U R E 2.4.  84  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 A N D IN VITRO  T R U E DIGESTIBILITY O F B A R L E Y GRAIN  (Y = 87.75 + 0.07*MOISTURE S . E . = 0.018)  LEVEL -  0.0014*MOISTURE L E V E L : N = 4, R = 0.998, 2  2  F I G U R E 2.5.  85  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 ( P ) A N D X Y L A N A S E (X)  APPLICATION (% V O L / W T ) O N T H E IN VITRO T R U E DIGESTIBILITY O F B A R L E Y GRAIN F I G U R E 2.6.  86 T H E E F F E C T OF P-GLUCANASE, PROTEASE ( P ) AND XYLANASE  APPLICATION (% V O L / WT) O N T H E C O N T E N T O F ACID D E T E R G E N T FIBER IN B A R L E Y GRAIN F I G U R E 2.7.  87 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 ( P ) APPLICATION (% V O L /  WT) A N D T H E DURATION O F H E A T T R E A T M E N T S  (55°C)  O N T H E C O N T E N T O F ACID  D E T E R G E N T FIBER IN B A R L E Y GRAIN F I G U R E 2.8.  88  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 A N D P R O T E A S E (% V O L /  WT) O N T H E C O N T E N T O F N E U T R A L D E T E R G E N T FIBER IN B A R L E Y GRAIN F I G U R E 2.9.  89  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 A N D X Y L A N A S E (% V O L /  W T ) O N T H E C O N T E N T O F N E U T R A L D E T E R G E N T FIBER IN B A R L E Y GRAIN F I G U R E 2.10.  90  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 /  WT) O N T H E C O N T E N T O F N E U T R A L D E T E R G E N T FIBER IN B A R L E Y GRAIN F I G U R E 3.1  91  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 / WT)  O N T H E 48 H IN VITRO DRY 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 F I G U R E 3.2.  118  Q U A D R A T I C RELATIONSHIP B E T W E E N MOISTURE L E V E L A N D T H E 48 H IN  (Y = 78.62 + 0.165* 4, R = 0.998, S . E . = 0.105). ...119  VITRO DRY 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 MEAL MOISTURE L E V E L F I G U R E 3.3.  0.003*  MOISTURE L E V E L , N = 2  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 (% V O L / WT)  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 F I G U R E 3.4.  120  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) A N D  (Y = 87.25 + 0.073* 4, R = 0.991, S . E . 0.0355)  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 MOISTURE L E V E L F I G U R E 3.5.  0.002*  MOISTURE L E V E L : N = 2  2  T H E E F F E C T O F T H E APPLICATION O F P R O T E A S E ( P ) , X Y L A N A S E (X)  121  (%  V O L / WT) A N D DURATION 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 ACID D E T E R G E N T FIBER IN C A N O L A M E A L F I G U R E 3.6.  122  T H E E F F E C T O F T H E APPLICATION O F MOISTURE, P R O T E A S E ( P ) A N D  X Y L A N A S E (X) (% V O L / WT) O N T H E C O N T E N T O F ACID D E T E R G E N T FIBER IN CANOLA MEAL F I G U R E 4.1.  123  T H E C O M P U T E R I Z E D G A S PRODUCTION 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)  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 TUBING A P P A R A T U S F O R VFA SAMPLING F I G U R E 4.3.  159  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  INCUBATION P E R I O D WITH R U M E N FLUID. F I G U R E 4.4. VITRO  160  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 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  F I G U R E 4.5.  : 161  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 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 FLUID F I G U R E 4.6.  162  T H E E F F E C T O F T H E A D D I T I O N 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 )  ON T H E PRODUCTION OF  VFAS  IN C A N O L A M E A L F O L L O W I N G  A6H/N  VITRO  INCUBATION WITH R U M E N FLUID F I G U R E 4.7.  163  LINEAR RELATIONSHIP B E T W E E N LEVEL O F P R O T E A S E APPLICATION AND  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 VITRO INCUBATION WITH R U M E N FLUID  (Y = 1 7 . 5 5 * P R O T E A S E L E V E L  4, R = 0.839, S . E . = 0.17)  +  2.851:  N =  164  2  xii  ACKNOWLEDGMENTS I w o u l d like 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 . f o 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 this project. I w o u l d a l s o like 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 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 . f o 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 h e s u c c e s s o f this project a n d this 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 . ) is gratefully a c k n o w l e d g e d f o r i n i t i a t i n g t h i s u n i q u e p r o j e c t a n d f o r h e r i n p u t into t h e 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 t o D r . J . W . H a l l o f t h e 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 . ) f o 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 h e s t a t i s t i c a l a n a l y s e s a n d interpretation o f t h e d a t a . I w o u l d a l s o like to t h a n k Dr. J o h n B a a h f o r h i s s u p p o r t . T h a n k y o u t o D r . L. J . F i s h e r o f t h e 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 Station ( A g a s s i z , B . C . ) for his constructive 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 h e writing o f t h e t h e s i s . I w o u l d a l s o like 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 , D r . R . M . T a i t o f t h e 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 D r . J . V a n d e r s t o e p o f t h e 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 . f o r t h e i r helpful c o m m e n t s o n the thesis. The  expertise  of  Gilles Galzi  is  gratefully  acknowledged  in  putting  t o g e t h e r t h e 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 t o 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 initial s t a g e s o f t h e e x p e r i m e n t a n d t o S a r a M u i r f o r h e r a s s i s t a n c e in s e t t i n g u p t h e g a s e x p e r i m e n t a n d t o t h e 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 s t a f f at U . B . C . is a l s o t h a n k e d f o r t h e i r h e l p in m a i n t a i n i n g t h e a n i m a l s f o r this e x p e r i m e n t . T h a n k y o u to all 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 .  who  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 for this  research was  provided  by  B.C. Science  C o u n c i l - T e c h n o l o g y B . C . with P r o F o r m F e e d s a s the Industrial partner. C S . Gill w a s s u p p o r t e d by a n N S E R C Industrial S c h o l a r s h i p with P r o F o r m F e e d s a l s o a s t h e i n d u s t r i a l partner. T h e e n z y m e s u s e d in this project w e r e s u p p l i e d by Finn F e e d s  International (Wiltshire, 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 like to t h a n k m y h u s b a n d R i c k for his l o v e , p a t i e n c e a n d s u p p o r t throughout this work. T h e c o m p l e t i o n of this t h e s i s w a s p o s s i b l e b e c a u s e of the c o n t i n u o u s e n c o u r a g e m e n t from N i n a , m y family my husband Rick.  xiii  and  CHAPTER 1 1.0 INTRODUCTION  F e e d c o m p a n i e s in B r i t i s h 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 t h dairy p r o d u c e r s with affordable, high quality feedstuffs. T w o major  supplying  developments  that h a v e affected the f e e d m a n u f a c t u r i n g , dairy a n d other livestock industries  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 the recent l o s s of rail s u b s i d i e s . T h e m a j o r i t y o f c o n c e n t r a t e s f e d t o 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 i g n i f i c a n t c o s t f o r u s e b y B . C . farmers.  Feed  represents  approximately  60%  of  dairy  farm  costs,  with  grain  a c c o u n t i n g f o r 3 0 % o f t h i s e x p e n s e in t h e a r e a o f t h e L o w e r F r a s e r V a l l e y ( B a r k e r and  Cheatley,  1992).  Therefore,  feed  companies  are  constantly  challenged  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 efficiently u s e d by dairy cattle. In a r e a s l i k e t h e intensive played  L o w e r F r a s e r V a l l e y region of B . C . a n d  monogastric animal  an  increasing  role  in  production reducing  is p r e v a l e n t , the  excretion  Europe,  exogenous enzymes of  polluting  nutrients  where have and  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 t h a t 8 0 % o f t h e w o r l d ' s poultry diets a r e n o w e n z y m e treated ( T r e a c h e r a n d Hunt, 1996). T h e f e e d i n d u s t r y h a s primarily f o c u s e d o n t h e u s e of 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 n u t r i t i v e 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 p o u l t r y d i e t s . T h e C a n a d i a n f e e d i n d u s t r y h a s a p p l i e d e n z y m e s to poultry d i e t s s i n c e t h e late 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 z a t i o n a s a r e s u 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  1  have  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 h e m a r k e t p l a c e . R e s e a r c h i n t o t h e 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 w a s briefly 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 , r e s u l t s w e r e t o o i n c o n s i s t e n t t o w a r r a n t f u r t h e r i n v e s t i g a t i o n , r e s u l t i n g in t h e l a c k o f 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 h e 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 i n t e r e s t into t h e 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 i n t o d a i r y c a t t l e d i e 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 the l e v e l s of i n c l u s i o n of t h e s e f e e d s a r e limited b e c a u s e high fiber content d e c r e a s e s their 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 of t h e s e feedstuffs with e x o g e n o u s e n z y m e s h a s the potential to  alter the  fermentation  profile  of the  c a r b o h y d r a t e fraction  and  improve  the  digestibility of t h e s e f e e d s . T h e intent of this t h e s i s project w a s to investigate the feasibility of improving the  nutritional  v a l u e of c a n o l a m e a l a n d  t e c h n o l o g y a n d mild hydrothermal treatment.  2  barley for dairy cattle  using  enzyme  1.1 EXOGENOUS ENZYMES 1.1.1 Sources of Enzymes E n z y m e s u s e d in t h e f e e d i n d u s t r y a r e p r i m a r 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  the food  and beverage  industries  (Chesson and Austin,  1996).  Enzyme  preparations a r e g e n e r a l l y not s p e c i f i c but contain a n array o f e n z y m e activities ( C a m p b e l l a n d B e d f o r d , 1992). A l t h o u g h t h e food a n d b e v e r a g e industries require g r e a t e r s p e c i f i c i t y in t h e 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 f o r t h e f e e d i n d u s t r y a r e r a r e l y r e q u i r e d t o b e s o s p e c i f i c in n a t u r e . T h e d e v e l o p m e n t o f a n enzyme  c o m p l e m e n t to a specific ingredient  requires a careful match  between  e n z y m e and substrate a s well a s consideration of storage and processing conditions in o r d e r t o o b t a i n t h e 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 o m f u n g i ( e . g . Trichoderma  a n d Aspergillus  y e a s t s ( e . g . Saccharomyces  s p p . ) , b a c t e r i a ( e . g . Bacillus  spp.) and  cerevisiae).  C o m m e r c i a l e n z y m e products o r " e n z y m e cocktails" usually contain a mixture of  specific e n z y m e  activities  (Newman,  1994).  E n z y m e "cocktails", have  been  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 a c t i v i t y (Mulder et al., 1 9 9 1 ; C h e s s o n , 1993). K n o w l e d g e of the type of e n z y m e , whether 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 " , it's s p e c i f i c i t y a n d m o d e o f a c t i o n i s 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 e c t s a n d m i n i m i z e n e g a t i v e e f f e c t s t h e e n z y m e may have o n the substrate (Amado, 1993).  3  1.1.2 Mode of Action Improvements  o b s e r v e d with e x o g e n o u s e n z y m e s , with both  monogastric  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 u t i l i z a t i o n a n d r e d u c e d v a r i a t i o n in t h e a v a i l a b i l i t y o f n u t r i e n t s 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 t h a t e n z y m e s a i d in t h e disruption of c o m p l e x plant cell w a l l s w h i c h c a u s e s intracellular nutrients, 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 h e 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 f e c t i v e  in d e g r a d i n g t h e c e l l  matrix  ( G r u p p e n , 1 9 9 6 ) . F o r e x a m p l e , the s y n e r g i s t i c effect of 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 rate of h y d r o l y s i s a n d p r o d u c e t h e d e s i r e d p r o d u c t s  when  a p p l i e d t o w h e a t a n d s o r g h u m f l o u r , 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 ( D u s t e r h o f t et al.,  1993).  The  improved  digestion  of  fiber  observed  with  some  enzyme  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 result of 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 p a r t i c l e s b y 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 e s u l t s w i t h enzyme  supplements  are  inconsistent.  o r c h a r d g r a s s h a y diet w h e r e  When  calves were  a cellulolytic e n z y m e w a s  fed  an  fibrolytic  alfalfa  sprayed onto  the  and grain  s u p p l e m e n t , the 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 lack of r e s p o n s e ( L e a t h e r w o o d al.,  1960).  On  the  other  hand,  a  multienzyme  supplementation  fed  to  et  calves  improved gain a n d feed efficiencies (Modyanov and Zel'ner, 1983). 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 t h a t s e v e r a l e n z y m e s w e r e r e q u i r e d in o r d e r t o 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 r o i l e r c h i c k s f e d a b a r l e y b a s e d d i e t . T h i s effect  was  attributed  to  the  increased  availability  of  intracellular  nutrients.  An  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 e s u l t e d in  4  the r e l e a s e of m o r e intracellular nutrients than w h e n p - g l u c a n a s e a l o n e w a s applied 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 The  successful application  research conducted  using  of e x o g e n o u s e n z y m e s m a y  monogastric  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  with  use  of  p-glucanases,  illustrated  animals. Several reviews on the  enzymes the  be  pentasonases  in a n i m a l  (xylanases)  and  by  use  of  performance  other  enzymes  ( 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 t o : d e s t r o y a n t i - n u t r i t i o n a l f a c t o r s , a s s i s t in t h e b r e a k d o w n o f b o n d s n o t hydrolyzed by the animal'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 , further e n h a n c e the e n d o g e n o u s e n z y m e activities of the a n i m a l a n d t h e r e b y i n c r e a s e the nutrient a n d e n e r g y availability of alternative feedstuffs  (Chesson,  1993; Close,  1 9 9 6 ) . It  has  b e e n reported that e n z y m e s r e d u c e the variability of 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 t h 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  efficiency a n d g r o w t h rates h a v e b e e n reported with poultry a n d s w i n e that w e r e fed enzyme  supplemented  diets  (Marquardt  et a l . , 1 9 8 7 ; R o t t e r et a l . , 1 9 8 7 ) .  c h i c k s , i n c r e a s e d rate of g a i n , efficiency of f e e d utilization, A M E a n d the  With  digestibility  of D M , fat a n d protein 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 primary g o a l s of the f e e d industry involving, current e n z y m e r e s e a r c h are mainly directed  towards  m o n o g a s t r i c s a n d a r e t o r e m o v e o r d e s t r o y t h e e f f e c t s o f a n t i - n u t r i t i o n a l f a c t o r s (ie. N S P s ) , i n c r e a s e overall digestibility  of the f e e d a n d the bioavailability of specific  5  nutrients  and  decrease  nutrient  pollution  (Newman,  1994).  For  example,  i m p r o v e m e n t s i n t h e d i g e s t i b 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 gut content viscosity, t h e r e b y i n c r e a s i n g the availability of  nutrients  and  improving  animal  performance  (Campbell  and  Bedford,  1992;  C h e s s o n , 1993).  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 h e d e m a n d t o m e e t  increasing  n u t r i t i o n a 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 r g e r a m o u n t s  of  d i f f e r e n t 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 large proportion of N S P s w h i c h a r e p o l y m e r s of s i m p l e s u g a r s a n d include cellulose, 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 not p o s e s s the e n d o g e n o u s e n z y m e s t o b r e a k d o w n N S P s . In t h e d i g e s t i v e t r a c t , t h e s e c o m p o u n d s b e c o m e soluble therefore  contributing  to a n anti-nutritional effect. 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 r e s u l t s in a r e d u c t i o n in t h e r a t e o f 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 e n c y o f f e e d u t i l i z a t i o n ; all o f w h i c h a r e r e f l e c t e d in p o o r a n i m a l p e r f o r m a n c e  (Bedford, 1993). T h e resulting  impaired gut  function  r e d u c e s t h e a v a i l a b i l i t y o f n u t r i e n t s s u c h a s g l u c o s e , fat a n d p r o t e i n ( F e n g l e r a n d Marquardt, 1988; Bedford, 1993). I n c r e a s e d viscosity of the gut a l s o c a u s e s "sticky d r o p p i n g s " , a  condition  a f f e c t i n g p o u l t r y w h i c h c a n r e s u l t in p o o r litter q u a l i t y ( C a m p b e l l a n d B e d f o r d , 1 9 9 2 ) . M e a t quality  may  be affected  if b r o i l e r  birds d e v e l o p  6  breast d a m a g e and  hock  problems  (Campbell  and  Bedford,  1992).  Poultry  diets  that  contain  a  large  p r o p o r t i o n 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 w i t h 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 h e d i e t ( A n n i s o n , 1 9 9 1 ) . T h e anti-nutritional  effects  of  NSPs  may  be  reduced  with  dietary  enzyme  s u p p l e m e n t a t i o n w h i c h will b e d i s c u s s e d f u r t h e r . S p e c i f i c e n z y m e s o f i n t e r e s t 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 f o r w h e a t in a d d i t i o n to a n d c e l l u l a s e s for barley a n d oats (Marquardt,  for  p-glucanases  1 9 9 4 ) . H o w e v e r , t h e i n c r e a s e in  production resulting f r o m the u s e of 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 the d e s t r u c t i o n of the g e l f o r m i n g  polysaccharides  ( C h e s s o n , 1 9 9 3 ) . I m p r o v e m e n t s in n u t r i e n t a b s o r p t i o n a n d g r o w t h r a t e s m a y a l s o b e a t t r i b u t e d to t h e  improved  conditions within the  gastrointestinal  tract  (Chesson,  1993).  1.1.3.2 p-glucanases A  l a r g e p o r t i o n o f t h e e n d o s p e r m c e 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 o f  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 h e m a j o r c e l l wall p o l y s a c c h a r i d e s are the p-glucans w h i c h b e c o m e soluble 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 g u t t h u s a f f e c t i n g p o u l t r y 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 h e 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 t h P-(1,3) l i n k a g e s b e t w e e n t h e 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 intermolecular bonding. T h e addition  o f p - g l u c a n a s e to  7  poultry  diets  hydrolyzes  the  s o l u b l e m i x e d linked p - g l u c a n s w h i c h m i n i m i z e s their anti-nutritional effects 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 h e g u t . T h e i n c r e a s e in t h e  production  r e s p o n s e of  broilers fed a barley diet s u p p l e m e n t e d with p - g l u c a n a s e c a n be e x p l a i n e d by the i n c r e a s e d availability of intracellular nutrients resulting f r o m t h e l o s s of t h e integrity of the cell wall ( H e s s e l m a n a n d A m a n , 1986).  1.1.3.3 Xylanases Like  p-glucans,  arabinoxylans  are  soluble  and  may  l e a d to  similar  n u t r i t i o n a l e f f e c 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 g n i f i c a n t a m o u n t s  anti-  in p o u l t r y  diets. A r a b i n o x y l a n s are the major cell wall c o m p o n e n t s of rye, w h e a t a n d  triticale  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 e c t s c a n b e e l i m i n a t e d b y s u p p l e m e n t i n g the  diet  with  xylanase  to  aid  in  the  solubilization  and  cleavage  of  the  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 h e a c t i v i t y o f t h e e n z y m e m a y b e limited  if t h e  xylan  chain  is highly  substituted  with glucuronic  acid residues  a r a b i n o s e m o l e c u l e s (Biely, 1985). T h e u s e of x y l a n a s e s h a s f o c u s e d o n  or  improving  d i e t a 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 h e d i g e s t i b i l i t y o f r y e , w h e a t a n d b a r l e y in b r o i l e 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 t h a t xylanase  applied  to  rye  diets  r e d u c e s the  i n c i d e n c e of  "sticky  droppings"  and  i m p r o v e s p e r f o r m a n c e in p o u l t r y ( 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 . , 1991; Bedford and C l a s s e n , 1992).  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 protein availability a n d e l i m i n a t e anti-nutritional  factors  and  potential  allergenic  proteins  (Classen,  protein  1996).  digestibility of protein 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 results  in  reduced  feed  utilization.  The  supplementation  of  canola  A  poultry  meal  p r o t e o l y t i c e n z y m e s h a s b e e n s h o w n t o i m p r o v e it's d i g e s t i b i l i t y b y p o u l t r y  low  with  (Bedford  and Morgan, 1995).  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 with m o n o g a s t r i c s h a v e r e n e w e d applications  of  exogenous  enzyme  e n z y m e s m a y benefit ruminant  in  ruminant  diets.  The  use  interest of  in  the  exogenous  nutrition by r e d u c i n g t h e current limitations 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 h e a d d i t i o n of 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 potential limited  to  improve  to t h e  efficiencies and  ensiling  h a s i n d i c a t e d t h a t t h e r e is t h e  performance,  process (Modyanov  and  their  Zel'ner,  applications 1983).  are  mainly  Interpretation  of  r e s u l t s f r o m e n z y m e r e s e a r c h is difficult 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 h e m o d e o f a c t i o n o f t h e e n z y m e s in t h e r u m i n a n t a n i m a l is l a r g e 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 , 1996). T h e potential u s e of alternate feedstuffs a n d g o a l s that a r e directed t o w a r d s m a x i m i z i n g n u t r i e n t e f f i c i e n c i e s , j u s t i f i e s f u r t h e r i n v e s t i g a t i o n into t h e u s e o f e n z y m e s with ruminant feeding s y s t e m s ( C h e s s o n a n d A u s t i n , 1996).  9  1.1.4.1 Sources of Variation in Results T h e v a r i a t i o n in t h e r e s p o n s e s t h a t h a v e b e e n f o u n d w i t h e n z y m e s u s e d in ruminant  feeding  s y s t e m s h a v e b e e n a t t r i b u t e d to a n u m b e r  of different  factors.  S o u r c e s of variation include: type a n d a g e of a n i m a l , diet c o m p o s i t i o n , e n z y m e type, level a n d c o m b i n a t i o n of e n z y m e s u s e d , e n z y m e stability a n d m e t h o d of application ( B e a u c h e m i n and R o d e , 1996). The  response  of  steers  fed  alfalfa,  timothy  hay  or  silage  based  diets  s u p p l e m e n t e d with 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 the f o r a g e source and the  level of e n z y m e applied  (Beauchemin  et a l . , 1 9 9 5 ) .  Substantial  improvements  in a v e r a g e d a i l y g a i n ( A D G ) w e r e f o u n d w h e n t h e e n z y m e s  added  alfalfa  to the  and  timothy  hay  diets,  however,  only  a  minor  effect  were was  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 i b r o l y t i c e n z y m e w a s a p p l i e d t o 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 , t h e i n c r e a s e in f i n a l w e i g h t w a s l i n e a r l y d e p e n d e n t o n t h e l e v e l o f e n z y m e ; w h e r e a s , ADG,  feed  conversion  ratio  ( F C R ) and  dry  matter  intake  (DMI)  all  remained  u n a f f e c t e d ( T r e a c h e r et a l . , 1 9 9 6 ) . A later s t u d y c o n d u c t e d by the s a m e r e s e a r c h e r s reported that w h e n finishing cattle w e r e f e d a total m i x e d ration c o n s i s t i n g of 7 0 % rye g r a s s silage a n d 3 0 % barley grain w h i c h  had b e e n treated  with a  fibrolytic  e n z y m e , A D G i n c r e a s e d w i t h o u 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 a k e ( 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 t h a t increased  enzyme  activity  was  necessary  with  silage  diets.  This  theory  was  supported by unpublished research c o n d u c t e d by the s a m e laboratory w h e r e feedlot  10  animals  were  fed  a  corn  (Beauchemin and Rode, correspond  with the  s i l a g e diet  that w a s  supplemented  with  an  enzyme  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  i n c r e a s e s in t h e  application  rate  of  the  fibrolytic  to  enzyme  complement.  1.1.4.2 Fibrolytic Enzymes 1.1.4.2.i) Forages The  majority  of  enzyme  research  conducted  in  the  ruminant  field  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 r a c t i o n o f f e e d s t u f f s w h i c h i s l e s s d i g e s t i b l e in  has the  r u m e n ; with the objective b e i n g to o p t i m i z e f e e d utilization by the entire a n i m a l . F o r e x a m p l e , the a p p l i c a t i o n of fibrolytic  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  digestibilities of both dry matter ( D M ) a n d fiber ( 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 the 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 t h a t w e r e f e d a f o r a g e b a s e d d i e t s u p p l e m e n t e d w i t h 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 e d 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 milk 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 dairy cattle w e r e f e d either h a y or barley w i t h s i l a g e d i e t s in w h i c h t h e f o r a g e r a t i o n w a s s u p p l e m e n t e d w i t h 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 milk 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 result of i m p r o v e d D M  nor  n e u t r a l d e t e r g e n t f i b e r ( N D F ) d i g e s t i b i l i t i e s but m a y b e a s a r e s u l t o f i n c r e a s e d r a t e o f 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 h e A D G a n d F C R o f  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  r a t i o n s ( 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 digestibilities w e r e o b s e r v e d w h e n fibrolytic e n z y m e s w e r e a d d e d to d r y f o r a g e s however, w h e n e n z y m e s w e r e a d d e d to fresh o r wilted forage, n o effects  were  o b s e r v e d ( F e n g e t a l . , 1 9 9 2 a ) . In c o n t r a s t , s t e e r s f e d f r e s h , w i l t e d o r d r y g r a s s supplemented  with  fibrolytic  enzymes  all elicited  a  positive  growth  response  a l t h o u g h i n c r e a s e s in D M I a s w e l l a s in D M a n d N D F t o t a f t r a c t d i g e s t i b i l i t i e s w e r e only found with dry g r a s s s u p p l e m e n t e d with e n z y m e s ( F e n g et al., 1992b).  1.1.4.2.H) Concentrates Grains  s u c h a s barley a n d sorghum  a n d oilseeds such a s canola  meal  c o n t a i n h i g h a m o u n t s o f fiber 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 fibrolytic 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 t h i s a r e a a r e l i m i t e d . In a r e c e n t s t u d y t h e e f f e c 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 r e p o r t e d t h a t h i g h e r xylanase levels improved feed efficiency of steers w h e r e a s lower x y l a n a s e levels had n o effect ( B e a u c h e m i n et al., 1996).  12  1.1.4.3 Other Enzymes and Enzyme Combinations 1 . 1 . 4 . 3 J ) Forages A n active a r e a of r e s e a r c h h a s b e e n the investigation of the effects of  a  f i b r o l y t i c e n z y m e u s e d in c o n j u n c t i o n w i t h 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 t o f o r a g e s in o r d e r t o e n h a n c e t h e i r d i g e s t i b 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 t h b e e f c a t t l e f e d e i t h e r c o r n o r o a t s i l a g e f o u n d n o c h a n g e s in  DMI  although  and  both gain and feed efficiencies  i n c r e a s e d with a m y l a s e , protease  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 . ( 1 9 6 6 ) f e d cattle  a c o r n or c o r n s i l a g e diet with the  B u r r o u g h s et a l . ( 1 9 6 0 ) a n d r e p o r t e d P e r r y et a l . ( 1 9 6 6 )  observed  no  s a m e fibrolytic  enzymes as used  beneficial effects o n digestibility.  r e s p o n s e in t h e  digestibility  by  However,  of the feed  when  p r o t e o l y t i c e n z y m e s w e r e a p p l i e d t o a s i m i l a r d i e t . In c o n t r a s t t o b o t h 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 digestibilities h a v e b e e n r e p o r t e d w i t h f o r a g e s with the a d d i t i o n o f e i t h e 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 b a c t e r i a l p r o t e a s e ( R u s t et a l . , 1965).  1.1.4.3M)  Concentrates Improvements  in  gain  and  feed  observed w h e n e n z y m e s containing  efficiencies  proteolytic,  of  steers  and  heifers  amylolytic a n d fibrolytic  were  activities  w e r e a d d e d to h i g h grain rations ( B u r r o u g h s et a l . , 1 9 6 0 ; C l a r k et a l . , 1 9 6 1 ; R o v i c s and  Ely,  1962).  Similar  results  were  produced  with  an  unspecified  enzyme  s u p p l e m e n t a p p l i e d t o 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 t o s t e e r s ( W e i c h e n t h a l et a l . ,  13  1996: cited by B e a u c h e m i n a n d  Rode,  1996). E n z y m e supplementation  of  corn  g r a 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 protein 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 colonization  and  by a protein  digestion  matrix w h i c h l e n d s r e s i s t a n c e to  ( M c A l l i s t e r et  al., 1990).  However,  the  microbial  response  to  e n z y m e s u p p l e m e n t a t i o n of corn b a s e d rations h a s b e e n inconsistent. 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 diet of s i m i l a r c o m p o s i t i o n to t h e  one  u s e d b y B u r r o u g h s et a l . ( 1 9 6 0 ) , w a s f e d t o c a l v e s a n d d i d n o t elicit 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 t h e c a l v e s were fed an e n z y m e supplemented  h i g h c o r n g r a i n d i e t ( P e r r y et a l . , 1 9 6 6 ) .  In  contrast, s e v e r a l s t u d i e s involving feedlot s t e e r s fed c o r n grain s u p p l e m e n t e d with 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 e d t o y i e l d r e s p o n s e s in A D G o r 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 , Some  studies  have  also  yielded  results  from  enzyme  research  1960).  that  were  i n c o n c l u s i v e ( T h e u r e r et 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 Recent  r e s e a r c h with m o n o g a s t r i c  animals  has  investigated  the  possible  marriage of e n z y m e s a n d feed p r o c e s s i n g w h e r e the e n z y m e s were applied  post  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 rolling a n d pelleting 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 utilization by i n c r e a s i n g digestibility a n d improving production  performance  in  poultry  (Bedford,  14  1993).  For  example,  subjecting  s o r g h u m g r a i n t o d r y r o l l i n g h a s b e e n s h o w n t o g i v e 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 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 s o l u b 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 t h e i r a n t i - n u t r i t i o n a l  increases the  effects m a y counteract  a n y benefit of the p r o c e s s i n g treatment ( C h e s s o n a n d A u s t i n , 1996). T h i s effect 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 h e d i e t w i t h 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 . , 1991). 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 benefit digestion  by  a c t i n g s y n e r g i s t i c a l l y in b o t h 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 latter c a s e , results from s t u d i e s investigating the c o m b i n e d effects of 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 relative to t h o s e o b t a i n e d with 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 t o c e r e a l g r a i n s h a s b e e n briefly investigated with varying results. Dairy c o w s fed e n z y m e treated dry rolled or s t e a m rolled s o r g h u m s h o w e d i m p r o v e d digestibilities of dry matter ( D M ) , o r g a n i c matter ( O M ) , c r u d e protein ( C P ) a n d N D F f r a c t i o n s , w i t h o u t a n y a d v e r s e e f f e c t s o n m i l k 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 ) reported that s t e e r s fed s t e a m f l a k e d s o r g h u m that w a s treated  with  an  unspecified  enzyme  had  increased DMI  and  an  even  greater  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 e n c y . In t h e s a m e study, w h e n dry rolled s o r g h u m w a s a l s o treated with a n unspecified e n z y m e a n d f e d t o 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 r e f l e c t e d in D M I a n d A D G w i t h a l a r g e 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 . In c o n t r a s t t o t h e a b o v e , d i g e s t i b i l i t y w a s n o t improved  in a s t u d y w h e r e  steers were fed  s o r g h u m d i e t ( R i c h a r d s o n et a l . , 1 9 9 0 ) .  15  a  processed and  enzyme  treated  The  above  review  summarizes many  of t h e results  from  research  with  e x o g e n o u s e n z y m e s a p p l i e d to diets f e d to ruminant a n i m a l s . T h e majority of t h e 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 t o b e a n i n e x p e n s i v e alternative samples.  to animal T h e work  trials  a n d invaluable  conducted  preliminary step to investigating  for this  in e v a l u a t i n g  thesis  required  of a large  n u m b e r of  in vitro a n a l y s e s a s a  a large n u m b e r o f s a m p l e s a n d t h e interactive  effects o f e n z y m e s a n d mild hydrothermal treatment o f barley 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 f o 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 effective alternatives 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 often expensive,  labor  intensive  a n d limited  in t h e 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 a t o n e t i m e . T h e m a j o r i t y o f in vitro m e t h o d s t h a t a r e s p e c i f i c t o e v a l u a t i n g r u m i n a n t f e e d s t u f f s i n v o l v e t h e i n c u b a t i o n o f a s a m p l e o f t h e f e e d w i t h r u m e n fluid 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 usually b a s e d o n a m e a s u r e o f e n d point digestion or  the appearance  detailing  t h e history,  of metabolic development  products  over  time.  a n d methodology  Comprehensive  reviews  o f in vitro r u m i n a n t  feed  evaluation techniques a r e given by J o h n s o n (1963; 1966). O n e of these reviews p r e s e n t s t h e 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 t h e a r e a o f 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 fluid f r o m a n o x . A n o t h e r s i g n i f i c a n t c o n t r i b u t i o n t o t h e 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 t h e  16  determination of the mineral c o m p o s i t i o n of s h e e p s a l i v a ( M c D o u g a l l ' s , 1948). T h i s d i s c o v e r y e n a b l e d the c h e m i c a l r e p r o d u c t i o n of 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 b u f f e r s o l u t i o n w h i c h i s still i n 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 t o e v a l u a t e f o r a g e d i g e s t i b i l i t y w h i c h h a s b e c o m e the standard.  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 i s a t w o s t a g e m e t h o d provides a n estimate  of the apparent  digestibility  o f a feedstuff.  that  T h e technique  i n v o l v e s t h e i n c u b a t i o n o f a feedstuff with r u m e n fluid i n o c u l u m for 4 8 h, f o l l o w e d b y 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  ruminant  d i g e s t i o n w h e r e t h e f e e d i s first f e r m e n t e d b y m i c r o b e s i n t h e r u m e n , r e f e r r e d t o a s t h e first s t a g e , a n d t h e n s u b j e c t e d t o p o s t r u m i n a l d i g e s t i o n , t h e s e c o n d s t a g e . A n end  point  d r y matter  measurement  digestibility  is o b t a i n e d .  e s s e n t i a l l y that t h e latter indigestible  in this  (IVDMD)  T h e only  or organic  difference  between  is corrected for a s h content  system.  IVDMD  represents  matter  IVDMD  which  t h e fraction  digestibility  (OMD)  a n d O M D is  is a s s u m e d to b e of the feed  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 b o t h t h e f i b r o u s a n d c e l l s o l u b l e fractions.  T h e undigested  plant  residue  however,  is c o n t a m i n a t e d  with  some  undigested microbial material. This technique has been s u c c e s s f u l b e c a u s e of the high  correlation  between  t h e in vitro r e s u l t s  a n d in vivo a p p a r e n t  digestibility  m e a s u r e m e n t s ( V a n S o e s t , 1982). Although the method w a s originally 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 i g e s t i b 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 with concentrates. F o r e x a m p l e , apparent I V D M D o f w h e a t w a s found to b e well c o r r e l a t e d ( R = 0 . 8 3 ) w i t h 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 e t a l . , 1 9 9 5 ) . 2  18  1.2.2 True Digestibility G o e r i n g a n d V a n S o e s t (1970) modified the s e c o n d step of the Tilley a n d Terry  method  to  arrive  at  an  estimate  of  "true  digestibility"  (IVTD).  The  true  digestibility 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 with r u m e n fluid for 4 8 h a n d then substituting the a c i d p e p s i n incubation with a neutral detergent fiber rinse. 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 all c e l l s o l u b l e s w h i c h , in e s s e n c e ,  represents  e n d point d i g e s t i o n .  1.2.3 Enzymatic Method A  digestibility  measurement  can  also  be  obtained  using  an  enzymatic  method. S e v e r a l enzymatic methods are available (Van S o e s t , 1994). For example, a p r o c e d u r e d e v e l o p e d by J o n e s a n d H a y w a r d (1975) involves incubating the feed 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 t h 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 t h t h i s 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 variety of e n z y m e s a n d m a y a d a p t to a variety of different 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 r e f e r r e d t o N o c e k ( 1 9 8 8 ) . E n z y m a t i c t e c h n i q u e s d i f f e r 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 not require t h e u s e of r u m e n fluid a n d t h u s t h e m a i n t e n a n c e of d o n o r a n i m a l s a n d an anaerobic environment.  19  1.2.4 Rumen Fluid Inoculum 1.2.4.1 Animal Diet Lindberg  (1985)  briefly  discussed a number  estimates of rumen fermentation factors  include:  solubility,  of factors  which  influence  b y v a r i o u s in vitro t e c h n i q u e s . S o m e o f t h e s e  endproduct  accumulation,  continuous  fermentation,  p r o t e o l y t i c e n z y m e s , o p t i m a l p H a n d t h e b a s a l d i e t f e d t o t h e a n i m a l . F i b e r s o u r c e in t h e d i e t h a s a l s o b e e n s h o w n t o a f f e c t t h e r e s u 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 e t al., 1 9 9 3 ) . T h e ratio o f c o n c e n t r a t e to f o r a g e affects the r u m e n microbial population a s t h e 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 different affinities f o r s u b s t r a t e s ( R u s s e l a n d Baldwin,  1979) a n d t h e products of fermentation  vary accordingly (Owens a n d  G o e t s c h , 1 9 8 8 ) . R u m e n fluid d o n o r a n i m a l s s h o u l d therefore b e m a i n t a i n e d o n a b a l a n c e d r a t i o n w i t h s i m i l a r d i e t a r y c o m p o n e n t s a s t o 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 r n a l v a r i a t i o n in r u m e n f l u i d a c t i v i t y c a n b e m i n i m i z e d b y increasing the feeding frequency  (Warner,  1965)  a n d conducting  collections at  similar t i m e s . T h e retention o f r u m e n d i g e s t a is d e c r e a s e d with i n c r e a s e d f e e d i n g frequency which  m a y b e relevant w h e n  a p p l y i n g t h e in situ t e c h n i q u e  1981).  20  (Warner,  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 fluid a f e w h o u r s after f e e d i n g to e n s u r e a m i x t u r e o f b a c t e r i a l s p e c i e s ( M o o r e e t a l . , 1 9 6 2 ) . It h a s b e e n r e p o r t e d t h a t post feeding  c o l l e c t i o n o f r u m e n fluid  r e d u c e s t h e variation  between  incubating  s a m p l e s in vitro ( T i l l e y 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 i n v o l v i n g t h e 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 fluid p r i o r t o f e e d i n g t o e n s u r e l o w r u m e n fluid activity  in b l a n k s ( 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 ) .  R e c e n t s t u d i e s i n v o l v i n g t h e 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 r e v e r t e d to c o l l e c t i n g t h e r u m e n fluid after f e e d i n g w h e n b a c t e r i a l activity is a s s u m e d t o 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 majority  o f r u m e n fluid 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 h e 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 fluid 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 buffer a n d mineral mixture (Quin, 1 9 3 8 , 1 9 4 3 ; H u n g a t e et al., 1955; P e a r s o n a n d S m i t h , 1943: cited b y J o h n s o n , 1966), although  a f e w researchers  u s e d u n s t r a i n e d r u m e n fluid ( 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 t o t h e p l a n t c e l l w a l l i n c r e a s e s l i n e a r l y u p t o 9 h ( L a t h a m e t a l . , 1 9 7 8 ) . It h a s b e e n s u g g e s t e d that approximately 7 0 - 8 0 % of the r u m e n m i c r o b e s are a s s o c i a t e d with  t h e particulate  matter  in t h e r u m e n  whereas  strained  rumen  fluid  alone  a c c o u n t s f o r 2 0 - 3 0 % o f m i c r o b i a l a c t i v i t y ( 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 fluid in a b l e n d e r t o d i s l o d g e m i c r o b e s a t t a c h e d t o f e e d p a r t i c l e s h a s p r o v e n t o b e b e n e f i c i a l i n o b t a i n i n g r e l i a b l e e s t i m a t e s o f in vitro digestion coefficients. 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 that blending affects t h e v i a b i l i t y 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 e t a l . , 1 9 8 4 ) . In t h e g a s p r o d u c t i o n s y s t e m , preliminary s t u d i e s c o n d u c t e d b y Pell a n d S c h o f i e l d (1993) indicated 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 l u i d g a v e s i m i l a r r e s u l t s . U n b l e n d e d r u m e n fluid is p r e f e r r e d 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 t o i n c r e a s e t h e e r r o r in g a s  production  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 h e a m o u n t o f s m a l l f e e d p a r t i c l e s in t h e fluid a n d exposing the microbes to more o x y g e n (Pell a n d Schofield, 1993).  1.2.5 Advantages of In Vitro Techniques The  greatest  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 simulate  animal  performance is that they are less intensive a n d costly than animal studies. 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 t o b e u s e f u l in s c r e e n i n g a l a r g e 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 t h in vivo r e s u l t s ( V a n S o e s t , 1982). 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 be faster a n d h a v e better replication, t h e 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 r e a l i t y .  22  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 t h 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 , r e q u i r e 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 t o s e r v e a s r u m e n f l u i d d o n o r s . It is this latter point w h i c h c r e a t e s o n e o f t h e 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  these  t y p e s o f s t u d i e s , t h e 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 l u i d . It is i m p e r a t i v e  that a n a n a e r o b i c environment  b e maintained  s u c c e s s of the experiment. T h e determination requires  long  periods  of time  a n d involves  in o r d e r t o e n s u r e  of apparent o r true many  preparatory  digestibilities  steps. A  major  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 information o n the kinetics o f d e g r a d a t i o n m a y be  obtained  unless  lengthy  a n d labor  intensive  time  course  incubations are  conducted.  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 first d e v e l o p e d b y Q u i n e t a l . , ( 1 9 3 8 ) ( c i t e d b y J o h n s o n , 1 9 6 6 ) a n d a l l o w s f o r a t h o r o u g h d e s c r i p t i o n o f the f e r m e n t a t i o n profile o f a feedstuff t h e r e b y e n a b l i n g the determination o f the rate a n d extent o f d i s a p p e a r a n c e of  a feedstuff.  Feedstuffs  are contained  within  small  nylon  bags  which are  s u s p e n d e d a n d incubated in t h e r u m e n for various periods o f time ( M e h r e z a n d Orskov, 1977; O r s k o v , 1982). T h e d i s a p p e a r a n c e of material from the bag indicates 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 , 1977). 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  23  determine fractional rates o f protein degradation e m p l o y i n g the e q u a t i o n s of O r s k o v and M c D o n a l d (1979). Although t h e method  most closely resembles the rumen  environment (Broderick et al., 1988), the method h a s also b e e n criticized for several technical problems. T h e in situ m e t h o d  is intensive, requires 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 a t o n e t i m e . In a d d i t i o n , f i n e f e e d particles m a y b e lost t h r o u g h t h e p o r e s o f the b a g s w h i c h a r e a s s u m e d to b e p a r t o f t h e i m m e d i a t e l y s o l u b l e f r a c t i o n b u t m a y n o t in f a c 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 p a r t i c l e s into t h e n y l o n b a g ( N o c e k , 1985) a n d bacterial c o n t a m i n a t i o n o f the s a m p l e ( N o c e k , 1987) h a v e b e e n s h o w n to affect degradability v a l u e s o f f e e d s . Microbial c o n t a m i n a t i o n is s m a l l with  protein  s u p p l e m e n t s , a n d highly digestible forages ( M e h r e z a n d O r s k o v , 1977; Varvikko and Lindberg,  1985). H o w e v e r , with rapidly fermentable  c e r e a l s s u c h a s barley a n d  poorly digested forages, contamination could b e significant (Varvikko a n d Lindberg, 1 9 8 5 ) . It s h o u l d b e n o t e d t h a t t h e p r e s e n c e o f m i c r o b i a l m a t t e r w i l l n o t g r e a t l y a f f e c 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 b u t will i n f l u e n c e n i t r o g e n 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 h e f e r m e n t a t i o n  kinetics of feedstuffs t h e greatest  d i s a d v a n t a g e o f this t e c h n i q u e is t h e a s s u m p t i o n that t h e s o l u b l e fraction feedstuff  is entirely 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 "true"  of a  fermentation  p r o f i l e o f a f e e d s t u 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 production technique.  24  1.2.8 The Gas Production Technique T h e kinetics o f digestion o f a feedstuff c a n b e d e s c r i b e d with t h e u s e o f the g a s p r o d u c t i o n t e c h n i q u e first d e v e l o p e d b y M e n k e e t 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 i n v o l v e s t h e i n c u b a t i o n o f a feedstuff with r u m e n fluid i n o c u l u m a n d a s fermentation  proceeds, the g a s produced  is recorded  at specific times. G a s  production is related t o the digestibility a n d m a y b e u s e d with other p a r a m e t e r s to estimate t h e metabolizable energy value of a feedstuff f e d to ruminant  animals  ( M e n k e et al., 1979; M e n k e a n d S t e i n g a s s , 1988). A n estimate of the energy value of a feedstuff is o b t a i n e d b y c o m b i n i n g t h e results 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 r o t e i n , c r u d e fat, c r u d e f i b e r a n d c r u d e a s h , w i t h t h o s e o b t a i n e d f r o m g a s production studies ( M e n k e and S t e i n g a s s , 1988). 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 b o t h f o u n d t o b e w e l l c o r r e l a t e d with t h e rates o f fermentation f o r w h e a t ( A d e s o g a n et al., 1995). H o w e v e r , results m a y d e p e n d o n the type of feedstuff a s there w a s n o significant correlation between g a s p r o d u c t i o n a n d t h e in situ o r in vivo d e g r a d a b i l i t i e s in t h e s t u d i e s c o n d u c t e d b y Piva et al., (1988) which  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 latter s t u d y  also  d e t e r m i n e d that nitrogen free extract ( N F E ) a n d A D F h a d the greatest influence o n feed fermentation. A strong linear relationship between N D F d i s a p p e a r a n c e and g a s production Blummel  h a s been and Orskov  shown (1993)  to exist found  with f o r a g e s a good  (Pell a n d Schofield,  correlation  between  1993).  in vitro g a s  production a n d the nylon b a g degradability o f f e e d w h i c h w a s c l o s e l y related to feed intake a n d g r o w t h rate in cattle.  25  Measurement  of the  gas produced  from  rumen  microorganisms  in  batch  c u l t u r e s w a s first 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 t e d b y 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 h e g a s a c c u m u l a t i o n o f a  feedstuff  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 t h r u m e n f l u i d 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 , gas  production  caused  the  syringe  plunger  to  rise  and  enabled  a  direct  m e a s u r e m e n t of g a s v o l u m e . T h i s author reported a high correlation between  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 production t e c h n i q u e w a s further refined 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 using a m a n u a l method w h e r e the g a s v o l u m e w a s determined visually  reading  advances  have  the  movement  allowed  for  the  of  the  syringe  changes  in  plunger.  gas  volumes  Recent to  be  by  technological electronically  m e a s u r e d b y 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 light diode voltmeter  a n d electronic 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  into f i x e d v o l u m e f e r m e n t a t i o n  emitting inserted  flasks. A s g a s a c c u m u l a t e d within the flasks, the  p r e s s u r e required to stabilize the 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  determined  at s p e c i f i e d t i m e i n t e r v a l s . T h i s t e c h n i q u e is b a s e d o n t h e a s s u m p t i o n t h a t m i c r o b i a 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 b y t h e a c c u m u l a t i o n o f g a s in t h e s y s t e m . It h a s b e e n recently  shown  that  if  pressure  is  maintained  within  the  range  of  a t m o s p h e r e s , f e r m e n t a t i o n will n o t b e a f f e c t e d ( P e l l a n d S c h o f i e l d , 1 9 9 3 ) .  26  0  to  0.6  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  results  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 t h i s 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 fermentation f l a s k s a n d interfaced with a d a t a logger 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 x e d container, the  resulting  pressure was  r e c o r d e d at d e t e r m i n e d  time  volume  intervals.  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  A  between  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 of  the  data.  1.2.8.1 Advantages of the Gas Production Technique The  m a i n a d v a n t a g e of the g a s production t e c h n i q u e  i s it m e a s u r e s t h e  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 r e f l e c t s t h e " t r u e " f e r m e n t a t i o n o f a f e e d s t u f f . It h a s b e e n s h o w n t h a t t h e o b s e r v e d f e r m e n t a t i o n p r o f i l e i s r e p r e s e n t a t i v e of the entire s a m p l e (Huntington,  1995). T h e g a s production technique d o e s  not  r e q u 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 , b o t h t h e s o l u b l e a n d  insoluble  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 volatile fatty a c i d s ( V F A ) by rumen microorganisms (Huntington, 1995). T h e procedure also requires a s m a l l s a m p l e s i z e w h i c h is r e f l e c t e d in t h e u s e o f l i m i t e d a m o u n t s o f supplies.  27  laboratory  1.2.8.2 Disadvantages of the Gas Production Technique There technique.  are  several  shortcomings  associated  Firstly, the c o m p u t e r i z e d t e c h n i q u e  requires highly sophisticated equipment.  with  the  gas  production  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  S e c o n d l y , a standard feedstuff  must  i n c u b a t e d w i t h i n e a c h r u n in o r d e r t o c o r r e c t f o r t h e v a r i a t i o n in r u m e n fluid  be  activity  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 ) . T h i r d l y , m i c r o b i a l c e l l s in t h e r u m e n fluid c o n t r i b u t e 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 b y o t h e r m i c r o o r g a n i s m s . T h i s latter p o i n t is o f p a r t i c u l a r i m p o r t a n c e w h e n  short  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 t h a t V F A s a r e a p o o r i n d i c a t i o n fermentation  rates  fermentation production  and  extent  of  digestion  (Van  Soest,  1982).  Lastly,  of the  o f r e a d 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 , r e s u l t s in t h e  of high a m o u n t s  of propionic a c i d w h i c h d o e s not contribute  to  gas  production, thus requiring careful interpretation of g a s d a t a .  1.2.8.3 The Fermentation Process of Carbohydrates in the Rumen. Dietary  carbohydrates  include  soluble  sugars,  starch  and  fiber.  Soluble  s u g a r s a r e r a p i d l y f e r m e n t e d in t h e r u m e n w h e r e t h e t y p e o f s u g a r d e t e r m i n e s t h e r a t e 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 h e 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 h e d i e t r e s u l t s in t h e  production  of high a m o u n t s  of propionic acid thus d e c r e a s i n g r u m e n  pH  ( 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 b e r d i g e s t i o n a s s o c i a t e d w i t h l o w r u m e n p H m a y be  caused  by  a  shift  in  the  types  of  microbes  from  cellulolytic  to  amylolytic  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 ) . Structural 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  28  cellulose and  hemicellulose fractions.  Starch, pectins and  polyuronids  are  quickly digested than fiber c o m p o n e n t s although m o r e slowly fermented by  more rumen  m i c r o o r g a n i s m s than the soluble s u g a r s ( V a n S o e s t , 1982). 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 o b t a i n a s o u r c e of e n e r g y . T h e m a i n e n d products of the a n a e r o b i c fermentation p r o c e s s are the short c h a i n volatile fatty 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 butyrate, a n d the g a s e s : carbon dioxide ( C 0 ) and methane ( C H ) (Wolin, 1975). Ruminants 2  utilize  4  V F A s a s a m a j o r s o u r c e of e n e r g y , for 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 metabolizable energy ( O w e n s and G o e t s c h , 1988), however, g a s produced as a result of c a r b o h y d r a t e 1966).  The  metabolism constitutes  equations  of  Wolin  (1960)  are  a major  l o s s of e n e r g y  presented  below  to  (Hungate,  illustrate  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 ) . hydrogen  produced  during  the  process  combines  with  C 0  2  to  the  Excess  produce  CH  ( 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 t h a t in t h e p r o d u c t i o n VFAs,  acetate  and  butyrate  participate  directly  in  gas  production  3  6  3  2  C H 0 + H 3  6  CH3COOH + C 0  + H 0  3  2  2  + 2H  => C H C H C O O H + H 0 3  2  2  2C H 0 ^ CH CH CH COOH + 2C0 3  C0  2  6  3  + 4H  3  2  2  2  2  2  + 2H  => C H + 2 H 0 4  2  (acetate)  ( E q u a t i o n 1)  (propionate)  ( E q u a t i o n 2)  (butyrate)  ( E q u a t i o n 3) (Equation 4)  2  29  of  whereas  p r o p i o n a t e d o e s not.  C H 0  4  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 h e proportion of p r o p i o n a t e i n c r e a s e s r e l a t i v e t o a c e t a t e a n d b u t y r a t e . T h i s i s , in p a r t , a s a r e s u l t o f a d e c r e a s e in e n e r g y loss through  decreased gas  production,  primarily  methane  (Van  Soest,  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 H  2  via  the  production  of  formate  (Owens  and  Goestch,  1988).  and  2  From  the  stoichiometric e q u a t i o n s of H u n g a t e (1966) a n d the e q u a t i o n s of W o l i n (1960), g a s production c a n b e e s t i m a t e d from the V F A c o m p o s i t i o n of the r u m e n fluid.  1.2.8.4 Estimation of Gas Volume Direct g a s production c a n b e explained by the a m o u n t a n d proportions VFAs  produced from  fermentation  (Menke  and  Steingass, 1988; Blummel  of  and  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 of V F A p r o d u c e d from the fermentation indirect e q u i v a l e n t m m o l of C 0  2  p r o c e s s a l s o r e s u l t s in t h e r e l e a s e o f a n  f r o m t h e in vitro b u f f e r s o l u t i o n a s t h e V F A s  are  neutralized ( M e n k e and Steingass, 1988; Beuvink and Sploestra, 1992; Blummel and  Orskov, 1993).  capacity  of  the  I n d i r e c t g a s p r o d u c t i o n will o c c u r a s l o n g a s t h e  incubation  medium  is  not  e x c e e d e d . Direct  and  buffering  indirect  gas  production resulting from e a c h m m o l of acetate, propionate a n d butyrate h a s b e e n d e t e r m i n e d to  be 4, 3 and 2 mmols C 0 , respectively (Beuvink and Sploestra,  1992). T h e following  2  equation  h a s b e e n s h o w n to d e s c r i b e t h e  amount  of  gas  directly p r o d u c e d f r o m t h e 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 content and proportions thereof (Beuvink and Sploestra, 1992):  30  mL gas = Mv mmol HAc + 2 Mv mmol HB + 0.87 Mv mmol total VFA  (Equation 4)  w h e r e HAc i s a c e t a t e , HB i s b u t y r a t e a n d Mv i s m o l a r g a s v o l u m e a t s t a n d a r d temperature and pressure (24 L*mol  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 h e 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 . , 1990; B e u v i n k a n d S p l o e s t r a , 1 9 9 2 ; Opatpatanakit et al., 1 9 9 4 , 1995). Q u i n (1943) (cited b y J o h n s o n , 1 9 6 6 ) m e a s u r e d t h e 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 found that t h e products of fermentation  w e r e c l o s e l y r e l a t e d t o d i g e s t i o n in t h e  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 t h e p H o f t h e 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 r e s u l t s in h i g h e r a m o u n t s o f a c i d production  putting a greater  load o n t h e buffering  capacity of the s y s t e m . T h e  b u f f e r i n g c a p a c i t y o f t h e s y s t e m will b e a f f e c t e d if t h e p H f a l l s b e l o w 6 . 0 ( M e n k e e t al., 1979). T h e p r e s e n c e of C 0  2  in t h e 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 b y  a c t i n g a s a b u f f e 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 " ) . A l l o f t h e V F A s c o n t r i b u t e t o 3  the proton pool. H o w e v e r , a s previously reported, the formation of propionate d o e s not  produce  C 0  2  in a d d i t i o n  to contributing  31  to t h e proton  pool, both  of which  c o n t r i b u t e t o 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 i g h c o n c e n t r a t e d i e t s (Van Soest, 1994). 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 e s u l t s in t h e f o r m a t i o n o f 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 h e 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 r e s u l t i n g in l e s s C 0  2  a n d C H 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 ratio will 4  v a r y d e p e n d i n g o n t h e c o n t e n t o f r e a d 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 h e s a m p l e . F o r this r e a s o n , interpretation of the fermentation profiles of c o n c e n t r a t e s m a y b e m o r e difficult 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 o m different s o u r c e s .  1.2.8.6 Kinetic Analysis of Gas Data Mathematical  models  kinetics of v a r i o u s feedstuffs. relationship b e t w e e n  provide  a  means  for  describing  the  fermentation  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 reflect  the  bacterial numbers and substrate d i s a p p e a r a n c e . Sigmoidal  m o d e l s u s e d t o d e s c r i b e t h e d i g e s t i o n o f f i b e r in t h e r u m e n w e r e p r i m a r i l y d e v e l o p e d f o r u s e in c o n j u n c t i o n w i t h d a t a o b t a i n e d f r o m t h e 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 , (1979); B r o d e r i c k a n d C r a i g , (1980); a n d V a n S o e s t , (1982)). T h e only n o n - s i g m o i d a l m o d e l is t h e 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 ( 1 9 7 9 ) . 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 s m s i n c e it p r o d u c e s h i g h r o o t  mean  s q u a r e v a l u e s w h e n fitted t o 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 t h a t a f t e r 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 Sploestra, 1992).  32  at a m a x i m u m  rate ( B e u v i n k  and  S c h o f i e l d et a l . ( 1 9 9 4 ) fit 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 w h i c h lag,  logistic a n d  both the  Gompertz single and  dual  pool  incorporated  m o d e l s to  gas  data  obtained from various substrates including bacterial cellulose, alpha-cellulose and m i x t u r e s of t h e t w o purified s u b s t r a t e s of fiber 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 logistic  model  fermentation  describing  bacterial  growth  that  incorporated  the  different  r a t e s o f t h e v a r i o u s c o m p o n e n t s in t h e i r m i x e d - f i b e r s u b s t r a t e  most  accurately d e s c r i b e d g a s production. U s i n g g a s production data from the incubation of g r a s s s i l a g e s with r u m e n fluid, B e u v i n k a n d K o g u t (1993) d e v e l o p e d a modified Gompertz  equation  to  d e s c r i b e the  fermentation  p r o c e s s . F r a n c e et a l .  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 lag to d e s c r i b e t h e of  leaves from  tropical  forage  trees.  The  simple  exponential  (1993)  fermentation  model  with  ( 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  lag  Loften  ( 1 9 8 0 ) . 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 h e m o d e l t o a c c u r a t e l y r e f l e c t t h e g a s production profiles of e n e r g y s u p p l e m e n t s a n d tropical feedstuffs. T h i s m o d e l was  found  to  accurately  reflect  the  gas  production  profile  of  canola  meal  in  experiment 3.  1.3 OBJECTIVES OF THE THESIS E v a l u a t i o n of e n z y m e a n d mild hydrothermal treatment of barley 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 with a s y s t e m e m p l o y i n g the u s e of I V D M D a n d I V T D results (Tilley a n d Terry, 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 of the e f f e c 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  33  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 t o t h e large n u m b e r o f s a m p l e s a n d time constraints, barley c o u l d not b e e v a l u a t e d to t h e s a m e extent. T h e u s e of t h e g a s production s y s t e m provided information rumen  in vitro f e r m e n t a t i o n  characteristics  of canola  meal  when  on the  treated  with  e n z y m e s and hydrothermal processing. T h e objectives o f this t h e s i s project w e r e to: 1) D e t e r m i n e o p t i m a l  treatment  combinations  of t h e e n z y m e s a n d hydrothermal  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 nutritive v a l u e of barley for dairy cattle. 2) D e t e r m i n e  t h e effects  of e n z y m e a n d hydrothermal  treatment  using  IVDMD,  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 nutritive v a l u e o f c a n o l a m e a l . 3) Further e v a l u a t e t h e treatment  effects o n t h e characteristics of fermentation of  c a n o l a m e a l u s i n g a s i n d i c e s : rate o f g a s p r o d u c t i o n , length o f the l a g p h a s e , t h e production o f V F A s a n d their proportions.  34  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 . 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Unit, A r m i d a l e , N S W , Australia.  45  CHAPTER 2  2.0 THE EFFECT OF ENZYME AND MILD HYDROTHERMAL TREATMENT ON THE IN VITRO DIGESTIBILITY OF B A R L E Y GRAIN.  2.1 ABSTRACT  B a r l e y g r a i n i s a m a j o r c o m p o n e n t o f l a c t a t i n g d a i r y c a t t l e d i e t s in t h e L o w e r F r a s e r V a l l e y region of British C o l u m b i a a n d m u c h of t h e Pacific Northwest. 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 ration, h o w e v e r , it's h i g h fiber 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 o b j e c t i v e o f the  present  study  w a s to e v a l u a t e t h e feasibility  of using  e n z y m e s a n d mild  hydrothermal treatment to improve the available energy content of barley grain. T h e experiment consisted o f a 4 x 4 x 4 x 2 x 2 following treatments: applied  at  (Trichoderma  heat applied at 5 5 ° C for 1 5 , 3 0 o r 6 0 m i n periods o r not  all; moisture sp.)  multi-factorial d e s i g n w h i c h included t h e  applied  at  0%,  10%, 2 0 % , or  3 0 % ; p-glucanase  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  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.)  sp.) a p p l i e d a t 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 t o d e t e r m i n e a p p a r e n t a n d t r u e d i g e s t i b i l i t i e s a n d fiber content. Nutrient c o m p o s i t i o n of t h e barley grain 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 significant third o r d e r interaction (P < 0.01) o n I V D M D w a s noted for the duration of the heat treatment, a n d the  46  addition  of moisture  a n d xylanase where  treatments  either  h a d n o effect o r  significantly 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 significant interaction ( P = 0.0001) between the e n z y m e s : p-glucanase, protease and xylanase where  both  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 interaction a m o n g t h e e n z y m e s w a s also determined to exist for IVTD v a l u e s ( P = 0.0001) w h e r e results paralleled t h o s e o f I V D M D . A quadratic effect ( P < 0.01) o f moisture 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 . In vitro 2  digestibility  results w e r e  decreased  digestibility  inconsistent, a s s o m e treatments relative  to t h e control.  increased a n d others  Fiber a n a l y s e s reflected  results  o b t a i n e d w i t h in vitro d i g e s t i b i l i t i e s . A D F w a s s i g n i f i c a n t l y ( P < 0 . 0 1 ) a f f e c t e d b y t h e significant  interaction  (P < 0.05) between  p-glucanase, protease a n d xylanase.  T h e r e w a s a l s o a significant interaction ( P < 0.05) noted for the duration of the heat treatment, a n d t h e application  of p-glucanase a n d protease e n z y m e s o n A D F  content. T w o - w a y interactions w h i c h w e r e f o u n d to h a v e a significant effect ( P < 0.05)  o n the N D F content  of barley  included:  p-glucanase  a n d p r o t e a s e , p-  glucanase a n d xylanase, protease a n d xylanase. T h e responses were  variable.  X y l a n a s e w a s detrimental o n the digestibility of barley. R e s u l t s indicated the addition 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 f u r t h e 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 n u t r i t i o n a l v a l u e o f b a r l e y g r a i n in ruminant feeding s y s t e m s .  47  2.2 INTRODUCTION  B a r l e y g r a i n is t h e m a j o r e n e r g y s o u r c e f e d t o d a i r y c a t t l e in t h e L o w e r F r a s e r V a l l e y region of British 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 % of the  concentrate  r a t i o n ( 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 t o c o r n is p a r t l y a r e s u l t o f h i g h 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 limit it's p e r f o r m a n c e in d a i r y c a t t l e d i e t s . T h e barley kernel c o n s i s t s of a hull, 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. The  hull c o n t a i n s t h e  majority of the fiber.  The  endosperm  is m a d e  up of  an  a l e u r o n e l a y e 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 c e l l l a y e r , t h e l a t t e r b e i n g o f little nutritional v a l u e to the ruminant a n i m a l ( N e w m a n a n d M c G u i r e , 1985). T h e a l e u r o n e l a y e r is primarily 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  McGuire,  1985). It  is  well  established  that  p-glucans  and  arabinoxylans,  when  fed  m o n o g a s t r i c s , exhibit anti-nutritional effects a s t h e s e a n i m a l s d o not p o s s e s s  to 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 t o 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 t o r e d u c e t h e e f f e c t s 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 led 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 p o u l t r y a n d s w i n e d i e t s w i t h o u t c o m p r i m i s i n g animal  performance.  because  rumen  These  microbes  NSPs  rarely  p o s s e s s the  pose  a  problem  e n z y m e s to  to  ruminant  a s s i s t in t h e i r  animals  digestion,  p-  g l u c a n s , for 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 . , 1992). H o w e v e r ,  48  in h i g h p r o d u c i n g c o w s , f e e d i n t a k e a n d f e e d p a s s a g e r a t e 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 t h a t a r e d u c t i o n in t h e 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  may  occur. T h e s u c c e s s f u l application of 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 i n t e r e s t in t h e 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 t o  ruminant  f e e d i n g s y s t e m s . R e s e a r c h into the addition of 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 t h a t a r e f e d t o 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 r e s u l t s ( r e f e r t o C h a p . 1, s e c t . 1.1.4) (Beauchemin  and  Rode,  1996).  Enzyme composites  containing  a  spectrum  different e n z y m e s , s u c h a s t h o s e with proteolytic, amylolytic a n d fibrolytic  of  activities,  e l i c i t e d 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 high grain rations ( B u r r o u g h s et al., 1 9 6 0 ; C l a r k et al., 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 c o n t r a s t , a l a c k o f response  in  investigated  DMI, A D G or feed the  effects  of  efficiency w a s  multi-enzyme  reported  preparations  in o t h e r added  to  studies a  which  variety  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 p a r t i c u l a r , h a s b e e n t h e f o c u s o f s e v e r a l r e s e a r c h experiments. supplemented  In  a  with  study  where  proteolytic  steers  and  were  amylolytic  fed  a  65%  enzymes,  barley  there  was  grain a  diet noted  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 r a i n d i e t s u p p l e m e n t e d w i t h 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 ) .  49  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 t o 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 digestibility digestibility  may  impact  digestion  in  several  ways.  m a y positively affect the production  Firstly,  increasing  of microbial protein  by  barley  providing  greater a m o u n t s of 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 dairy farmers 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 s h C o l u m b i a r e l y o n h i g h  quality  g r a s s s i l a g e a s the m a i n f o r a g e s o u r c e fed to their lactating dairy herds. T h e s e s i l a g e s t e n d t o b e h i g h in s o l u b l e n i t r o g e n ( v o n K e y s e r l i n g k et a l . , 1 9 9 6 ) . T h i s l a r g e s u p p l y of s o l u b l e nitrogen to the r u m e n m u s t b e b a l a n c e d with a  complementary  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 i s i m p o r t a n t in o r d e r t o o p t i m i z e t h e use  of  rumen  microbial  degradable  protein  nitrogen  (Krishnamoorthy  and  energy  et a l . , 1 9 9 1 )  s o u r c e s for a n d to  the  production  m a x i m i z e the  supply  of of  microbial protein to the intestine ( S t o k e s et a l . , 1991). M a i n t e n a n c e of this 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 t o r e d u c e t h e e x c r e t i o n o f e x c e s s n i t r o g e n a n d m a x i m i z e f e e d utilization (Huntington,  1995).  S e c o n d l y , i n c r e a s i n g t h e ruminal d e g r a d a b i l i t y of b a r l e y m a y i m p r o v e total tract  digestibility.  Although  ruminal  acidosis  is  a s s o c i a t e d with  high  ruminally  digestible c e r e a l g r a i n s , L e n v e n t i n i et a l . , (1990) f o u n d that the effect of low r u m e n p H w a s m i n i m i z e d with t h e a d d i t i o n of a buffer to t h e ration. B o t h a l o w r u m e n and  a  shift  in t h e  rumen  digestion. T h e addition  microbial  of fibrolytic  population e n z y m e s to  d i s a d v a n t a g e to b e o v e r c o m e .  50  may  contribute  particular  to  reduced  diets m a y  enable  pH fiber this  T h e digestibility o f barley 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 digestibility of t h e s t r u c t u r a l c a r b o h y d r a t e s , t h e h u l l f r a c t i o n in p a r t i c u l a r , a n d b y e n h a n c i n g t h e 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 within t h e p r o t e i n m a t r i x . R e s e a r c h i s w a r r a n t e d in o r d e r t o f u r t h e r i m p r o v e t h e d i g e s t i b i l i t y (Hunt et al., 1995) a n d s u b s e q u e n t l y t h e digestible energy content of barley. T h e Alberta  Barley  Commission  (1995)  reports  that  for every  1% increase  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 n d u s t r y will a c c r u e 1 0 million d o l l a r s in benefits. T h e r e f o r e , t h e objective o f this s t u d y w a s to investigate the  feasibility  of using  exogenous e n z y m e s applied  in conjunction  with  mild  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 t o i n c r e a s e 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 f e d t o d a i r y cattle.  2.3 MATERIALS AND METHODS 2.3.1 Feed Treatments T h e experiment consisted of a completely randomized 4 x 4 x 4 x 2 x 2  multi-  factorial d e s i g n to e v a l u a t e t h e effects o f e n z y m e s a n d mild hydrothermal treatment 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 m a t t e 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 r u e d i g e s t i b 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 v e s t i g a t i o n w e r e 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 (Trichoderma  sp) 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 t 0 % o r 0 . 0 0 2 5 % , x y l a n a s e (Trichoderma  sp.) a t 0 % o r  0 . 0 0 1 % , the addition of moisture at 0 % , 1 0 % , 2 0 % or 3 0 % and the application of heat at 5 5 ° C for 0 , 1 5 , 3 0 o r 6 0 m i n periods (Table 2.1). T r e a t m e n t levels w e r e  51  p r i m a r 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 h e e n z y m e s u p p l i e r , F i n n F e e d s Intl. (Wiltshire, U . K ) . E n z y m e s w e r e not pure cultures a n d s u b s t r a t e activity of the 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 Barley grain, ground through a 3 m m screen w a s obtained from Pro Form 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 time m o i s t u r e a n d / or e n z y m e s w e r e a d d e d during mixing a s required for e a c h t r e a t m e n t . M o i s t u r e w a s a p p l i e d w i t h a m a n u a l s p r a y e r in o r d e r t o f a c i l i t a t e e q u a l d i s t r i b u t i o n o v e r t h e e n t i r e 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 t e d in 5 m L o f d i s t i l l e d w a t e r a n d a p p l i e d (% v o l / w t ) v i a s y r i n g e t o 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 f o r a n a d d i t i o n a l 5 m i n 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 within  a  4  L  glass vessel and  s e a l e d for  further  processing  in  a  simulated  hydrothermal cooker. 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 fitting r o l l e r s 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 h e t r e a t e d  material  w e r e p l a c e d ( F i g u r e 2 . 1 ) . T h e r o t a t i o n o f t h e v e s s e l s w a s s e t at 1 5 r p m t o s i m u l a t e commercial  Inc.,  personal  communications). T h e incubator a n d v e s s e l s w e r e both preheated a n d  maintained  at 5 7 ° C  hydrothermal  ± 2°C.  Feed  conditions.  s a m p l e s treated  (Pro  Form  Feeds  with 2 0 % or 3 0 %  52  moisture  levels  took  a p p r o x i m a t e l y 7 m i n t o a t t a i n 5 5 ° C , w h e r e a s all 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  min. F o l l o w i n g r e m o v a l of the s a m p l e s from the c o o k e r , individual treatments w e r e s p r e a d o u 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 f o r 3 h. Drying w a s facilitated with a large fan w h i c h s i m u l a t e d the f o r c e d air 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 f a c i l i t i e s . S a m p l e s w e r e t h e n s e a l e d in p l a s 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 f o r s u b s e q u e n t l a b o r a t o r y a n a l y s e s .  2.3.3 Chemical Analyses A representative s a m p l e from e a c h feed treatment w a s collected and ground t h r o u g h a 1 m m s c r e e n f o r I V D M D a n d I V T D u s i n g t h e 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 . , Fairport, 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 fiber ( A D F ) a n d n e u t r a l d e t e r g e n t f i b e r ( N D F ) w e r e d e t e r m i n e d o n all 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 b e r a n a l y z e r ( # F 2 0 0 , A n k o m C o . , F a i r p o r t , N Y ) . F i l t e 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 filter material with a p o r e s i z e of 4 0 m i c r o n s (#F57, A n k o m C o . , Fairport, 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 the u s e of a heat stable a l p h a - a m y l a s e ( # F A A , A n k o m C o . , F a i r p o r t , N Y ) a s s u g g e s t e d b y V a n S o e s t et a l . , ( 1 9 9 1 ) f o r u s e in f e e d s c o n t a i n i n g high a m o u n t s of s t a r c h . R e s i d u a l dry matter ( D M ) determinations w e r e c o n d u c t e d by d r y i n g 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 until c o n s t a n t weight w a s achieved.  53  2.3.4 Animals and Feeding T w o H o l s t e i n c o w s fitted w i t h 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 fluid. T h e c o w s w e r e fed a 6 0 % g r a s s h a y a n d 4 0 % grain diet ( A p p e n d i x 6 . 2 ) t w i c e d a 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 e e s t a l l s f o r t h e duration of the 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 for a c c o r d i n g to the g u i d e l i n e s set; forth b y t h e C a n a d i a n C o u n c i l of A n i m a l C a r e (1993).  2.3.5 Rumen Fluid Inoculum Rumen  fluid  was  collected 3  h post feeding  from  both  animals  and  the  c o l l e c t i o n s p o o l e d . R u m e n fluid a n d s o l i d s f r o m t h e 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 airtight c o n t a i n e r for t r a n s p o r t to t h e l a b o r a t o r y w h i l e  maintaining  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 l u i d 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 f o r 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 filtered t h r o u g h  5-8 m m  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 l o t h a n d t h e filtrate u s e d a s i n o c u l u m m i x e d with mineral a n d buffer solutions ( G o e r i n g a n d V a n S o e s t , 1970).  54  of  when  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 t o t h e first s t e p o f t h e T i l l e y a n d  Terry  ( 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 . , Fairport, N Y ) w a s u s e d to d e t e r m i n e I V D M D of the s a m p l e s ( A n k o m C o . , F a i r p o r t , N Y ; T i l l e y 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  39.5°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 r o t a t e d at 1 r p m t o s i m u l a t e r u m e n conditions. 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 o u t into filter b a g s ( # F 5 6 , A n k o m C o . , Fairport, 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 t o e n z y m e t r e a t m e n t , w e r e p l a c e d in e a c h r u n . 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 i s t i l l e d H 0 a n d d r i e d at 6 0 ° C until c o n s t a n t 2  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 b y 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 t o N D F a n a l y s i s in o r d e r t o o b t a i n t r u e d i g e s t i b i l i t y by difference from the original s a m p l e weight (Goering a n d V a n S o e s t , 1970).  2.3.7 Statistical Analysis Statistical a n a l y s e s w e r e c o n d u c t e d using the G e n e r a l Linear M o d e l ( G L M ) procedure  of  Statistical  Analysis System  ( S A S Institute  Inc.,  1990)  using  p r o g r a m l i s t e d 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 h e m o d e l s t a t e m e n t . present  study,  it w a s  a s s u m e d the  mean  square  error  of  the  highest  the  In t h e order  interaction w a s representative of the e x p e r i m e n t a l error ( C o c h r a n a n d C o x , 1950).  55  M e a n s e p a r a t i o n s w e r e obtained using the least s q u a r e m e a n s for I V D M D , I V T D , A D F and N D F measurements.  2.4 RESULTS AND DISCUSSION: Dry matter ( D M ) content of the untreated barley grain s a m p l e w a s 8 7 . 3 % and the level of c r u d e protein ( C P ) w a s 1 2 . 7 % of D M . A c i d detergent fiber ( A D F ) w a s 7 . 3 2 % of D M a n d neutral detergent fiber ( N D F ) w a s 2 2 . 8 % a s a percent of D M . T h e b a r l e y u s e d in t h i s e x p e r i m e n t h a d s i m i l a r n u t r i e n t c o m p o s i t i o n , w i t h t h e e x c e p t i o n o f N D F , to t h e v a l u e s cited by N R C ( 1 9 8 9 ) for b a r l e y g r a i n w h i c h h a s a typical D M content of 8 8 % , C P content of 1 3 . 5 % , A D F a n d N D F content of 7 % a n d  19%,  respectively.  2.4.1 IVDMD T h e r e w a s a significant t h r e e w a y interaction ( P < 0.05) n o t e d for duration of heat  treatment,  moisture  and  xylanase  application  (Figure  2.2).  However,  no  t r e a t m e n t s i g n i f i c a n t l y i n c r e a s e d I V D M D ( P < 0 . 0 5 ) w h e n c o m p a r e d to t h e u n t r e a t e d s a m p l e (Table 2.2). All treatment m e a n s w e r e either similar or lower than the I V D M D v a l u e of 8 1 . 8 % for t h e control. A p r e v i o u s s t u d y reported a c o m p a r a b l e D M l o s s for b r o k e n barley following a 4 8 h ruminal nylon b a g incubation of 8 5 % (Nordin  and  C a m p l i n g , 1 9 7 6 ) . In t h e p r e s e n t s t u d y , t h e 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 t h t h e 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 .  56  F r o m T a b l e 2 . 2 , it is a p p a r e n t t h e m a j o r i t y o f t r e a t m e n t  combinations decreased  I V D M D r e l a t i v e t o 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 n o t s u p p o r t  the  s u g g e s t i o n t h a t m o i s t u r e m a y p l a y a r o l e in d e t e r m i n i n g t h e e f f i c a c y o f 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 r e s u 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 a r e difficult t o interpret d u e to the large n u m b e r of interactions p r e s e n t . F i g u r e 2.3 illustrates the significant interaction ( P = 0 . 0 0 0 1 ) noted for  the  addition of 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 addition of x y l a n a s e d e c r e a s e d I V D M D with the e x c e p t i o n of the c o m b i n a t i o n of both 0 . 0 0 2 5 % p r o t e a s e a n d 0 . 0 0 1 % xylanase  where  IVDMD  increased from  82.4%  to  82.9%.  The  addition  of  p-  g l u c a n a s e a l o n e did not significantly ( P < 0.05) affect I V D M D results. T h e addition 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 t h p r o t e a s e s i g n i f i c a n t l y i n c r e a s e d I V D M D r e l a t i v e to the u n t r e a t e d s a m p l e ( P < 0.05). T h e addition 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 level of 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 , the addition of p r o t e a s e did not further i n c r e a s e I V D M D ( P < 0.05). T h e addition of p r o t e a s e a l o n e a l s o did not h a v e a positive effect on I V D M D . The  addition  of  protease  has  been  previously  shown  to  increase  the  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 o w i n g 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 . , 1 9 9 3 ) . T h e s e results led the a u t h o r s to s p e c u l a t e that structural c a r b o h y d r a t e s m a y a l s o p l a y a r o l e in t h e l i m i t e d 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 within a protein  matrix  ( 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 h e u s e o f  57  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 n d i n g c e r e a l g r a i n s e x p o s e d t h e e n d o s p e r m to e n z y m a t i c attack, the starch g r a n u l e s r e m a i n e d e m b e d d e d within the protein The  difference  in t h e  degradability  of  cereals may  be  partly  matrix.  attributed to  the  p r e s e n c e o f a p r o t e i n m a t r i x w h i c h s u r r o u n d s t h e 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 r u c t u r e a n d c o m p o s i t i o n , in a d d i t i o n t o it's i n t e r a c t i o n protein,  is i m p o r t a n t  in d e t e r m i n i n g  it's d i g e s t i b i l i t y .  The  efficiency  of  with  microbial  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 a c t o r in d e t e r m i n i n g t h e r a t e a n d e x t e n t of  degradation  in  the  rumen  (McAllister,  1990b).  Enhancing  the  microbial  colonization of f e e d particles h a s b e e n s u g g e s t e d a s a m e a n s of improving  fiber  d i g e s t i b 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 , t h i s w a s n o t m e a s u r e d in t h e p r e s e n t e x p e r i m e n t . W a l d o (1973) reported that barley starch w a s 9 4 % ruminally d e g r a d a b l e . This v a l u e is h i g h e r t h a n t h a t 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 t h a t barley starch w a s 9 1 % rumen degradable. T h e s e s a m e authors determined barley grain w a s 7 8 % ruminally degradable, a s s u m i n g a 6 % h  1  that  rate of p a s s a g e , .  T h i s l a t t e r v a l u e i s c o m p a r a b l e t o 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 h e  present  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 t o b e intermediately r a n k e d with r e g a r d s to starch a n d D M degradabilities 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 milo; digestibility  experiments  o f t e n d o n o t r e c o g n i z e t h e p o o r d i g e s t i b i l i t y o f t h e h u l l f r a c t i o n ( 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 % of t h e b a r l e y hull d i s a p p e a r e d f o l l o w i n g 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 e t a l . , 1 9 9 1 ) . In f e e d i n g a h i g h p r o d u c i n g d a i r y c o w  58  it is i m p o r t a n t t o c o n s i d e r t h e f a s t e r r a t e o f r u m e n p a s s a g e w h i c h m a y  negatively  influence digestibility. A s p r e v i o u s l y m e n t i o n e d , r e s u l t s f r o m t h e t h r e e w a y i n t e r a c t i o n s a r e difficult to interpret. A l t h o u g h s i g n i f i c a n t l y l o w e r ( P < 0 . 0 5 ) I V D M D v a l u e s w e r e o b t a i n e d with a v a 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 pattern 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 t h 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 % xylanase  which  resulted  compared  to  the  significant  d e c r e a s e of  in  control,  a  6.5  digestibility  p-glucanase  5.0  digestibility  unit  decrease  applied  at  units  I V D M D from  in  0.01%  in  alone  IVDMD  when  resulted  8 2 . 4 % to  in  a  77.4%;  w h e r e a s the l o w e s t a n d h i g h e s t level of a p p l i c a t i o n of p - g l u c a n a s e did not  affect  I V D M D ( P < 0.05) (Table 2.3). Although the  results of this s t u d y a r e not a s definitive  a s the  results  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 yet 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 r a i n w h e r e t h e r e should  be  a  r e s p o n s e in t e r m s  of digestibility.  Past  research has  shown  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 e v e l i s 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 e d 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 t h 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 r a t i o n , 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 ; cited by B e a u c h e m i n a n d  Rode,  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 r o l l e d b a r l e y g r a i n d i e t s u p p l e m e n t e d w i t h 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  59  d a t a ; c i t e d 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 r u e in t h e 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 . When investigated supplemented  Krause  et a l . , (1996)  t h e in  situ  barley  (cited  degradability  straw  or  of  silage  by Beauchemin barley  diet  in  and Rode, 1996)  steers  the authors  fed an  reported  enzyme  increased  degradability of barley grain only with the silage b a s e d diets. T h i s led to speculation t h a t 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 r o l e in i m p r o v i n g t h e o b s e r v e d d i g e s t i b i l i t y in t h e s i l a g e d i e t s ( B e a u c h e m i n e t 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 play a role in the current study w h e r e results are inconsistent a n d trends cannot b e established. A combination of a number of factors m a y provide a n environment c o n d u c i v e o r detrimental to e n z y m e action.  2.4.2 IVTD A quadratic relationship ( P < 0.01) w a s found  between the application of  moisture a n d t h e IVTD of barley grain described by t h e following equation: Y = 8 7 . 7 5 + 0.071 *moisture level - 0 . 0 0 1 3 6 * m o i s t u r e l e v e l  2  (Figure 2.4). M e a n s from  treatment with 2 0 % a n d 3 0 % moisture w e r e significantly higher ( P < 0.01) than the control but w e r e not different from e a c h other (Table 2.4). T h e optimal moisture  application  in o r d e r  equation w a s determined  to m a x i m i z e  IVTD,  derived  to b e 2 6 % with a resulting  from  level of  the regression  IVTD of 88.7%; a small  i n c r e a s e o f 0 . 9 % relative to t h e control. Moisture treatment o f c e r e a l grains h a s g e n e r a l l y n o t 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 h e a b s e n c e o f h e a t ( T a i t  60  and  Beames,  1988).  However, soaking cereal has  been  s u g g e s t e d to  release  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 within the grain ( N e w m a n , 1994). A s previously 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 affect o n e n z y m e activity ( B e a u c h e m i n a n d R o d e , 1996). T h e r e w a s a significant three w a y interaction (P = 0.0001) noted a m o n g the addition of the 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 (Figure 2.5). T h e s e results a r e similar to t h o s e  obtained  with  I V D M D determinations.  N o n e of  the  e n z y m e c o m b i n a t i o n s significantly i n c r e a s e d (P < 0.05) the I V T D of barley grain 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 for t h e control ( T a b l e 2.3). I V T D did not reflect a p o s i t i v e r e s p o n s e of b a r l e y g r a i n to t h e a d d i t i o n of e i t h e 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 of barley grain with x y l a n a s e either d e c r e a s e d or h a d no effect o n I V T D relative 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  barley  grain w a s treated with the c o m b i n a t i o n of 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 e s u l t s p r e v i o u s l y o b s e r v e d with t h e a d d i t i o n o f x y l a n a s e to b a r l e y w e r e m a i n l y a t t r i b u t a b l e t o it's h i g h f i b e 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 ) . Hristov et a l . ( u n p u b l i s h e d  data; cited  by  Beauchemin and  Rode,  1996)  c o m p a r e d t h e a p p l i c a t i o n of cellulolytic 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 and  found  xylanase  that  reducing  is a n e f f e c t i v e  sugars were e n z y m e for  only  released  u s e with  barley.  in  barley.  Further  This  suggests  investigation  into  w h e t h e r fibrolytic e n z y m e t r e a t m e n t of a barley or c o r n b a s e d ration i n c r e a s e d the digestible e n e r g y content of the f e e d w a s c o n d u c t e d by B e a u c h e m i n a n d  61  Rode  (1996).  T h e s e authors  found  that a g a i n only  barley  r e s p o n d e d to  the  enzyme  s u p p l e m e n t a t i o n . T h e application of high levels of 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 ratio ( F C R ) o f s t e e r s f e d a h i g h b a r l e y d i e t . 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 result o f i m p r o v e d total tract d i g e s t i b 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 i b u t e d t o its l o w e r f i b e r content. 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 t o e n z y m e t r e a t m e n t t h a n t h e I V D M D v a l u e s . T h i s is r e f l e c t e d 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 a d d i t i o n to t h e 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 r o r in t h e I V T D r e s u l t s . T h e p o o l e d s t a n d a r d error ( S E p ) for the I V D M D m e a n s for the interaction  between  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 the S E p for true digestibility w a s 0 . 2 6 . T h e s a m e t h i r d o r d e r i n t e r a c t i o n 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 t h e evaluation  of  IVTD,  however,  the  spread  of  means  was  reduced  from  8.3  p e r c e n t a g e units with I V D M D a n d a reduction of 3.0 p e r c e n t a g e units with I V T D . The  large  spread  contamination digestion  in v a l u e s o b t a i n e d  of the  with  IVDMD  results  may  reflect  microbial  s a m p l e s . D i f f e r e n c e s t h a t a r e p r e s e n t at t h i s s t a g e o f  process may  not  b e detected with a s u b s e q u e n t  IVTD  the  determination  b e c a u s e t h i s l a t t e r p r o c e d u r e e l i m i n a t e s all c e l l s o l u b l e s i n c l u d i n g all  undigested  microbial cell c o m p o n e n t s ( V a n S o e s t , 1982). T h e results of the present e x p e r i m e n t s u g g e s t that I V T D m a y be a  better  indication of the effect of e n z y m e action a s I V D M D results m a y be m a s k e d by other compounding  factors.  Cheng  et  al., (1995)  62  indicated  that  dietary  p-glucanase  s u p p l e m e n t a t i o n w o u l d benefit d i g e s t i o n b y d e g r a d i n g the cell w a l l c o m p o n e n t s of b a r l e y . P r e v i o u s r e s e a r c h h a s s h o w n t h a t t h e 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 b y pg l u c a n a s e h o w e v e r , d u e t o 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 effective ( C h e s s o n , 1993). 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 offered for  IVDMD  a n d I V T D v a l u e s f o 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 n o t a b l y t h o s e in combination with 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 g n i f i c a n t i n t e r a c t i o n ( P < 0 . 0 5 ) s h o w n in F i g u r e 2 . 6 , n o t e d f o r 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 exist with I V D M D a n d I V T D determinations. T a b l e 2.5 s h o w s that n o n e of the treatments significantly d e c r e a s e d t h e A D F fraction of b a r l e y ( P < 0.05). T h e A D F c o n t e n t of the c o n t r o l s a m p l e w a s 8 . 2 % o n a D M b a s i s . A s i g n i f i c a n t i n c r e a s e ( P < 0 . 0 5 ) in A D F w a s f o u n d with a n u m b e r of treatments. T h e application of x y l a n a s e either had no e f f e c 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 r e l a t i v e t o t h e c o n t r o l . 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 (to 9 . 3 p e r c e n t a g e u n i t s ) w a s o b t a i n e d w i t h t h e a p p l i c a t i o n of 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 the results of a l o w I V D M D v a l u e o b s e r v e d for the s a m e treatment. M a n y of the treatments with high 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 shift 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 h e f e e d s u b j e c t e d to p a r t i c u l a r t r e a t m e n t s , w o u l d r e s u 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 h e p r o p o r t i o n 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 r e f l e c t e d  63  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 g h m o l e c u l a r w e i g h t m a t e r i a l s u s u a l l y r e s u l t s in a n i n c r e a s e in t h e s o l u b 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 , 1 9 7 5 ) . A  significant  interaction  on A D F content  (P < 0.05) w a s also noted  for:  d u r a t i o n 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 i n t e r e s t t h a t t h i s t e n d e d t o 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 h e control ( T a b l e 2.6). T h e untreated s a m p l e w a s not significantly different from  the  s a m p l e with the l o w e s t A D F content ( 8 . 1 % of D M ) o b t a i n e d by treating barley with h e a t f o 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 content w a s found w h e n barley w a s treated with heat for 15 min, 0 . 1 % p - g l u c a n a s e and 0.0025% protease. It is o f p a r t i c u l a r i n t e r e s t t o n o t e t h a t t h e i n t e r a c t i o n b e t w e e n t h e 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 h e A D F v a l u e s i n c r e a s e d a s d i g e s t i b 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 w i t h t h e n e g a t i v e a s s o c i a t i o n o f f i b e r w i t h d i g e s t i b 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 affect t h e A D F fraction but m a y elicit their effects by a l t e r i n g t h e h e m i c e l l u l o s e f r a c t i o n , w h i c h w o u l d b e r e f l e c t e d in t h e 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 g n i f i c a n t i n t e r a c t i o n w a s f o u n d t o e x i s t ( P < 0 . 0 1 ) b e t w e e n h e e n z y m e s pg 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 this interaction, N D F c o n t e n t of the control w a s 2 1 . 8 % of D M , w h i c h w a s the  l o w e s t o f all t r e a t m e n t  means. The  following three treatment c o m b i n a t i o n s significantly i n c r e a s e d ( P < 0.05) N D F (Table  64  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 % pglucanase. A n o t h e r s i g n i f i c a n t s e c o n d o r d e r i n t e r a c t i o n ( P < 0 . 0 1 ) w a s f o u n d b e t w e e n pg 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 addition of 0 . 0 0 1 % P - g l u c a n a s e significantly i n c r e a s e d (P < 0.05) N D F from 2 3 . 0 % t o 2 4 . 5 % a s a p e r c e n t a g e o f D M . In c o n t r a s t , t h e a p p l i c a t i o n o f t h e h i g h e s t c o n c e n t r a t i o n of p - g l u c a n a s e significantly d e c r e a s e d ( P < 0 . 0 5 ) N D F f r o m 2 3 . 0 % to 2 1 . 8 % . T h e s e r e s u l t s d o n o t p a r a 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 t h a t the addition of both the l o w e s t a n d h i g h e s t l e v e l s of p - g l u c a n a s e did not significantly affect  IVDMD  (P  <  0.05).  The  observed  decrease  in  NDF  content  with  the  application of 0 . 1 % p - g l u c a n a s e m a y be e x p l a i n e d by the b r e a k d o w n of cell wall c o m p o n e n t s w h i c h m a y c o n t r i b u t e t o t h e N D F f r a c t i o n . It a p p e a r s t h a t t h e a d d i t i o n o f P - g l u c a n a s e m a y h a v e h a d a n affect o n t h e h e m i c e l l u l o s i c fraction of b a r l e y a n d not the A D F fraction a s indicated by the interactions investigated. T h e r e w a s a l s o a significant interaction (P = 0.01) found b e t w e e n protease and xylanase which determined  that  the  is i l l u s t r a t e d treatment  in F i g u r e 2 . 1 0 . W i t h i n of  barley  with  0.001%  this  interaction,  xylanase  it  was  significantly  d e c r e a s e d ( P < 0.05) t h e N D F c o n t e n t f r o m 2 3 . 1 % to 2 1 . 9 % of the D M content of barley, w h e r e a s all 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 control ( T a b l e 2.9).  T h i s s p e c i f i c r e s u l t w a s r e f l e c t e d in a s l i g h t i n c r e a s e in I V T D b u t n o t w i t h a h i g h e r IVDMD value.  65  R e s u l t s f r o m f i b e r a n a l y s e s a r e difficult t o i n t e r p r e t , h o w e v e r , t h e y r e f l e c t t h e lack of r e s p o n s e o b t a i n e d with I V D M D a n d I V T D determinations. T h e variation  in  N D F w a s m u c h greater than the variation o b s e r v e d with A D F . C o n s e q u e n t l y , fewer 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 t h a t t h e h i g h 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 t e r f e r e s w i t h t h e N D F f i l t r a t i o n p r o c e s s a n d may  contaminate  the  sample  (Van  Soest  et  al.,  1991).  The  present  study  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 h e N D F p r o c e d u r e in o r d e r t o r e d u c e t h e interference  from  starch  however,  it  is  possible  that  the  elimination  of  the  interference 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 Inconclusive results from the present study a n d inconsistencies from  other  s t u d i e s e x e m p l i f y t h e d i f f i c u l t i e s 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 h e r u m i n a n t f e e d i n d u s t r y . T h e p r e s e n t s t u d y r e v e a l e d t h a t f u r t h e r i n v e s t i g a t i o n 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 interest p u r s u i n g t h e a p p l i c a t i o n of e n z y m e s to barley. 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 f u r t h e r r e s e a r c h . In t e r m s o f d i g e s t i b i l i t y , r e s u l t s of t h e 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 of 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 of 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 the g r e a t e s t positive r e s p o n s e . F i b e r a n a l y s e s reflected similar  results  that  were  obtained  with  the  digestibility  measurements.  The  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 n o t a t t e m p t e d in t h i s s t u d y . 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Effects of e n z y m e s o n nitrogen  supplemental  b a l a n c e , digestibility of e n e r g y a n d nutrients  and  on  growth a n d f e e d efficiency of cattle. 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 . Utilization of steam-flaked  grain  s o r g h u m with a d d e d "grain  sorghum  specific"  m i x t u r e 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 ( A b s t r a c t ) . 70  enzyme  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 f e c t i n g s t a r c h d i g e s t i b i l i t y w i t h special emphasis on sorghum and corn. J . Anim. Sci., 63:1607. 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 cattle 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 (Abstract). S t a t i s t i c a 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 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 z a t i o n 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 protein. J . Dairy S c i . , 7 5 : 3 4 5 - 3 5 7 . Tait, 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 preservation of c e r e a l s a n d p r o t e i n 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 e y , 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 f o 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 . J . Brit. G r a s s l . S o c , 1 8 : 1 0 4 - 1 1 1 . V a n S o e s t , P . J . 1 9 8 2 . Nutritional Corvallis, Oregon.  e c o l o g y of the  ruminant.  O & B Books  Inc.,  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 of dietary fiber, n e u t r a l d e t e r g e n t f i b e 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 r e l a t i o n t o a n i m a l nutrition. 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 . ; Swift,  M . L.; P u c h a l a , R . a n d J . A . S h e l f o r d .  Degradability characteristics of dry matter a n d c r u d e protein  1996.  of f o r a g e s  in  ruminants. A n i m . F e e d S c i . Technol., 57:291-311. W a l d o , D . R . 1 9 7 3 . E x t e n t a n d p a r t i t i o n 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 . Anim. Sci., 37:1062-1074. Ward,  J . K.,  R i c h a r d s o n , D.  and  W . S . Tsien. 1960. Value  of  added  enzyme  p r e p a r a t i o n s in b e e f c a t t l e r a t i o n s . J . A n i m . S c i . , 1 9 : 1 2 9 8 ( A b s t r a c 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 P e 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 for  finishing  steers.  Pages  68-69  in  B e e f cattle  report,  Univ.  Nebraska-  Lincoln, Lincoln, N E , U S . W i s e m a n , A . 1 9 7 5 . E n z y m e utilization  in i n d u s t r i a l p r o c e s s e s . P a g e s  111-124  H a n d b o o k of e n z y m e biotechnology. A . W i s e m a n , E d . Ellis H o r w o o d Chichester. 71  in  Ltd.,  W i n g , J . M . a n d W i l c o x , C . J . 1 9 6 0 . Effect of s u p p l e m e n t a r y digestive e n z y m e s on growth of dairy heifers. J . Dairy S c i . , 4 3 : 1 6 5 5 - 1 6 7 5 .  72  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 w i t h 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 d i g e s t i b i l i t y o f b a r l e y g r a i n .  Factor  Level of application  Heat  c o n t r o l (0), 1 5 , 3 0 , 6 0 m i n  1  Moisture  0%, 10%, 20%, 3 0 %  2  P-glucanase  3 , 4  0%, 0.001%, 0.01%, 0.1%  Protease  3 , 5  0%, 0.0025%  Xylanase  3 , 6  0%, 0.001%  1  2  Applied at 55°C. Applied o n a % vol / wt basis.  3  S u p p l i e d b y F i n n F e e d s International Ltd., Wiltshire, U.K.  4  Avizyme 1110  5  Heptex sb-protease  6  Avizyme 1310  73  T a b l e 2.2. T h e effect of the duration of heat treatment a n d the application of 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 basis). Feed Treatment Duration of heat treatment (min)  Moisture  2  (%)  Xylanase  2  (%)  IVDMD  3  1  0 " 0 0 0 0 10 0 10 0 20 0 20 0 30 0 30 15 0 15 0 15 10 15 10 15 20 15 20 15 30 15 30 30 0 30 0 30 10 30 10 30 20 30 20 30 30 30 30 60 0 60 0 60 10 60 10 60 20 60 20 60 30 60 30 S.E.M. Heat was applied at 55°C. Applied on a % vol / wt basis. IVDMD, in vitro dry matter disappearance. S.E.M., pooled standard error of the mean. ' Means in a column with different superscripts are  0 0.001 0 0.001 0 0.001 0 0.001 0 0.001 0 0.001 0 0.001 0 0.001 0 0.001 0 0.001 0 0.001 0 0.001 0 0.001 0 0.001 0 0.001 0 0.001  81.8 79.3 82.2  c d e f g  a b  ef9  g-| gcdefg  82.2 80.0 82.5 80.5 82.1 79.5 8  e f g  a b  fg  a b c d  d e f g  a b  gcdefg  2  79.6 82.1 80.0 8 2 3  a b  e f g  a b  efg  80.4  a b c  79.2  a  79.7  a b  g l gcdefg g-| gcdefg g  0  gbcdef  80.3 82.2 g  0  a b c  e f g  gabcde  79.9  a b  8 0  gbcdef  8  1  g  gcdefg 0 4  abc  82.6 79.3 82.8 80.5 0.56  9  a b  9  a b c  1  2  3  4 a  9  74  significantly different (P < 0.05).  (%)  CD  O rc z o  Q  r— >  a T *  E .0  "8 ro"8 "8 TO <u  CD CM O  O  CT> CT> 1 ^ oi 00 CO 00 00 OO 00 00  CD  0 CO  TO J3  .0 CM  0  CD "D  E  X—  O  co  0 0 co C O 00 00 00  00 CO  a)  CD CO  OO  03  co 0 0 00  CD 00  CD CN O  LO  O  V  a. a>  Q >  CO  CO  00  00  TO  c  •0 LO  CD  I  o c o c fc l o co co n s c o e d >«t c d so r--co CD  Ti-  CN  CD  L O  00  •4—'  C CO  o 4= 'c cp CD CO CD  c  _C0  CO CD 1— CO  0 0 0 0 p 0 0 0 0 p o o o o p o 000000000000000  CO -*-» Q .  • i—  c o ca co  CD O CD  E  CD  E N C LU  g E a.  c ca ca  c  CD  CD  CD Q . Q .  CD  co CO CD  2 IQ-  CO  LO CN  LO CN  O O  LO CN  LO CN  O O  O O  LO CN  LO CN  LO CN  O O  O O  O O  O O  O O O  O  O O  O O  O O O O  'co C0  L—  CD  O O  O  O  ca co  5  CD  CD  ^  + = -n -c to C .Ii  .CO "D  o  D CO  ^  -a c c  > T3  CD  -2 §  -fc T3 O  CO  CD CO CO  C  S  C  o •== . £ •a r f  c CO  o  ll  s 5O  O p  O O  p  O p  O p  p  T ~ T - T ~  p  -  r  -  ;  T  -  T  -  T  -  000000000000000 75  f  Q  2  <>  ® ° O TO  8.-E  . co  ^ CD  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 vitro t r u e d i g e s t i b i l i t y o f b a r l e y grain ( D M basis).  M o i s t u r e (%)  I V T D (%)  0  87.8  a  10  88.3  a b  20  88.6  b  30  88.6  b  1  S.E.M.  1  2  0.14  Applied o n a % vol / wt basis.  2  I V T D , in vitro t r u e d i g e s t i b i l i t y .  3  S . E . M . , pooled standard error of the m e a n .  a b  M e a n s in a c o l u m n w i t h d i f f e r e n t s u p e r s c r i p t s a r e s i g n i f i c a n t l y d i f f e r e n t  76  (P < 0.05).  T a b l e 2.5. T h e effect of 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 application o n t h e A D F fraction of barley grain ( D M basis).  E n z y m e treatment  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 (%)  ADF (%)  0  0  0  8.2  a  0  0  0.001  8.4  a b  0  0.0025  0  8.4  a b  0  0.0025  0.001  9.1  2  d e  0.001  0  0  g abc  0.001  0  0.001  8.2  a  0.001  0.0025 0.0025  0 0.001  8.4  a b  0  0  9.3  e  0.01  0  0.001  9.1  d e  0.01  0.0025  0  8.6  a b c  0.01  0.0025  0.001  8.5  a b  0.1  0  0  8.3  a  0.1  0  g gbcd  0.1  0.0025  0.001 0  9.1  d e  0.0025  0.001  9.0  0.001 0.01  0.1 S.E.M.  9.0°*°  d e  0.15  3  1  Applied o n a % vol / wt basis.  2  A D F , a c i d d e t e r g e n t fiber.  3  S . E . M . , p o o l e d standard error of t h e m e a n .  a _ e  5  M e a n s in a c o l u m n w i t h d i f f e r e n t s u p e r s c r i p t s a r e s i g n i f i c a n t l y d i f f e r e n t  77  (P < 0.05).  T a b l e 2.6. T h e effect of heat treatment a n d the application of p - g l u c a n a s e a n d x y l a n a s e o n the A D F fraction of barley grain ( D M b a s i s ) .  E n z y m e treatment Duration of heat t r e a t m e n t (min)  p-Glucanase  0 0 0 0 0 0 0 0 15 15 15 15 15 15 15 15 30 30 30 30 30 30 30 30 60 60 60 60 60 60 60 60 S.E.M.  0 0 0.001 0.001 0.01 0.01 0.1 0.1 0 0 0.001 0.001 0.01 0.01 0.1 0.1 0 0 0.001 0.001 0.01 0.01 0.1 0.1 0 0 0.001 0.001 0.01 0.01 0.1 0.1  (%)  1  P r o t e a s e (%)  ADF (%)  0 0.0025 0 0.0025 0 0.0025 0 0.0025 0 0.0025 0 0.0025 0 0.0025 0 0.0025 0 0.0025 0 0.0025 0 0.0025 0 0.0025 0 0.0025 0 0.0025 0 0.0025 0 0.0025  g gabcd  3  2  g ybcdefghij  g -| abc g abc 2  g gefghijk g ^abcdef g ycdefghij g yabcdefghij g gabcd g gabcdefghi g ^abcdefgh g gabcdefgh  9.2  jkl  g ycdefghij  8.1 9.6'  a b  g gabcde g gdefghijk  8.0 9.1 9.4  a  jkl  kl  g gabcd g gdefghijk  9.1  ijkl  g gabcd g_ hijkl 0  g gdefghijk g fj9 i hi  9.2  kl  ikl  g gcdefghij g ^abcdefg g o 9 'i f  h  kl  0.21  4  A p p l i e d o n a % vol / wt b a s i s . . H e a t w a s a p p l i e d at 5 5 ° C . A D F , a c i d d e t e r g e n t fiber. S . E . M . , p o o l e d s t a n d a r d error of the m e a n . "' M e a n s in a c o l u m n with different s u p e r s c r i p t s a r e significantly different  1  2  3  4  a  78  (P < 0.05).  T a b l e 2.7. T h e effect of p - g l u c a n a s e a n d p r o t e a s e application o n the N D F content of barley grain ( D M basis).  Enzyme  treatment  1  P - G l u c a n a s e (%)  P r o t e a s e (%)  NDF (%)  0  0  21.8  a  0  0.0025  23.2  b c  0.001  0  22.8  a b c  0.001  0.0025  23.7  C  0.01  0  23.4  C  0.01  0.0025  22.1  a b  0.1  0  22.0  a  0.1  0.0025  22.1  a  S.E.M.  1  A p p l i e d o n a % vol / wt b a s i s . N D F , neutral d e t e r g e n t fiber.  3  a  0.37  3  2  2  S . E . M . , p o o l e d s t a n d a r d error of the m e a n . "° M e a n s in a c o l u m n w i t h d i f f e r e n t s u p e r s c r i p t s a r e s i g n i f i c a n t l y d i f f e r e n t  79  (P < 0.05).  T a b l e 2 . 8 . T h e effect of 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 fraction of barley grain ( D M basis).  E n z y m e treatment  p-Glucanase  (%)  1  Xylanase  (%)  NDF (%) 2  0  0  23.0  b c  0  0.001  22.1  a b  0.001  0  24.5  d  0.001  0.001  22.0  a b  0.01  0  22.4  a b c  0.01  0.001  23.2°  0.1  0  21.8  0.1  0.001  22 2 '  S.E.M.  a  a  3C  0.37  3  1  A p p l i e d o n a % vol / wt b a s i s .  2  N D F , neutral d e t e r g e n t fiber.  3  S . E . M . , p o o l e d s t a n d a r d error of the m e a n .  ~° M e a n s in a c o l u m n w i t h d i f f e r e n t s u p e r s c r i p t s a r e s i g n i f i c a n t l y d i f f e r e n t  a  80  (P < 0.05).  T a b l e 2.9. T h e effect of p r o t e a s e a n d x y l a n a s e application o n the N D F content of barley grain ( D M basis).  Enzyme  Protease  (%)  treatment  1  Xylanase  (%)  NDF (%) 2  0  0  23.1  b  0  0.001  21.9  a  0.0025  0  22.7  b  0.0025  0.001  22.9  b  S.E.M.  1  0.26  3  A p p l i e d o n a % vol / wt b a s i s .  2  N D F , neutral d e t e r g e n t fiber.  3  S . E . M . , p o o l e d standard error of the m e a n .  a b  M e a n s in a c o l u m n w i t h d i f f e r e n t s u p e r s c r i p t s a r e s i g n i f i c a n t l y d i f f e r e n t  81  (P < 0.05).  Figure 2.1. Laboratory scale hydrothermal cooker.  82  84  - • - 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  20  M o i s t u r e l e v e l (% v o l / w t )  F i g u r e 2 . 2 . T h e effect of the addition of moisture, x y l a n a s e (X) (% v o l / w t ) a n d d u r a t i o n o f t h e h e a t t r e a t m e n t ( 5 5 ° C ) o n t h e 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 .  83  30  F i g u r e 2.3. T h e effect of 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 (% v o l / wt) o n t h e in vitro d r y m a t t e r d i s a p p e a r a n c e of barley grain.  84  0  10  20  30  M o i s t u r e l e v e l (% v o l / wt)  Figure 2.4. Q u a d r a t i c relationship b e t w e e n level of moisture a n d in vitro t r u e d i g e s t i b i l i t y o f b a r l e y g r a i n ( Y = 8 7 . 7 5 + 0 . 0 7 1 * moisture level - 0.0014 * moisture l e v e l : n = 4, R 2  = 0.018).  85  2  = 0.998, S . E .  F i g u r e 2.5. T h e effect of 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 / w t ) o n t h e in vitro t r u e digestibility of barley grain.  86  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 e v e l (% v o l / w t )  F i g u r e 2.6. T h e effect of b e t a - g l u c a n a s e ,  protease (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 / w 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 b e r in b a r l e y g r a i n .  87  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 . 0 0 2 5 % P, 15 heat H—0.0025% P, 30 heat — 0.0025% P, 60 heat —I 0  0.001  0.01  0.1  B e t a - g l u c a n a s e l e v e l (% v o l / w t )  F i g u r e 2 . 7 . T h e effect of 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 (% v o l / w t ) a n d d u r a t i o n o f t h e 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 b e r in b a r l e y g r a i n .  88  (55°C)  0% Protease 0.0025% Protease  0  0.02  0.04  0.06  0.08  0.1  B e t a - g l u c a n a s e l e v e l (% v o l / wt)  F i g u r e 2.8. T h e effect of the addition of b e t a - g l u c a n a s e a n d protease  (% v o l / w t ) o n t h e c o n t e n t o f n e u t r a l d e t e r g e n t f i b e r in  barley grain.  89  0  0.02  0.04  0.06  0.08  0.1  B e t a - g l u c a n a s e l e v e l (% v o l / w t )  F i g u r e 2.9. T h e effect of the addition of b e t a - g l u c a n a s e a n d xylanase  ( % v o l / w t ) o n t h e c o n t e n t o f n e u t r a l d e t e r g e n t f i b e r in  barley grain.  90  25 24.5 24  vP L_  CD  23.5  - Q M— -4—'  23  c  CD  E>  22.5  Q)  "CD "O  22 21.5  CD  z  21 20.5 20 0  0.0005  0.001  0.0015  0.002  0.0025  P r o t e a s e l e v e l (% v o l / w t )  F i g u r e 2 . 1 0 . T h e effect of the addition of 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 c o n t e n t o f n e u t r a l d e t e r g e n t f i b e r in b a r l e y grain.  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 h e primary alternative protein 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 i s r e s t r i c t e d in l a c t a t i n g d a i r y c a t t l e 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 a v a i l a b i l i t y p a r t l y a t t r i b u t a b l e t o it's h i g h f i b e r c o n t e n 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 h e f e a s i b 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 t h m i l d  hydrothermal  treatment in o r d e r to i m p r o v e t h e nutritional v a l u e o f c a n o l a m e a l f o r dairy cattle. 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 m u l t i - f a c t o r i a 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 r u e in vitro d i g e s t i b i l i t i e s a n d f i b e r a n a l y s e s w e r e u s e d t o e v a l u a t e t r e a t m e n t effects. T r e a t m e n t s i n c l u d e d t h e application of heat at 5 5 ° C for 1 5 , 3 0 o r 6 0 min periods o r not a p p l i e d at all; moisture applied at 0 % , 1 0 % , 2 0 % o r 3 0 % ; protease (Bacillus sp.)  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  a p p l i e d at 0 % o r 0 . 0 0 1 % . Nutrient c o m p o s i t i o n of t h e untreated c a n o l a m e a l  sample was 89.3% D M ; 36.6% C P , 3.25% E E , 17.67% A D F and 21.83% N D F on a D M basis. T h e r e w a s a significant interaction ( P = 0.0001) b e t w e e n protease a n d x y l a n a s e o n I V D M D . T h e addition of protease w a s the major factor responsible for increased I V D M D . A highly significant quadratic relationship ( P = 0.0001) between moisture  and  IVDMD  w a s described  92  by:  Y  =  78.62  +  0.165*moisture  -  0.003*moisture . I V T D w a s affected b y the significant interaction ( P < 0.01) between 2  protease  a n d xylanase, where  results  parallel  those  obtained  with  IVDMD. A  quadratic relationship ( P = 0.0001) w a s also found between moisture a n d IVTD described b y Y = 8 7 . 2 5 + 0.073*moisture - 0.002*moisture . T h e addition of 1 0 % 2  moisture w a s sufficient to significantly i n c r e a s e both I V D M D a n d I V T D ( P < 0.05). H o w e v e r , w i t h I V T D n o f u r t h e r i n c r e a s e in d i g e s t i b i l i t y w a s o b s e r v e d w i t h a f u r t h e r 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 t h i r d o r d e r i n t e r a c t i o n s w h i c h a f f e c t e d fiber content:  duration  of t h e heat treatment, a n d the addition of protease a n d  x y l a n a s e ( P < 0.05) a n d the addition of moisture, 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 significantly affected ( P < 0.05) b y the addition of 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 m i l d h e a t t r e a t m e n t u s e d in t h e p r e s e n t s t u d y d i d n o t e n h a n c e e n z y m e a c t i v i t y 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 , using t h e s e l e c t e d indices, provided results from which several c o n c l u s i o n s m a y b e drawn. T h e application of x y l a n a s e w a s not effective,  although  t h e addition of  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 t e d a p o s i t i v e r e s p o n s e . T h i s r e s p o n s e w a s s i m i l a r to that o b t a i n e d with t h e 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 indicated a possible improvement  in t h e d i g e s t i b 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 t h e  application of protease o r moisture  a l o n e o r in c o m b i n a t i o n  with other  factors.  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 r o d u c t i o n m a y reveal t h e effects of the treatments o n fermentation characteristics which w a s u n a v a i l a b l e i n t h e p r e s e n t s t u d y a n d i s c e r t a i n l y j u s t i f i e d in o r d e r t o f a c i l i t a t e t h e selection of treatments for c a n o l a m e a l .  93  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 r n a t i v e t o s o y b e a n m e a l f o r u s e a s a p r o t e i n s u p p l e m e n t in r a t i o n s f o r l a c t a t i n g d a 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 h e c o n c e n t r a t e ration d e p e n d i n g o n t h e c o s t of s o y b e a n m e a l a n d the protein  content  of the  forage  (Pro  Form  Feeds  Inc.,  personal  communication).  C a n o l a m e a l i s 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 o w i n g t h e oil e x t r a c t i o n p r o c e s s . T h e h u 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 u p t o 3 0 % b y w e i g h t a n d a r e r i c h in f i b e r a n d l i g n i n ( B e l l a n d S h i r e s , 1 9 8 2 ) . T h e h i g h f i b e r c o n t e n t o f c a n o l a m e a l is t h e  major  contributing  factor  to  it's  relatively  lower  energy  availability  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 h e  when energy  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 i n t e r e s t t o t h e f e e d a n d d a i r y i n d u s t r i e s . I n c r e a s i n g t h e nutritive v a l u e of feedstuffs for r u m i n a n t s t h r o u g h the u s e of 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 i n t e r e s t . T h i s is p r i m a r i l y a r e s u l t o f t h e recent  advances  in  monogastric  feeding  regimes  using  exogenous  enzymes  ( 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 t h e u s e o f 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 results ( 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 h e l i t e r a t u r e , it is e v i d e n t t h a t 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 h e 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 o f improving  it's d i g e s t i b i l i t y f o r r u m i n a n t s . F o r f u r t h e r d e t a i l r e g a r d i n g t h e r e s u l t s o f  e n z y m e r e s e a r c h w i t h r u m i n a n t s , t h e r e a d e r m a y b e r e f e r r e d t o 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 f o r 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 f o r r u m i n a n t s ( T i l l e y a n d T e r r y , 1 9 6 3 ; G o e r i n g  94  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 i s t h a t a l a r g e n u m b e r of s a m p l e s m a y b e evaluated at o n e time. T h e objective of the current study was  to identify  treatments  that w o u l d  i m p r o v e t h e digestibility  of canola meal,  p a r t i c u l a r l y o f t h e f i b e r c o m p o n e n t , in o r d e r t o i m p r o v e it's n u t r i t i o n a l v a l u e f o r d a i r y cattle.  3 . 3 MATERIALS AND METHODS 3.3.1 Feed Treatments T h e experiment consisted of a completely randomized 4 x 4 x 4 x 2  multi-  factorial d e s i g n to e v a l u a t e the effects of e n z y m e s a n d hydrothermal treatment 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 m a t t e 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 r u e d i g e s t i b 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 n v e s t i g a t i o n 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 a t 0 % , 0 . 0 0 5 % , 0 . 0 1 % o r 0 . 0 5 % (Bacillus s p . ) ; x y l a n a s e a t 0 % o r 0 . 0 0 1 % (Trichoderma  sp.); m o i s t u r e a t 0 % , 1 0 % , 2 0 % o r 3 0 %  a n d heat a p p l i e d at 5 5 ° C for 0 , 1 5 , 3 0 o r 6 0 m i n p e r i o d s (Table 3.1). E n z y m e s w e r e n o t p u r e c u l t u r e s a n d s u b s t r a t e a c t i v i t y o f t h e 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 meal, ground through a 3 m m screen, w a s obtained from P r o Form F e e d s I n c . 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 t o t h e p r o c e d u r e o u t l i n e d in Chapter 2, section 2.3.2.  95  3.3.3 Chemical Analyses Chemical  analyses  were  conducted  on  the  samples  according  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 l u i d 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  outlined  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 IVTD a n a l y s e s w e r e determined on the feed s a m p l e s according to t h e 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 . 6 .  3.3.7 Statistical Analysis Statistical a n a l y s e s w e r e c o n d u c t e d using the G e n e r a l Linear M o d e l ( G L M ) p r o c e d u r e o f S t a t i s t i c a l A n a l y s i s S y s t e m ( S A S Institute Inc., 1 9 9 0 ) u s i n g t h e p r o g r a m l i s t e d 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 h e m o d e l s t a t e m e n t . In t h e  96  p r e s e n t s t u d y , it w a s a s s u m e d t h a t t h e h i g h e s t o r d e r i n t e r a c t i o n d i d n o t e x i s t , but it's m e a n s q u a r e error w a s representative of the e x p e r i m e n t a l error ( C o c h r a n a n d C o x , 1950). Statistically significant differences between 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 the u s e of 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 . Further investigation of the relationship b e t w e e n a significant m a i n effect a n d a m e a s u r e d p a r a m e t e r with orthogonal 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 the C O N T R A S T statement of S A S P R O C G L M ( S A S , 1990) a n d reported w h e n the relationship could be defined.  3.4 RESULTS AND DISCUSSION Dry matter ( D M ) content of the untreated 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 r o t e i n ( 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 % , both p r e s e n t e d a s a portion of D M . A c i d detergent fiber ( A D F ) w a s 1 7 . 7 % of D M a n d the neutral detergent fiber ( N D F ) w a s 2 1 . 8 % of the D M fraction. T h e s e results are c o m p a r a b l e with the v a l u e s reported by Bell a n d Keith (1991) from a s u r v e y of 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 23.5% NDF.  97  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 significantly affected ( P = 0.0001) by the i n t e r a c t i o n 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 incubation, 7 9 . 8 % o f the untreated c a n o l a meal s a m p l e had d i s a p p e a r e d (Table 3 . 2 ) . T h i s i s n o 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 f o r in situ d r y  matter  d i s a p p e a r a n c e ( I S D M D ) o f 72.1 % for a ruminal incubation o f 2 4 h ( M o s h t a g h i a n d Ingalls, 1995). T h e control s a m p l e w a s intermediately ranked with respect to the s a m p l e s treated with 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 applied a l o n e at 0 . 0 1 % decreased I V D M D from 7 9 . 8 % to 77.8%. T h e combination of 0.005% protease and 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 applied 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 % t o 8 2 . 4 % . F r o m T a b l e 3 . 2 , it is evident that there is a synergistic effect b e t w e e n the two 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 a t 0 . 0 1 % o r 0 . 0 5 % in c o n j u n c t i o n w i t h x y l a n a s e . H o w e v e r , p r o t e a s e a t certain l e v e l s h a d a positive effect w h e n a p p l i e d with 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 the two e n z y m e s is s h o w n with the application of either 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 with 0 . 0 0 1 % x y l a n a s e (Table 3.2). T h e c o m b i n a t i o n of the two higher 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 with x y l a n a s e significantly i n c r e a s e d ( P < 0.05) the I V D M D f r o m 7 9 . 8 % to greater than 8 1 % . T h i s synergistic e f f e c t w o u l d b e e x p e c t e d t o elicit a n e f f e c t b y d e g r a d i n g t h e p r o t e i n m a t r i x a n d o t h e r r e s i s t a n t p l a n t c e l l w a l l c o m p o u n d s . T h e p r o t e i n m a t r i x in c e r e a l s h a s b e e n s h o w n t o b e r e s p o n s i b l e f o r d i c t a t i n g t h e r a t e a n d e x t e n 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 ) .  98  T h e c o m b i n a t i o n of the lowest level of p r o t e a s e with x y l a n a s e significantly r e d u c e d ( P < 0 . 0 5 ) I V D M D relative to t h e c o n t r o 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 effect 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 e v e l ( 0 . 0 1 % ) produced a significantly lower (P < 0.05) I V D M D value than the I V D M D v a l u e s o b t a i n e d with the control s a m p l e a n d the lowest a n d highest levels of p r o t e a s e application. T h e e f f e c t o f m o i s t u r e a p p l i e d in t h e a b s e n c e o f h e a t o n t h e d i g e s t i b i l i t y c h a r a c t e r i s t i c s of c e r e a l g r a i n s , with t h e e x c e p t i o n of 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 ( T a i t a n d B e a m e s , 1 9 8 8 ) . I n t e r e s t i n g l y , 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 that moisture h a d a highly significant quadratic relationship (P < 0.01) with the I V D M D of 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 * moisture level 0 . 0 0 3 * m o i s t u r e l e v e l ( F i g u r e 3 . 2 ) . F r o m t h e 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 2  d e t e r m i n e d t h a t a n o p t i m a l l e v e l o f 2 6 % m o i s t u r e w o u l d r e s u l t in a D M d e g r a d a b i l i t y of 8 1 % . E v e n t h e a p p l i c a t i o n of t h e l o w e s t level of m o i s t u r e ( 1 0 % ) w a s sufficient to elicit a s i g n i f i c a n t i n c r e a s e ( P < 0 . 0 5 ) in I V D M D f r o m 7 8 . 6 % t o 7 9 . 9 % ( T a b l e 3 . 3 ) . T h e application of 2 0 % or 3 0 % moisture both p r o d u c e d I V D M D v a l u e s of 8 0 . 7 % , w h i c h w a s significantly greater than the control a n d than that o b t a i n e d with the 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 e c t s o f m o i s t u r e a d d i t i o n m a y b e attributed to the r e l e a s e of e n d o g e n o u s e n z y m e s within the c e r e a l grain ( 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 h e o p p o r t u n i t y for t h e c e r e a l to b e partly d e g r a d e d p r i o r t o b e i n g e x p o s e d t o m i c r o b i a l a t t a c k in t h e r u m e n . A r e c e n t s t u d y reported the 12 h ruminal  in situ d r y m a t t e 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  99  from 4 8 . 9 % to 5 5 . 4 % with the addition of 1 5 % moisture ( W e u r d i n g , 1996). T h e lack o f r e s p o n s e t o t h e m i l d 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 t h i s s t u d y , m a y b e a t t r i b u t e d to t h e 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 effects or effects from hydrothermal treatment u s e d p r o c e s s i n g technologies including s t e a m rolling, s t e a m flaking, p r e s s u r e c o o k i n g , e x p l o d i n g , extruding or pelleting 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 ( T a i t a n d B e a m e s , 1 9 8 8 ) .  3.4.2 IVTD T h e results of I V T D d e t e r m i n a t i o n s w e r e similar to the results o b t a i n e d for I V D M D . T h e interaction 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 of c a n o l a m e a l w a s highly significant ( P < 0.01) (Figure 3.3). H o w e v e r , n o n e of the treatments significantly i n c r e a s e d I V T D ( P < 0.05) from the v a l u e of 8 7 . 9 % o b t a i n e d for the control ( T a b l e 3.4). T h e application of 0 . 0 1 % p r o t e a s e significantly 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 for this t r e a t m e n t reflect 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 i d n o t h a v e a n e f f e c t o n I V T D w h i c h is a l s o r e f l e c t e d in t h e r e s u 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 h e sensitivity o f t h e r e s u 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 attributed to the c o m p o n e n t s e a c h o n e estimates. T h e major difference between the IVTD and I V D M D p r o c e d u r e s is in t h e l a t t e r in vitro t e s t , all c e l l s o l u b l e s a r e e l i m i n a t e d , including undigested microbial matter (Van S o e s t , 1982). T h e r e w a s a quadratic relationship between moisture level and IVTD ( P = 0.0001) d e s c r i b e d by: Y = 8 7 . 4 5 + 0.73 * moisture level - 0.002 * moisture l e v e l  100  2  (Figure 3.4). T h e optimal level of moisture, d e t e r m i n e d from the r e g r e s s i o n equation, w a s 2 4 % with a c o r r e s p o n d i n g I V T D v a l u e of 8 8 % . T h e application of the lowest m o i s t u r e l e v e l ( 1 0 % ) w a s sufficient to significantly i n c r e a s e ( P < 0 . 0 5 ) t h e I V T D of c a n o l a m e a l f r o m 8 7 . 3 % t o 8 7 . 8 % ( T a b l e 3 . 5 ) . I n t e r e s t i n g l y , f u r t h e r i n c r e a s e s in m o i s t u r e l e v e l d i d n o t f u r t h e 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 i s in c o n t r a s t t o t h e f i n d i n g s of the effect of moisture o n I V D M D w h e r e the application of 2 0 % a n d 3 0 % moisture b o t h 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 promote the r e l e a s e of e n d o g e n o u s e n z y m e s within the grain allowing further 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 t h i s 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 b o t h I V D M D a n d I V T D to m i l d 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 t h e e n z y m e s u p p l i e r ( F F I , W i l t s h 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 a c t i v i t y u n d e r h i g h m o i s t u r e a n d optimal h e a t c o n d i t i o n s . H o w e v e r , the results of the 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 n o f u r t h e r i n c r e a s e in e n z y m e a c t i v i t y w a s a t t a i n e d w i t h t h e a p p l i c a t i o n o f 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 activity is limited by o t h e r factors w h i c h c o u l d not b e o v e r c o m e with this mild h y d r o t h e r m a l treatment. T h e s e r e s u l t s m a y a l s o b e a t t r i b u t e d t o t h e l a c k o f a n y i n c r e a s e in e n z y m e a c t i v i t y o n t h e hydrothermally treated ingredient a n d with the concentrations a n d / or combinations 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 of h e a t at 5 5 ° C for a n y of t h e s e l e c t e d t i m e p e r i o d s did not a f f e c t d i g e s t i b 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 h e d i g e s t i b 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 , the r e s p o n s e to e n z y m e addition w a s inconsistent, h o w e v e r , the positive effects that w e r e o b s e r v e d w e r e primarily a s a result of the addition of 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 r a c t i o n s w e r e f o u n d in t h e i n t e r p r e t a t i o n o f f i b e r r e s u l t s w h i c h reflected t r e a t m e n t d i f f e r e n c e s . A significant third o r d e r interaction ( P < 0.05) w a s noted for duration of the heat treatment, a n d the application of 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 t h a t 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 treatment m e a n s w e r e significantly different f r o m o n e a n o t h e r ( P < 0.05). T h e application of h e a t for 1 5 min r e d u c e d A D F from 2 0 . 1 % to 1 8 . 9 % of D M . T h e a p p l i c a t i o n of 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 % of t h e D M a n d t h e c o m b i n a t i o n of 6 0 m i n of h e a t with 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 t o g r e a t e r t h a n 2 1 % o f t h e D M in t h e A D F c o n t e n t w a s o b s e r v e d with two t r e a t m e n t s w h e r e heat w a s a p p l i e d for 3 0 o r 6 0 min p e r i o d s a n d the addition of 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 of either 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 a d d i t i o n t o a n y l e v e l o f h e a t , p r o d u c e d A D F v a l u e s that w e r e not significantly different ( P < 0.05) f r o m the control (Table 3.6).  102  p r o d u c e d A D F v a l u e s that w e r e not significantly different ( P < 0.05) f r o m the control (Table 3.6). A significant interaction ( P < 0.01) w a s a l s o noted for the addition of moisture, p r o t e a s e a n d x y l a n a s e (Figure 3.6). T h e A D F content of the untreated s a m p l e w a s 1 8 . 3 % of D M , w h i c h w a s significantly lower ( P < 0.05) than a n y other treatment ( 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 t h a t w e r e s i g n i f i c a n t l y d i f f e r e n t than the untreated s a m p l e with the e x c e p t i o n of the application of 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 e v e l of 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 content with a f e w e x c e p t i o n s ( T a b l e 3.7). T h e addition of 2 0 % or 3 0 % moisture, i r r e g a r d l e s s o f e n z y m e t r e a t m e n t , s i g n i f i c a n t l y i n c r e a s e d A D F ( P < 0 . 0 5 ) r e l a t i v e to the control. 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 i b u t e d t o a r t i f a c t f i b e 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 , t h i s e x p l a n a t i o n is u n l i k e l y a s p r e l i m i n a r y 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 n d i c a t o r of artifact fiber, f o u n d n o effect of t r e a t m e n t s o n this p a r a m e t e r . A n o t h e r m o r e likely e x p l a n a t i o n is t h a t t h e p r o p o r t i o n o f 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 t h o u t a c h a n g e in f i b e 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 h e n o n - f i b r o u s c o m p o n e n t s of c a n o l a m e a l . A s fiber results a r e p r e s e n t e d a s a portion of the D M , t h i s w o u l d r e s u l t in p r o p o r t i o n a l l y g r e a t e r a m o u n t s o f f i b e 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 e n d o b s e r v e d in d i g e s t i b i l i t y r e s u 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 h e f i b e r r e s u l t s .  103  3.4.4 NDF T h e N D F fraction of c a n o l a m e a l w a s significantly affected (P < 0.05) by p r o t e a s e treatment. T r e a t m e n t of c a n o l a m e a l with 0 . 0 0 5 % p r o t e a s e significantly (P < 0 . 0 5 ) 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 , treatment with h i g h e r l e v e l s of this e n z y m e did not significantly alter ( P < 0 . 0 5 ) t h e N D F c o n t e n t r e l a t i v e t o t h e c o n t r o l . It is o f i n t e r e s t t o n o t e t h e r e s p o n s e o f N D F to t h e l o w e s t l e v e l of e n z y m e w a s significant, w h e r e a s h i g h e r l e v e l s did not elicit 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 t o t h e o n e o f f e r e d f o 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 f o r t h e i n c r e a s e in t h e N D F c o n t e n t : t h a t 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 t h o u t a c h a n g e in t h e f i b e 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 p o r t i o n w o u l d b e r e f l e c t e d in h i g h e r f i b e r v a l u e s c o m p a r e d t o t h e c o n t r o l . It is u n l i k e 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 h e 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 h e s m a l l p o r e s i z e . N D F content w a s a l s o affected (P = 0.0001) by the level of moisture applied t o 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 g n i f i c a n 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 of c a n o l a m e a l ( T a b l e 3.9). T h e addition 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 of e i t h e r 1 0 % o r 3 0 % moisture p r o d u c e d N D F v a l u e s of 1 8 . 6 % a n d 1 8 . 2 % of D M , respectively w h i c h w e r e significantly lower than the control a s well a s the 2 0 % moisture treatment. 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 h e 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 the v a r i o u s t r e a t m e n t s a r e unlikely to h a v e a n y practical 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 significant d u e to t h e s m a l l s t a n d a r d error of the  104  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 0.16 for c a n o l a m e a l obtained from s e v e r a l c r u s h i n g plants w h i c h are c o m p a r a b l e t o 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 t h e r e s u l t s o f t h i s s t u d y , it w a s d e t e r m i n e d t h a t x y l a n a s e w a s n o t e f f e c t i v e in t e r m s o f i m p r o v i n g t h e d i g e s t i b 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 p o t e n 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 h e h i g h e s t l e v e l ( 0 . 0 5 % ) . A l t h o u g h the c o m b i n a t i o n of p r o t e a s e a p p l i e d at 0 . 0 1 % or 0 . 0 5 % l e v e l s with x y l a n a s e often g e n e r a t e d positive r e s p o n s e s , the m a g n i t u d e of the r e s p o n s e w a s no greater than 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 h e s a m e l e v e l s . H e a t t r e a t m e n t for a n y length o f t i m e w a s n o t e f f e c t i v e in i m p r o v i n g e n z y m e a c t i v i t y , n o r d i d it i n d e p e n d e n t l y elicit a n y r e s p o n s e from a n y of the 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 the other h a n d , h a d a n effect o n both the digestibility a n d the fibrous c o m p o n e n t s of 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 i g e s t i b i l i t y o r t h e f i b e r c o n t e n t m a y b e a t t r i b u t a b l e t o a s h i f t in t h e 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 t o b e f u r t h e r investigated. T h e h i g h n u m b e r o f i n t e r a c t i o n s 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 i n t e r p r e t a t i o n o f t h e r e s u l t s difficult. It is o b v i o u s t h e e f f e c t s o f e n z y m e t r e a t m e n t c o m b i n a t i o n s p r o d u c e d b o t h p o s i t i v e a n d n e g a t i v e r e s p o n s e s . It is t h i s i n c o n s i s t e n c y in t h e r e s u l t s f r o m t h e r e s e a r c h w i t h e n z y m e s w i t h r u m i n a n t d i e t s , c o m b i n e d w i t h t h e h i g h c o s t o f e n z y m e s t h a t will c o n t i n u e t o r e s t r i c t e n z y m e  105  a p p l i c a t i o n s in t h i s a r e a . T h e p r e s e n t s t u d y i s n o t c o n c l u s i v e b u t 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 t h e field of r e s e a r c h with r u m i n a n t e n z y m e r e s e a r c h applications. Further evaluation of the effects of the treatments using other 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 , w i l l a l l o w e x a m i n a t i o n o f t h e f e r m e n t a t i o n p r o f i l e s 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 will p r o v i d e m o r e information o n the effects of e n z y m e addition a n d mild hydrothermal treatment of canola meal.  106  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 u m i n a n t n u t r i t i o n . 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 Development: Meeting Future Challenges. Proc. 1996 C a n . S o c . Anim. Sci. A n n . Mtg., Lethbridge, Alberta, C a n a d a . B e l l , J . M . 1 9 9 3 . F a c t o r s affecting the nutritional v a l u e of c a n o l a m e a l : A C a n . J . Anim. Sci., 73:679-697.  review.  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 v a r i a t i o n 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 l a n t s . C a n . J . Anim. Sci., 71:469-480. 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 i g e s t i b i l i t y b y p i g s o f hull f r a c t i o n s from r a p e s e e d cultivars with yellow or brown s e e d coats. C a n . J . A n i m . S c i . , 62:557-565. 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 . and J . A. Shelford.  1995. Biotechnology  in t h e f e e d l o t .  J . Anim. Sci.,  46:600-605. Chesson, A. 1993. Feed enzymes. Anim. Feed Sci. Technol., 45:65-79. Cochran, W . G . and G . M. C o x . 1950. Experimental designs. P a g e s 154-156; 2412 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 t h f e e d i n g r a p e s e e d m e a l to d a 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 N u t r i t i o n .  Garnsworth,  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  Univ.  Press. G o e r i n g , H . K. a n d  P. J . V a n Soest. 1970. Forage Fiber Analyses.  Reagents, Procedures, and s o m e Applications). Agric. Handbook  (Apparatus, No. 379.,  A R S - U S D A , Washington, 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 . , 70:581-589. 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 . 1990b. Effect of ruminal colonization on cereal grain digestion. C a n . J . A n i m . Sci., 70:571-579.  107  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 study of the digestion of barley a n d m a i z e grain  by  rumen microorganisms. Anim. Feed Sci. Technol., 31:91-105. M o s h t a g h i N i a , S . A . a n d J . R. Ingalls. 1 9 9 5 . E v a l u a t i o n of 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 h e r u m e n , s m a l l i n t e s t i n e , l a r g e i n t e s t i n e a n d total d i g e s t i v e tract of 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 United S t a t e s market for 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 h e F e e d I n d u s t r y . P r o c . o f A l l t e c h ' s Tenth A n n u a l S y m p o s i u m . Nottingham University P r e s s , pp.99. S t a t i s t i c a 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 . Tait, 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 of c e r e a l s a n d p r o t e i n 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 (B4), 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 e y , 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 f o 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 . Brit. J . G r a s s l . S o c , 1 8 : 1 0 4 - 1 1 1 . V a n S o e s t , P . J . 1 9 8 2 . Nutritional Corvallis, Oregon.  e c o l o g y of the  ruminant.  O &  B Books  Inc.,  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 of dietary fiber, n e u t r a l d e t e r g e n t f i b e 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 r e l a t i o n to a n i m a l nutrition. 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 effect of heat a n d moisture o n the digestibility matter  and  crude  gastrointestinal Wageningen  protein  tract Institute  of of  of  barley  ruminants. Animal  Netherlands.  108  and M.  canola Sc.  Science  in t h e  Thesis. Agricultural  rumen Dept.  and  of dry lower  Anim.  Sci.  University,  The  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 i n o r d e r to 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 w i t h 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 d i g e s t i b i l i t y o f c a n o l a m e a l .  Factor  Level of application  Heat  c o n t r o l (0), 1 5 , 3 0 , 6 0 m i n  1  Moisture  2  Protease  1  Xylanase  0%, 10%, 20%, 3 0 %  ,2, 3, 4  0%, 0.005%, 0.01%, 0.05%  ,2, 3, 5 :  0%, 0.001%  1  Applied at 55°C.  2  Applied on a % vol * wt  3  S u p p l i e d b y F i n n F e e d s International Ltd., Wiltshire, U.K.  4  Heptex sb-protease.  5  Avizyme 1310.  1  basis.  109  T a b l e 3.2. T h e effect o f the addition of protease a n d x y l a n a s e to c a n o l a m e a l on the 4 8 h r u m e n in vitro dry 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 ( D M b a s i s ) .  E n z y m e Treatment  1  P r o t e a s e (%)  X y l a n a s e (%)  I V D M D (%)  0  0  79.8  C  0.005  0  79.1  b c  0.01  0  77.8  a  0.05  0  82.4  e  0  0.001  79.4  b c  0.005  0.001  78.7  a b  0.01  0.001  81.5  d  0.05  0.001  81.1  S.E.M.  2  0.31  3  1  Applied o n a % vol * wt " basis.  2  I V D M D , in vitro d r y m a t t e r d i s a p p e a r a n c e .  3  S . E . M . , pooled standard error o f the m e a n .  a d  d  1  M e a n s in a c o l u m n w i t h d i f f e r e n t s u p e r s c r i p t s a r e s i g n i f i c a n t l y d i f f e r e n t  110  (P < 0 . 0 5 ) .  T a b l e 3.3. T h e effect of moisture a d d e d to c a n o l a m e a l at the time of 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 d i g e s t i b i l i t y o f c a n o l a m e a l ( D M b a s i s ) .  Moisture  1  (%)  I V D M D (%) 2  0  78.6  a  10  79.9  b  20  80.7  C  30  80.7  C  S.E.M.  3  0.22  1  Applied o n a % vol * wt " basis.  2  I V D M D , in vitro d r y m a t t e r d i s a p p e a r a n c e .  3  S . E . M . , pooled standard error of the m e a n .  a _ c  1  M e a n s in a c o l u m n w i t h d i f f e r e n t s u p e r s c r i p t s a r e s i g n i f i c a n t l y d i f f e r e n t  111  (P < 0.05).  T a b l e 3.4. T h e effect o f the addition 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 meal on the r u m e n in vitro t r u e d i g e s t i b 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 Treatment  1  P r o t e a s e (%)  X y l a n a s e (%)  I V T D (%)  0  0  87.9  b c  0.005  0  87.6  a b  0.01  0  87.4  a  0.05  0  88.2  C  0  0.001  87.8  a b c  0.005  0.001  87.5  a b  0.01  0.001  88.2  C  0.05  0.001  87.9  b c  2  S.E.M.  0.18  1  Applied o n a % vol * wt  2  I V T D , in vitro t r u e d i g e s t i b i l i t y .  3  S . E . M . , pooled standard error of the m e a n .  ^  1  basis.  M e a n s in a c o l u m n w i t h d i f f e r e n t s u p e r s c r i p t s a r e s i g n i f i c a n t l y d i f f e r e n t  112  (P < 0.05).  T a b l e 3.5. T h e effect o f moisture a d d e d t o c a n o l a m e a l at the time of 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 d i g e s t i b i l i t y o f c a n o l a m e a l ( D M b a s i s ) .  Moisture  1  (%)  I V T D (%) 2  0  87.3  a  10  87.8  b  20  88.1  b  30  88.1  b  S.E.M.  3  0.13  1  A p p l i e d o n a % vol * wt  2  I V T D , in vitro t r u e d i g e s t i b i l i t y .  3  S . E . M . , p o o l e d standard error o f the m e a n .  a b  1  basis.  M e a n s in a c o l u m n with different s u p e r s c r i p t s a r e significantly different  113  (P < 0.05).  T a b l e 3.6. T h e effect of the duration of heat treatment a n d the application of 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 A D F fraction of c a n o l a m e a l ( D M basis). Feed Treatment Duration of heat treatment (min)  Protease  2  (%)  Xylanase  2  (%)  ADF  (%  3  1  2 0 1 def 0 0 0 15 0 0 18.9 30 0 0 20.0 •j g ^abcd 60 0 0 0 0.005 0 19.3 15 0.005 0 20.3 19 6 d 30 0.005 0 60 0.005 0 19.1 19 8 0 0.01 0 20 o 15 0.01 0 30 0.01 0 20.0 19 8 60 0.01 0 0 0.05 0 20.2 2 0 1 def 15 0.05 0 19 6 ' 30 0.05 0 60 0.05 0 0 0 0.001 2 0 . 3 ef 15 0 0.001 20.2 30 0 0.001 20.3 60 0 0.001 20.5 .j g yabcdef 0 0.005 0.001 15 0.005 0.001 20.0 30 0.005 0.001 21.0 60 0.005 0.001 21.2 0 0.01 0.001 20.0 •| g yabcde 15 0.01 0.001 -| g ybcdef 30 0.01 0.001 60 0.01 0.001 20.0 1 g yabcdef 0 0.05 0.001 •j g gbcdef 15 0.05 0.001 30 0.05 0.001 20.3 60 0.05 0.001 20.5 S.E.M. 0.27 Applied at 55°C. Applied on a % vol * wt ' basis. ADF, acid detergent fiber. S.E.M., pooled standard error of the mean. Means in a column with different superscripts are significantly different a  c d e f  a b c  e f 9  a b c  e  a b  b c d e f  c d e f  c d e f  b c d e f  e f g  a  ) c d e  20.49 e f g  d e f g  e f g  f g  c d e f  9  g  c d e f  c d e f  e f g  f g  4  1  2  1  3  4  3 9  114  (P < 0.05).  T a b l e 3.7. T h e effect of moisture, protease a n d x y l a n a s e application to c a n o l a meal on the A D F fraction of c a n o l a meal ( D M basis).  Feed Treatment  M o i s t u r e (%)  P r o t e a s e (%)  1  X y l a n a s e (%)  ADF  2  (%)  0 0 0 18.3 10 0 0 19.3 20 0 0 20.5 30 0 0 20.5 0 0.005 0 19.3 10 0.005 0 19.4 20 0.005 0 19.7 -| g gdefghi 30 0.005 0 0 0.01 0 18.0 10 0.01 0 19.5 20 0.01 0 20.9 30 0.01 0 21.2 0 0.05 0 19.0 -|g gcdefg 10 0.05 0 20 0.05 0 20.6 30 0.05 0 21.1 0 0 0.001 19.2 10 0 0.001 19.6 20 0 0.001 20.6 30 0 0.001 21.8' 0 0.005 0.001 19.4 -|g gdefghi 10 0.005 0.001 20 0.005 0.001 21.2 30 0.005 0.001 21.4 cde 0 0.01 0.001 •j g gdefgh 10 0.01 0.001 20 0.01 0.001 19.7 30 0.01 0.001 20.5 0 0.05 0.001 19.2 20 i 9 10 0.05 0.001 20 0.05 0.001 20.5 30 0.05 0.001 20.6 S.E.M. 0.27 Applied on a % vol * wt " basis. A D F , acid detergent fiber. S . E . M . , pooled standard error of the mean. "' Means in a column with different superscripts are significantly different a b  cd  fghij  fghij  c d e  c d e  c d e  a  c d e  ikl  ikl  bo  hifk  ikl  cd  c d e  ijk  c d e  ikl  kl  1 9 4  c d e f  9hij  cd  e f  h i  9 h i i  hijk  3  1  1  2  3  a  115  (P < 0.05).  T a b l e 3 . 8 . T h e e f f e c t o f t h e 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 t o c a n o l a m e a l at t h e 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 r a c t i o n in c a n o l a m e a l ( D M b a s i s ) .  Protease  1  (%)  NDF  0  19.6  0.005  20.5  0.01  19.4  a  0.05  19.4  a  S.E.M.  3  2  (%)  a  b  0.25  1  A p p l i e d o n a % vol * wt " b a s i s .  2  N D F , neutral d e t e r g e n t fiber.  3  S . E . M . , p o o l e d s t a n d a r d error of the m e a n .  a b  1  M e a n s in a c o l u m n w i t h d i f f e r e n t s u p e r s c r i p t s a r e s i g n i f i c a n t l y d i f f e r e n t  116  (P < 0.05).  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 a d d i t i o n to c a n o l a m e a l at t h e 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 r a c t i o n in c a n o l a m e a l ( D M b a s i s ) .  Moisture  1  (%)  NDF  2  0  22.1°  10  18.6  a  20  20.3  b  30  18.2  a  S.E.M.  3  (%)  0.25  1  A p p l i e d o n a % vol * wt  1  2  N D F , neutral detergent  fiber.  3  S . E . M . , p o o l e d s t a n d a r d error of the m e a n .  a c  basis.  M e a n s in a c o l u m n w i t h d i f f e r e n t s u p e r s c r i p t s a r e s i g n i f i c a n t l y d i f f e r e n t  117  (P < 0.05).  0  0.01  0.02  0.03  0.04  0.05  P r o t e a s e l e v e l (% v o l / wt)  F i g u r e 3 . 1 . T h e effect of the addition of x y l a n a s e a n d  protease  (% v o l / w t ) 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 of c a n o l a m e a l .  118  81  -r  77.5  J  1  1  h-  0  10  20  30  M o i s t u r e l e v e l (% v o l / wt)  Figure 3.2. Quadratic relationship between moisture level a n d the 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 c a n o l a m e a l ( Y = 7 8 . 6 2 + 0.165 * moisture level - 0.003 * moisture level , n = 4, R 2  0.988, S . E . = 0.105).  119  2  =  F i g u r e 3.3. T h e effect of the addition of 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 r u m e n in vitro t r u e d i g e s t i b i l i t y o f c a n o l a m e a l .  120  8 8 . 2 -r  8 6 . 8 -\  1  1  0  10  20  ;  :  h 30  M o i s t u r e l e v e l (% v o l / w t )  F i g u r e 3 . 4 . Q u a d r a t i c r e l a t i o n s h i p b e t w e e n m o i s t u r e l e v e l (% v o l / w t ) a n d t h e r u m e n in vitro t r u e d i g e s t i b i l i t y o f c a n o l a m e a l ( Y = 8 7 . 2 5 + 0.073 * moisture level - 0.002 * moisture level : n = 4, R 2  = 0.991, S . E . = 0.0355).  121  2  F i g u r e 3.5. T h e effect of the application of p r o t e a s e (P), x y l a n a s e ( X ) (% v o l / w t ) a n d d u r a t i o n 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 b e r in c a n o l a m e a l .  122  23  22  M o i s t u r e l e v e l (% v o l / w t )  F i g u r e 3.6. T h e effect of the application of moisture, p r o t e a s e (P) a n d x y l a n a s e ( X ) (% v o l / w 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 b e r in c a n o l a m e a l .  123  CHAPTER 4  4.0 THE EFFECT OF ENZYME AND MILD HYDROTHERMAL TREATMENT ON THE IN VITRO G A S PRODUCTION IN CANOLA MEAL.  4.1 ABSTRACT T h e h i g h f i b e r c o n t e n t o f c a n o l a m e a l l i m i t s i t ' s i n c l u s i o n in d a i r y c a t t l e d i e t s . Abundant  in C a n a d a  a n d grown  worldwide,  canola  meal  is a c o s t  effective  alternative protein 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 objective o f this 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 feasibility 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 mild  hydrothermal  t r e a t m e n t in o r d e r t o i m p r o v e t h e n u t r i t i o n a l 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 consisted o f a 4 x 4 x 4 x 2  m u l t i - f a c t o r i a 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  technique a n dV 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 h e r e s p o n s e o f c a n o l a meal subjected to t h e following treatments: heat applied at 55°C for 1 5 , 3 0 or 6 0 min periods o r not applied at all; moisture applied at 0 % , 1 0 % , 2 0 % o r 3 0 % ; protease {Bacillus sp.) a p p l i e d a t 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 a t 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 fitting t h e g a s  production d a t a a c q u i r e d o v e r 1 8 h, to t h e substrate limited e x p o n e n t i a l growth m o d e l w i t h l a g (Y = A ( 1 - e' ' ). b<t  l)  Total V F A production a n d V F A ratios w e r e  determined from o n e of the incubating s a m p l e s for e a c h treatment. T h e application o f h e a t ( 5 5 ° C ) d i d n o t i m p r o v e e n z y m e a c t i v i t y a s d e t e r m i n e d b y 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 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  124  not  affected  by  the  addition  of  xylanase. The  application  of  0.01%  protease  significantly i n c r e a s e d ( P = 0 . 0 0 0 1 ) the rate of g a s production f r o m 0 . 0 7 7 m L / h 0.108 m L / h . T h e length of the lag p h a s e w a s affected by the interaction 1  1  to  between  protease 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 significant interaction (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 ratio o f a c e t a t e to 0.0001)  propionate w a s affected  application. T h e  ratio  of  by protease (P < 0.01)  butyrate  to  propionate  and xylanase (P  was  protease (P < 0.01) and x y l a n a s e application (P = 0.0001), 0.05%  protease  treatment  significantly  increased (P  <  0.05)  also  affected  independently. the  = by  The  proportion  of  a c e t a t e to p r o p i o n a t e o r butyrate 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 h e  proportion  of a c e t a t e relative  to  p r o p i o n a t e o r b u t y r a t e . In vitro r e s u l t s o b t a i n e d in 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 that e n z y m 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 m a y  be an alternative  p r o c e s s in  i m p r o v i n g t h e r a t e 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 t o d e c r e a s i n g t h e l e n g t h o f t h e l a g p h a s e . In p a r t i c u l a r , t h e r e s u l t s o f t h i s s t u d y i n d i c a t e t h a 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 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 t o i m p r o v e t h e v a l u e of c a n o l a m e a l for dairy cattle.  125  nutritional  4.2  INTRODUCTION C a n o l a m e a l a s a f e e d s t u f f is h i g h in p r o t e i n m a k i n g it t h e p r i m a r y  alternative  t o s o y b e a n m e a l in d i e t s f o r l a c t a t i n g 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 i m i t e d h o w e v e r , d u e t o it's h i g h f i b e r c o n t e n t r e l a t i v e t o t h a 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 of c a n o l a m e a l is related to fiber c o n t e n t , c o m p o s i t i o n of cell wall 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 of t h e s e fractions o n the utilization of both t h e protein a n d e n e r g y c o m p o n e n t s of t h e feedstuff ( B r e n e s et a l . , 1 9 9 3 ) . Slominski canola  meal  hydrolysis  and  and  was  C a m p b e l l (1990)  found  derived  that from  87%  investigated  of  the  cellulose.  the  glucose  High  carbohydrate  released  amounts  of  content  during  xylose  of  enzymatic  indicated  the  p r e s e n c e of x y l a n s a n d x y l o g l u c a n s a n d large a m o u n t of uronic acid residues (30%) s u g g e s t e d t h a t t h e p o l y s a c c h a r i d e s in t h e c e l l u l o s i c f r a c t i o n a l s o i n c l u d e d a l a r g e a m o u n t of pectins (Slominski a n d C a m p b e l l , 1990). Pectins are generally  regarded  a s r e a d 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 i g h 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 r a t e i s o f t e n g r e a t e r t h a n 8 % h" 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 1  r u m e n is l e s s t h a n 1 0 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 u n l i k e l y ( V a n Soest,  1982).  For the  s a m e reasons, xylans and xyloglucans also may  c o m p l e t e l y d e g r a d e d w i t h i n t h e s h o 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  not  be  addition,  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 highly substituted x y l a n s are less readily hydrolyzed ( V a n S o e s t , 1994). T h e fibrous carbohydrate  fraction  of  canola  meal  has  126  been  determined  to  be  the  main  c o n t r i b u t i n g f a c t o r t o i t ' s l o w e n e r g y a v a i l a b i l i t y in r u m i n a n t a n i m a l s a n d i s t h e r e f o r e a n i m p o r t a n 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 ) . Many  studies  have  shown  t h e benefit  of adding  e x o g e n o u s e n z y m e s to  concentrates fed to ruminants, a s well a s deleterious effects. S e v e r a l studies were i n c o n c l u s i v e in their results. F o r a brief r e v i e w o f the literature the r e a d e r is referred t o 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 h e l i t e r a t u r e r e v e a l s t h e r e h a s b e e n limited i n v e s t i g a t i o n into t h e 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 p a r t i c u l a r t o c a n o l a m e a l in o r d e r t o i m p r o v e it's n u t r i t i o n a l v a l u e w h e n f e d to  ruminants.  Preliminary  a s s e s s m e n t of t h e effects  o f mild  hydrothermal a n d  e n z y m e t r e a t m e n t o n t h e n u t r i t i o n a 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 ( r e f e r t o C h a p t e r 3 ) . In vitro  fermentation  techniques  have  been  successfully  used  in t h e  e v a l u a t i o n o f f o r a g e s f o r r u m i n a n t s for d e c a d e s (Tilley a n d Terry, 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 p a r t i c u l a r h a s b e e n s h o w n t o b e i n d i c a t i v e o f the  digestibility  a n d fermentation  a p p e a r a n c e of fermentation  of a variety  products  of feedstuffs  a s it m e a s u r e s t h e  rather than their d i s a p p e a r a n c e o v e r  (Menke et al., 1979; M e n k e a n d Steingass, 1988; Blummel a n d Orskov,  time 1993;  Opatpatanakit et al., 1994, Krishnamoorthy et al., 1995). T h e production of g a s ( C 0  2  a n d C H ) a r i s e s a s a result o f the fermentation o f c a r b o h y d r a t e s b y r u m e n m i c r o b e s 4  t o v o l a t i l e f a t t y a c i d s ( V F A ) , p r i m a r i l y a c e t a t e , b u t y r a t e a n d p r o p i o n a t e , t h e latter o f which d o e s not directly contribute to the production of 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  127  were  indicative of the g a s p r o d u c e d a n d therefore the fermentation feedstuffs.  The  present  study  investigated  the  u s e of  a  profile of v a r i o u s  similar system  in  the  e v a l u a t i o n of the nutritive v a l u e of 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 this 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 investigate the feasibility of u s i n g e n z y m e s a n d  mild  hydrothermal  treatment  a p p l i e d t o c a n o l a m e a l , in o r d e r t o i m p r o v e it's n u t r i t i o n a l v a l u e f o r d a i r y c a t t l e .  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 of 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  multi-  factorial d e s i g n w h i c h e v a l u a t e d the effects of e n z y m e s a n d hydrothermal treatment o n c a n o l a m e a l u s i n g t h e in  vitro g a s p r o d u c t i o n  technique. Treatments  under  i n v e s t i g a t i o n 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 h e p r o c e d u r e outlined in C h a p t e r 2 , s e c t i o n 2 . 3 . 2 .  128  4.3.3 Chemical Analyses Chemical  analyses  were  conducted  on  the  samples  according  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 fluid w a s c o l l e c t e d  p r i o r to t h e  morning  feeding  as suggested  by  M e n k e a n d S t e i n g a s s (1988). F o l l o w i n g collection, r u m e n fluid w a s filtered through 4 layers of c h e e s e c l o t h a n d a layer of g l a s s w o o l c o n t a i n e r f o r t r a n s p o r t to t h e  into a p r e w a r m e d  and  airtight  laboratory.  4.3.6 Gas Production System Gas  production  measurements  were  made  using  a computerized  a d a p t e d from Pell a n d S c h o f i e l d (1993) (Figure 4.1) with the following  system  modifications.  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  32  customized  for  Erlenmeyer  flasks  (50  mL).  A  rotating  bottom  plate  allowed  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 fitted w i t h a s m a l l port in w h i c h  a vacutainer  stopper was  placed which  129  allowed  for s y s t e m  calibration  ( A p p e n d i x 6.5), r u m e n fluid injection 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 maintained by moistening the s e a l with water. P r e s s u r e t r a n s d u c e r s ( P X 1 7 0 series, 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 h e t o p o f silicone stoppers which contained a 0.5 c m hole. T h e stoppers a n d  transducers  w e r e s e c u r e d w i t h e l a s t i c b a n d s a n c h o r e d t o t w o s m a l l " e a r s " l o c a t e d at t h e n e c k o f the flask t h r o u g h o u t the acquisition of g a s m e a s u r e m e n t s (Figure 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 t o 1 0 3 k P a (0 t o 1 5 p s 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 the p r e s s u r e within a f l a s k a n d g e n e r a t e d a differential output v o l t a g e proportional to that 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  using  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 i g h  speed  m e a s u r e m e n t s y s t e m set 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 with a d i r e c t t r a n s f e r t o 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 t o a c q u i r e d a t a at 5 m i n intervals o v e r a n 18 h incubation period. T h e following  r e g r e s s i o n e q u a t i o n (r  2  =  0.994) w a s u s e d to d e t e r m i n e the 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*Y  1  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 .  130  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 i p l i c a t e into 5 0 ml_ e r l e n m e y e r f l a s k s t o which  12 m L of p r e h e a t e d phosphate-bicarbonate m e d i u m a n d reducing  solution  ( G o e r i n g a n d V a n S o e s t , 1970) w a s a d d e d . T h e flasks w e r e flushed with 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 prior t o s e a l i n g . R u m e n fluid (3 m L ) w a s t r a n s f e r r e d v i a a n 1 8 g a u g e n e e d l e t h r o u g h t h e p o r t into t h e f l a s k s t o m a k e u p 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 h e flasks w e r e a l l o w e d to equilibrate for 5 min a n d the e x c e s s g a s r e l e a s e d with a n e e d l e ( 2 6 . 5 g a u g e ) t h r o u g h t h e port prior 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 fluid a n d buffer) w h i c h w e r e a v e r a g e d in o r d e r t o c o r r e c t e a c h c h a n n e l f o r r u m e n f l u i d a c t i v i t y . 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 d a i l y v a r i a t i o n a n d d e t e c t a b n o r m a l  rumen  fluid a c t i v i t y . T h e b u f f e 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  using  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 o f f l a s k s at t h e e n d o f e a c h g a s run. T h e buffer c a p a c i t y of the s y s t e m w a s m a i n t a i n e d within a p H r a n g e of 6.7-7.0.  131  4.3.9 Data Processing All voltage m e a s u r e m e n t s a c q u i r e d with t h e d a t a logger w e r e recorded using TempWindows  3 . 1 ® software  (Omega  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 . ) .  Determination of g a s production for a specific channel required: 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 a t 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 a t a n y time. 2) C o n v e r s i o n o f t h e v o l t a g e o u t p u t t o g a s v o l u m e u s i n g t h e c a l i b r a t i o n  correction  factor (corrected to 1 0 3 k P a , 3 9 ° C ) , ( A p p e n d i x 6.5). 3) Normalization of the g a s production d a t a to 1 g dry s a m p l e weight. 4) U s e of s t e p s 1 a n d 2 to calculate g a s production of t h e blank, h a y a n d c a n o l a standards.  4.3.10 Kinetic Analysis of Gas Data T h e g a s p r o d u c t i o n p r o f i l e o f a c a n o l a m e a l s a m p l e i s i l l u s t r a t e d in F i g u r e 4 . 3 . T h e s i m p l e limited 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 with lag w a s u s e d to d e s c r i b e the g a s production data. T h i s m o d e l h a s b e e n previously u s e d to determine g a s production  parameters  in  tropical  feedstuffs  (Krishnamoorthy  Krishnamoorthy et al., 1995):  Y =A(1-e  b ( t l >  ),  132  (Equation 2)  et  al.,  1991;  w h e r e V i s 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 initial l a g f o r t h e 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 p o t e n t i a 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 r a t e b ( m L / h ) . 1  Rates regression  of  and  fermentation the  and  lag  Secant method  periods  to  were  minimize  the  estimated residual  using sums  of  nonlinear squares  a s s o c i a t e d with the r e g r e s s i o n m o d e l (Statistical A n a l y s i s S y s t e m s , ( S A S ) ,  1990)  ( 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 i s a l i n e a r i t e r a t i v e c u r v e fitting p r o c e d u r e o f t h e P R O C N L I N p r o c e d u r e s of S A S (1990).  4.3.11 V F A 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 t h a t 6 h t o t a l gas production  m e a s u r e m e n t s of c a n o l a m e a l treatments  w e r e r e f l e c t e d in a 6 h  s a m p l i n g t i m e f o 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 n v e s t i g a t i o n o f t h e c h a n g e s in the ratios of V F A s p r o d u c e d a l s o indicated this w a s a n a d e q u a t e s a m p l i n g time. I n h e r e n t a n a l y t i c e r r o r a s s o c i a t e d w i t h V F A a n a l y s e s in t h i s 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 f o r t o t a l a n d individual V F A s  (acetate,  propionate,  isobutyrate,  v a l e r a t e ) a c c o r d i n g t o t h e p r o c e d u r e o f E r w i n et al.,  n-butyrate,  iso-valerate and  n-  ( 1 9 6 1 ) . A l u e r l o c k fitted w i t h a  t u b e (I.D. = 1 . 1 9 m m , O . D . 1.70 m m ) w a s i m m e r s e d b e l o w t h e r u m e n fluid 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 a s k u s i n g a s y r i n g e . A f t e r 6 h of i n c u b a t i o n , a 0.5 mL representative  sample w a s withdrawn  from the fermentation  flasks. Samples  w e r e c e n t r i f u g e d at 3 0 0 0 g for 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 with a 2 0 %  133  phosphoric  acid  chromatograph fused  silica  solution  (v/v). T h e  supernatant  was  later  injected  into a  gas  ( 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 with a  capillary  column  (30m  x  0.25  I.D.  Stabilwax-DA,  Restek  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 t o i n c r e a s e at 1 0 ° m i n  Corp., 1  from  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 for the internal s t a n d a r d .  4.3.12 Statistical Analysis Statistical a n a l y s e s w e r e c o n d u c t e d using the G e n e r a l Linear M o d e l ( G L M ) procedure program  of  Statistical  Analysis System  l i s t e d in A p p e n d i x 6 . 4 . In t h e  ( S A S Institute  present study,  Inc.,  1990)  using  the  it w a s a s s u m e d t h a t  the  h i g h e s t o r d e r i n t e r a c t i o n d i d n o t e x i s t , but it's m e a n s q u a r e e r r o r w a s r e p r e s e n t a t i v e of  the  experimental  error  (Cochran  and  Cox,  1950).  Statistically  significant  differences (P < 0.05) between m e a n s w e r e determined using the program which e m p l o y e d t h e u s e of l e a s t s q u a r e m e a n s for rate of g a s p r o d u c t i o n , length of the lag p h a s e , total 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 ratios a n d b u t y r a t e to p r o p i o n a t e ratios. F u r t h e r investigation of the relationship b e t w e e n a significant m a i n effect a n d a  measured  parameter  with  orthogonal  polynomials  C O N T R A S T statement of S A S P R O C G L M ( S A S , 1990).  134  was  conducted  using  the  4.4 RESULTS AND DISCUSSION N u t r i e n t a n a l y s i s o f t h e c o n t r o 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 with 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 variety 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 the  exponential  model  including  treatment w a s determined  lag. T h e  rate  to b e 0 . 0 7 7 ± 0 . 0 0 4  of g a s mL/fr  1  production  of the  of  using control  a n d t h e length of the  lag  p h a s e w a s d e t e r m i n e d t o 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  appropriate  model  parameters  has  been  shown  to  affect  the  estimation  of g a s  production  ( K r i s h n a m o o r t h y et a l . , 1 9 9 1 ) , r a t e s 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 t h o t h e r f e e d s have  produced  comparable  results  to  those  obtained  in  the  present  study  irrespective of w h e t h e r single or d u a l - p o o l e d logistic m o d e l s w e r e u s e d . R a t e s of g a s p r o d u c t i o n in a s t u d y w i t h 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 l o g i s 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  and 0.0192  m L / h " f r o m d a t a o b t a i n e d w i t h 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 ) . 1  A  review of the  production  are  computerized  literature r e v e a l s that m a n u a l  inherently  system. The  associated present  with  study  135  greater  employed  methods  of recording  experimental the  u s e of a  error  than  gas a  computerized  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 by S c h o f i e l d a n d Pell (1995) with the  minor  modifications  previously  noted. T h e  computerized  system  has  been  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 , timothy 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 error r a n g i n g f r o m 0 . 7 % to 2 . 6 % of t h e 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 h e r s i n g l e or 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 h e p r e s e n t s t u d y , t h e s t a n d a r d error for rate w a s ± 0 . 0 0 4 mL/h" 0.077 mL/h"  1  1  pooled  with v a l u e s for treatments ranging  from  to 0 . 1 0 8 m L / h " . T h e error a s s o c i a t e d with the length of the lag p h a s e 1  w a s ± 0 . 0 4 6 h with 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 t o 0 . 4 4 9 h. T h e  v a l u e s f o 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 r o r a s s o c i a t e d w i t h t h e m t h a n most other reported f e e d v a l u e s with other s y s t e m s . T h i s m a y  b e attributed  to  variation within a feedstuff b e i n g investigatedor the large n u m b e r of 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 interval w h i c h is u n i q u e to this t y p e of s y s t e m . T h i s variation h o w e v e r , is n o t r e f l e c t e d in t h e v a l u e s f o r r a t e . 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 ) t o 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 errors 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 % t o 5 7 % f o r l a g p e r i o d . In t h e p r e s e n t s t u d y , the s p r e a d of observations a s a proportion of the estimated value w a s on a v e r a g e 5 . 2 % for rate of g a s production a n d 1 0 . 2 % for length of the lag p e r i o d . T h e larger e r r o r a s s o c i a t e d w i t h t h e l e n g t h o f t h e l a g p h a s e in b o t h m e t h o d s m a y b e a t t r i b u t e d t o 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 f a c t o 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 fluid activity, population a n d c h e m i c a l c o m p o s i t i o n of the feedstuff.  136  microbial  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 h e p r e s e n t s t u d y w i t h c a n o l a m e a l , u s i n g a computerized production  system,  are  comparable  to  those  obtained  with  a  manual  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  gas  et a l . ,  1991). E m p l o y i n g the e x p o n e n t i a l m o d e l with lag, the rates of g a s production wheat,  barley,  m a i z e , tapioca, oat,  rye, s o r g h u m w e r e  0.1196, 0.1193,  for  0.0767,  0 . 1 3 4 9 , 0 . 0 8 6 6 , 0 . 0 8 7 5 a n d 0.1101 mL/h" . T h e lag periods for the s a m e f e e d s w e r e 1  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 the rate of g a s production w a s 0 . 1 2 5 m L / h  1  and  t h e l e n g t h 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 f o r rate of g a s p r o d u c t i o n o b t a i n e d by t h e s e a u t h o r s w a s faster t h a n the v a l u e of 0 . 0 7 7 mL/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 c o n t r a s t , t h e v a l u e f o r l a g o b t a i n e d in t h e  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 f o r t h e l a r g e 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 t w o 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 a n t i - n u t r i t i o n a l  factors  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 t o c a n o l a m e a l , m a y h a v e a d e t r i m e n t a l  effect  on lag time through s o m e action on rumen microbes. A n o t h e r possible explanation may  be that m a n u a l  system.  In  estimation  addition, of  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 error t h a n the  parameters  frequency from the  of  gas  model  measurements which  may  is d e p e n d e n t  a  computerized  impact the  on  accuracy  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 i k e l y t o d e t e c t  the of the  p o i n t o f m a x i m u m r a t e 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 with f e w m e a s u r e m e n t s .  137  4.4.2 Rate of In Vitro Gas Production E v a l u a t i o n of the rates of g a s production from the c a n o l a m e a l  treatments  e s t a b l i s h e d that the application of p r o t e a s e h a d a significant impact ( P <  0.05).  P r o t e a s e a p p l i e d at 0 . 0 1 % significantly i n c r e a s e d the rate of g a s production ( P < 0.05) from highest  0.077  levels of  mL/h"  1  to 0 . 1 0 8  protease  mL/h"  produced  1  (Table 4.2).  rates  of  0.075  However, the mL/h"  1  and  lowest  0.089  and  mL/h" , 1  r e s p e c t i v e l y w h i c h w e r e not significantly different ( P < 0.05) f r o m the control. T h e i n c r e a s e d rates of g a s production o b s e r v e d with the addition of 0 . 0 1 % protease m a y r e f l e c t t h e a c c e s s i b i l i t y a n d t h e 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 meal by rumen microbes. With forages, increased bacterial colonization density has b e e n a s s o c i a t e d w i t h 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 ( H i d a y a 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 b i l i t y o f t h e f i b r o u s c a r b o h y d r a t e f r a c t i o n o f c a n o l a m e a l is i m p o r t a n t in o r d e r  to  improve  it's  energy  value  and  optimize  the  use  of  an  expensive  commodity. T h e n o n l i n e a r e f f e c t o f p r o t e a s e is i l l u s t r a t e d in F i g u r e 4 . 4 . I n v e s t i g a t i o n into t h e n a t u r e o f t h i s e f f e c 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  effects of c a n o l a m e a l treated with levels of 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 non linear effect of e n z y m e treatment of a feedstuff h a s b e e n previously reported. W h e n s t e e r s w e r e f e d c u b e d alfalfa h a y that h a d b e e n s p r a y e d with v a r i o u s levels 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 t h e a p p l i c a t i o n d o s e t h a n at 8 o r 1 6 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 . ,  138  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 attributed to the saturation of e n z y m e binding sites w h e r e a n optimal rather than 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 of r e s p o n s e of rate f r o m x y l a n a s e treatment 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 e v e 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 t o m i l d h e a t a n d moisture treatments recommended  which were  by the  a p p l i e d in o r d e r t o  benefit  e n z y m e activity  e n z y m e supplier (FFI, personal communication).  as  Current  research of hydrothermal treatment of c a n o l a meal now f o c u s e s o n moderate heat processes which employ temperatures ruminal  escape  protein  (Mir  et  al.,  in e x c e s s o f 1984;  100°C  Moshtaghi  Nia  in o r d e r t o and  increase  Ingalls,  1992).  T h e r e f o r e , the lack of r e s p o n s e o b s e r v e d with the mild h y d r o t h e r m a l treatments w a s n o t 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 t h a t h y d r o t h e r m a l t r e a t m e n t d i d n o t e n z y m e activity a s there w e r e n o interactive effects of e n z y m e a n d  improve  hydrothermal  treatments. T h e r e a r e relatively few e x p e r i m e n t s w h i c h h a v e u s e d the g a s  production  t e c h n i q u e in o r d e r t o e v a l u a t e t h e e f f e c t o f e n z y m e s o n t h e f e r m e n t a t i o n k i n e t i c s o f a f e e d s t u 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 h e u s e o f c e l l w a l l d e g r a d i n g e n z y m e s to improve the quality of g r a s s silage using g a s production p a r a m e t e r s (Beuvink 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 production w a s affected  but total g a s p r o d u c t i o n  remained the s a m e , were  attributed to a shift in t h e solubility o f f e e d c o m p o n e n t s a s a result 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 t h a t in g r a s s s i l a g e t h a t w a s e n z y m e t r e a t e d , t h e e n z y m e s  139  a p p e a r e d to m o d i f y t h e c e l l w a l l m a t e r i a l to m o r e rapidly f e r m e n t a b l e  components  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 r a c t i o n a s i n d i c a t e d b y t h e s h o r t e r initial p h a s e a n d l o w e r m a x i m u m r a t e o f g a s p r o d u c t i o n . In t h a t i n s t a n c e , t h e  lag  authors  s p e c u l a t e d t h a 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 , l a c t i c a c i d ) o f t h e f e e d w e r e r e s p o n s i b l e f o r t h e o b s e r v e d c h a n g e s in t h e r a t e o f g a s p r o d u c t i o n . A s i m i l a r explanation  may  be  offered  in t h i s  study  where  total g a s  production  between  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 r a t e s of f e r m e n t a t i o n a n d lag times were  significantly affected  by e n z y m e pretreatment  ( A p p e n d i x 6.8).  Feed  t r e a t m e n t s h a d n o effect o n total g a s p r o d u c t i o n , this i n d i c a t e s the t r e a t m e n t s not affect t h e total a m o u n t of f e r m e n t a b l e  material but p e r h a p s c a u s e d a  did shift  t o w a r d s m o r e rapidly 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 production for the control 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 significantly different ( P < 0.05) f r o m a n y o t h e r of the 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 solubility of f e e d c o m p o n e n t s m a y h a v e a n effect o n the rate of g a s p r o d u c t i o n  however, a  similar 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 of 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 t h t h e e x t e n t of d i g e s t i o n of 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 extent of 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 t o b e d e t e r m i n e d b y 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 , 1977).  140  4.4.3 Lag Period T h e length of the  lag p h a s e w a s significantly (P < 0.05) affected  by  the  i n t e r a c t i v 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 w h i c h is i l l u s t r a t e d in F i g u r e 4 . 5 . T h e l e n g t h o f t h e l a g p h a s e o f t h e u n t r e a t e d s a m p l e in t h e 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 t h a t t h e c o n t r o l t r e a t m e n t significantly longer lag p h a s e than a n y of the e n z y m e treatments addition, a n y combination of e n z y m e treatment  had  ( P < 0.05).  shortened the length  of the  a In  lag  p h a s e o f c a n o l a m e a l t o l e s s t h a n 0 . 2 9 4 h. The  application  of  protease  s h o w e d the greatest reduction  at  0 . 0 1 % with  or without  in t h e l e n g t h o f t h e  0.001%  xylanase  lag p h a s e c o m p a r e d to  the  c o n t r o 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 a g t i m e f r o m 0 . 4 4 9 h to 0 . 1 4 0 h;  whereas  protease  d e c r e a s e d the  applied  lag p h a s e from  x y l a n a s e w a s a s effective  at  0.01%  0.449  in  conjunction  h to 0 . 1 3 6  with  0.001%  xylanase  h. T h e a p p l i c a t i o n o f  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  0.001%  where  an  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 i g n i f i c a n t l y i n c r e a s e d ( P < 0.05) t h e l e n g t h of t h e l a g p h a s e to 0 . 2 7 3 h f r o m a lag p e r i o d of 0 . 1 4 0 h for the 0 . 0 1 % protease treatment.  T h i s o b s e r v a t i o n is h o w e v e r , difficult t o e x p l a i n s i n c e  m a n y factors h a v e b e e n a s s o c i a t e d with lag. L a g t i m e m a y b e i n t e r p r e t e d a s t h e t i m e r e q u i r e d for: h y d r a t i o n o f t h e f e e d particle, fermentation  of  soluble sugars, attachment  and  c o l o n i z a t i o n of  rumen  m i c r o b e s to t h e f e e d a s w e l l a s t h e l e n g t h of t i m e r e q u i r e d for t h e 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 a g p h a s e r e f l e c t s t h e 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 r u c t u r e a n d c o m p o s i t i o n o f t h e f e e d , t h e 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 i g n i f i c a n t l y impact the length of the lag 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 t h a n u m b e r o f t r e a t m e n t m e a n s in t h i s 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 lag 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 from rapidly fermentable solubles  ( 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 p a r t i c l e s 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 h e r u m e n fluid a r e f e r m e n t e d a n d c o n t r i b u t e to t h e g a s v o l u m e . It h a s b e e n s h o w n t h a t t h e r a t e a n d e x t e n 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 attributed to t h e p r e s e n c e of a protein matrix w h i c h r e n d e r s s t a r c h a n d  protein  l e s s r e a d 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 results of the p r e s e n t s t u d y s h o w that rate of g a s production m a y b e i n c r e a s e d from 0.077 m L / h  1  to a m a x i m u m of 0 . 1 0 8 m L / h " with t h e a d d i t i o n of p r o t e a s e . T h e length 1  of t h e 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 of 0 . 1 3 6 h with the addition of a c o m b i n 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 . T h e c o m b i n a t i o n of e n z y m e s w o u l d b e e x p e c t e d t o elicit a s y n e r g i s t i c e f f e c t b y d e g r a d i n g t h e p r o t e i n m a t r i x a n d other resistant plant 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 s u f f i c i e n t t o e l i c i t a p o s i t i v e e f f e c t o n r a t e 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 results  that  the  lag  phase  is  more  sensitive  to  the  effects  of  these  enzyme  s u p p l e m e n t a t i o n t h a n r a t e s 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 r e a t m e n t m e a n s w e r e often lower with the application of the c o m b i n a t i o n of p r o t e a s e a n d  142  x y l a n a s e , t h e y w e r e not significantly different f r o m the m e a n s o b s e r v e d with either 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 at 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 n e t i c s h a s b e e n s h o w n t o b e a useful a n d rapid m e t h o d to d e t e r m i n e the effects of e n z y m e s u p p l e m e n t a t i o n  on  grass silage (Beuvink and Sploestra, 1994). T h e s e s a m e principles m a y be applied t o t h e c u r r e n 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 t o elicit t h e i r r e s p o n s e in t e r m s o f increasing the fermentability of c a n o l a m e a l . G a s production parameters s u c h a s rate a n d lag p r o v i d e information a b o u t the fermentation pattern of v a r i o u s feedstuffs w h i c h m a y b e u s e f u l i n f o r m u l a t i n g r a t i o n s t o m a x i m i z e t h e u t i l i z a t i o n o f n u t r i e n t s in the rumen.  4.4.4 Volatile Fatty Acids I n f o r m a t i o n 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 t h e i r a m o u n t s is e s p e c i a l l y important  in  fermentation  the  evaluation  of  gas  production  results  from  concentrates.  The  of c o n c e n t r a t e s p r o d u c e s a large proportion of propionic acid w h i c h  d o e s not contribute to the formation of C 0 u n d e r e s t i m a t e d . T h e r e f o r e , interpretation  2  nor C H  4  and thus g a s volume may be  of results from  involving c o n c e n t r a t e s requires c o n s i d e r a t i o n of the  g a s production  production  studies  of total V F A s  in  addition to their proportions. It h a s b e e n r e p o r t e d t h a t 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 c a n o l a m e a l in o r d e r t o d e g r a d e t h e c e l l w a l l r e s u l t s in t h e r e l e a s e o f s t a r c h a n d p r o t e i n t h a t was  previously unavailable (Slominski a n d C a m p b e l l , 1990). This m a y explain the  143  s i g n i f i c a n t i n c r e a s e ( P < 0 . 0 5 ) 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 the untreated mmol/g  DM  xylanase  1  for  - 1  s a m p l e to V F A production 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  for s a m p l e s treated with a n effective c o m b i n a t i o n of p r o t e a s e  and  (Table  4.4).  Figure 4.6  illustrates  protease a n d x y l a n a s e (P < 0.01) which  the  significant  affected the  interaction  production  between  of V F A s .  The  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 f o 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 h e c a u s e of the  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 h e  effect  of  protease  s u p p l e m e n t a t i o n o n t h e r a t e o f g a s p r o d u c t i o n , t h e e f f e c t o f e n z y m e a d d i t i o n is n o n linear  with  the  combination  of  enzymes. A  possible explanation  for  this  was  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 binding sites o n the 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  the  present  study,  treatment  means  for  VFA  production  that  were  s i g n i f i c a n t l y h i g h e r t h a n t h e c o n t r o l w e r e , (in i n c r e a s i n g o r d e r ) : 1) 0 . 0 0 1 % x y l a n a s e , 2)  0.005%  p r o t e a s e , 3)  0.001% xylanase and  0.01%  protease  and  4)  0.01%  protease. O n l y the 0 . 0 0 1 % x y l a n a s e treatment w a s significantly (P < 0.05)  lower  than the other treatment m e a n s listed. The treatments  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 with a n u m b e r of the  enzyme  m a y b e a t t r i b u t e d t o a n i n c r e a s e in t h e a v a i l a b i l i t y o f s u b s t r a t e s  microbial fermentation  for  resulting from e n z y m a t i c hydrolysis of 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 n o t i n c l u d e d in t h e c e l l u l o s i c f r a c t i o n 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 r g e 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 s o l u b i l i t y o f f e e d c o m p o n e n t s i s k n o w n t o b e r e l a t e d to t h e d i g e s t i b i l i t y o f a f e e d s t u 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 t h a t  144  t h e 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 of 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 h e 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 . It h a s a l s o been  suggested  polysaccharides  that from  carbohydrases the  cell  wall  result by  in  the  release  systematically  of  removing  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 DM  1  short the  chain surface  2.58 mmol / g  to 3 . 8 8 m m o l / g D M " o b s e r v e d with the c o m b i n a t i o n of 0 . 0 1 % p r o t e a s e a n d 1  0.001%  xylanase treatment  availability.  Proteolytic  may  enzymes  b e attributed to have  been  increased  shown  to  microbial  increase the  substrate  total  water  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 t o c a n o l a m e a l ( S l o m i n s k i et a l . , 1 9 9 3 ) . It s h o u l d  be  noted  protease  applied  however, alone.  The  that x y l a n a s e a l o n e effectiveness  of  resulted  xylanase  in is  higher V F A evident  than  from  it's  c o n t r i b u t i o n t o t h e 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 f e c 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 of the c a r b o h y d r a t e s c o n t a i n e d within c a n o l a m e a l s u c h a s the high a m o u n t s of  xylans  and  xyloglucans  xylanolytic attack  (Slominski  and  Campbell,  1990)  (Biely, 1985). Therefore, improvements  are  susceptible  to  in t h e f e r m e n t a b i l i t y  of  c a n o l a m e a l a s reflected with i n c r e a s e d V F A production w o u l d b e e x p e c t e d with the addition of 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 of c a n o l a m e a l did not significantly the  production  of V F A s  nor did  it i m p r o v e  enzyme  activity.  These  results  c o m p a r a b l e to t h o s e o b t a i n e d with the e s t i m a t e d g a s production p a r a m e t e r s .  145  affect are  4.4.5 V F A 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 t h i s 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 i k e l y t o r e f l e c t 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 r e f l e c t t h e t y p e of substrate 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 t h a t p r o t e a s e e l i c i t e d a  s i g n i f i c a n t l i n e a r e f f e c t ( P = 0 . 0 0 0 1 ) o n t h e ratio o f a c e t a t e r e l a t i v e t o p r o d u c t i o n ( F i g u r e 4 . 7 ) . T h e ratio of butyrate to p r o p i o n a t e w a s a l s o  highly  propionate significantly  affected by the p r o t e a s e treatment ( P = 0.0001), although the nature of the  effect  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 significantly i n c r e a s e d t h e a c e t a t e to p r o p i o n a t e ratio f r o m 2 . 7 9 t o 3 . 7 4 w h e r e a s t h e 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 n o t s i g n i f i c a n t l y a f f e c t t h e r a t i o ( 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 i d n o t a f f e c t t h e ratio o f a c e t a t e t o p r o p i o n a t e n o r t h e ratio o f b u t y r a t e t o p r o p i o n a t e . In c o n t r a s t , t h e ratio o f b u t y r a t e t o 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 t o 0 . 6 8 w i t h both the lowest a n d highest level of p r o t e a s e application (Table 4.5). T h e n o n linear effect of 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 with rate of 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 t h a t 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 h e ratio o f a c e t a t e t o 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 t o 3 . 5 0 , a n d t h e ratio o f b u t y r 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 0 . 5 7 t o 0 . 6 9 . G r e a t e r i n c r e a s e s in t h e ratio o f 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 with the addition of 0 . 0 5 % p r o t e a s e t h a n with 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 ratio o f b u t y r a t e w a s o b t a i n e d w i t h the s u p p l e m e n t a t i o n of 0 . 0 0 1 % x y l a n a s e . T h e cellulosic fraction of a feedstuff  is  o f t e n a s s o c i a t e d w i t h 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 , 1 9 9 4 ) .  146  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 t h t h e 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 that the  e n z y m e s elicited their  actions on  the  structural  carbohydrate  c o m p o n e n t s of 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 relative to a c e t a t e nor to butyrate 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  indicate  b e t t e r u t i l i z a t i o n o f e n e r g y a s a c e t i c a n d b u t y r i c a c i d s r e s u l t in a h i g h e r p r o d u c t i o n o f gases ( C 0  2  a n d C H ) , w h i c h a r e a s o u r c e of d i e t a r y e n e r g y lost to t h e a n i m a l ( V a n 4  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 the results of the present s t u d y w h e r e proteolytic a n d xylanolytic e n z y m e s elicited their effects o n the f i b r o l y t i c 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 p a r t i c u l a r i n c r e a s e d with specific treatments. T h e m e c h a n i s t i c action a n d limitations of p r o t e a s e a n d x y l a n a s e help explain s o m e of the o b s e r v e d results of the p r e s e n t study. T h e e n z y m e p r o t e a s e attacks the proteolytic  b o n d s w h e r e a s x y l a n a s e attacks the xylan chain a n d  both of  which  render m o r e c a r b o h y d r a t e s available for digestion by r u m e n m i c r o b e s . X y l a n a s e activity h o w e v e r , m a y b e limited by the n u m b e r of 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 arabinose' s i d e c h a i n s (Biely, 1985). T h i s m a y explain s o m e of the lack of 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 w i t h 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 i b u t e d t o a n i n c r e a s e in s u b s t r a t e a v a i l a b i l i t y f o r 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 r e f l e c t e d 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 of g a s p r o d u c t i o n a n d s h o r t e r lag p h a s e s relative to t h e u n t r e a t e d c a n o l a m e a l .  147  4.5 CONCLUSIONS Evaluation of the fermentation was  kinetics of c a n o l a m e a l u s i n g g 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 m i l d  production  hydrothermal  t r e a t m e n t of c a n o l a m e a l . T h e a d d i t i o n of 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 r a t e 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 a g 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 no  effect  on  the  ratio  of  VFAs.  Xylanase was  only  effective  when  used  in  c o m b i n a t i o n w i t h p r o t e a s e , h o w e v e r , it's e f f e c t s 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 for both the length of the lag 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 application of x y l a n a s e a n d of the production  of  acetate  relative  to  highest  propionate.  level of p r o t e a s e i n c r e a s e d  The  lowest  and  highest  level  the of  p r o t e a s e a p p l i c a t i o n o r t h e a d d i t i o n 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 ratio o f b u t y r a t e r e l a t i v e t o p r o p i o n a t e . T h e 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 f c a n o l a m e a l i m p o s e d in this e x p e r i m e n t did not benefit e n z y m e activity a s indicated by t h e l a c k of r e s p o n s e to a n y of t h e m e a s u r e d v a r i a b l e s . T h e results of t h e p r e s e n t s t u d y s u p p o r t further r e s e a r c h i n t o t h e e f f e c t i v e n e s s o f t h e 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 t o i m p r o v e t h e nutritional v a l u e of c a n o l a m e a l for dairy cattle.  148  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 . Fibrolytic e n z y m e s i n c r e a s e fiber digestibility a n d g r o w t h rate o f s t e e r s f e d dry f o r a g e s . C a n . J . Anim. Sci., 75:641-644. Bell, J . M . 1 9 9 3 . F a c t o r s affecting t h e nutritional v a l u e o f c a n o l a m e a l : A review. Can. J . Anim. Sci. 73:679-697. B i e l y , J . 1 9 8 5 . 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F o r a g e Fiber A n a l y s e s .  (Apparatus,  Reagents, Procedures, a n d s o m e Applications). Agric. Handbook No. 379., A R S - U S D A , Washington, D C .  149  Hidayat, K.; Hillman, N e w b o l d , C . J . a n d C S . Stewart. 1993. T h e contributions of b a c t e r i a a n d p r o t o z o a t o 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 by microbial g a s production. A n i m . F e e d S c i . Technol., 42:193-208. Hotten, 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 feed additive? M i s s e t - World Poult., 7: 1 3 - 1 5 . K r i s h n a m o o r t h y , U., Soller, 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 comparative 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 . Nutr., 6 5 : 2 8 - 3 5 . K r i s h n a m o o r t h y , U., Soller, 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 . 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A n i m . Sci., 70:571-579. Menke,  K. H. a n d H . Steingass. 1988.  Estimation  of the energetic feed  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  using  value rumen  fluid. 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 . , Fritz, D. a n d W . S c h n e i d e r . 1979.  T h e estimation o f the digestibility a n d m e t a b o l i s a b l e e n e r g y content of  ruminant feedingstuffs f r o m the g a s production w h e n they are incubated with r u m e n l i q u o 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 . Dietary fiber c o m p o n e n t s ; relationship to rate a n d extent of ruminal digestion. F e d . Proc. F e d . A m . S o c . E x p . Biol., 36:187-192. Mir, 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 . 1992.  M e t h o d s forproteacting s o y b e a n and c a n o l a proteins from degradation  in t h e r u m e n . C a n . J . A n i m . S c i . , 6 4 : 8 5 3 - 8 6 5 .  150  M o s h t a g h i N i a , S . A . a n d J . R . Ingalls.  1992. Effect of heating  o n canola  p r o t e i n d e g r a d a t i o n in t h e 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  meal  gastrointestinal  tract 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 r b y . 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 1263. 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 monitoring o f g a s production 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 . Schofield, P . a n d A . N . Pell.  1995. Validity of using a c c u m u l a t e d  g a s pressure  r e a d i n g s t o 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 n v o l v i n g  three  forages. J . Dairy S c i . , 78:2230-2238. S l o m i n s k i , B . A . a n d L. D. C a m p b e l l . 1990. N o n - s t a r c h p o l y s a c c h a r i d e s of c a n o l a meal: Quantification,  digestibility  in poultry a n d potential  benefit of dietary  enzyme supplementation. J . Sci. Food A g r i c , 53:175-184. 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 watersoluble carbohydrate determination  a s a tool f o r evaluation o f plant cell wall  degrading enzymes. J . Agric. Food C h e m . , 41:2304-2308. S t a t i s t i c a 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 . T h e o d o r o u , M . K., Williams, 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 . 1994.  A simple  determine  g a s production  t h e fermentation  method  kinetics  using  of ruminant  a pressure feeds.  t r a n s d u c e r to  Anim.  FeedS c i .  Technol., 48:185-197. T i l l e y , 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 f o 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 . Brit. J . G r a s s l . S o c , 1 8 : 1 0 4 - 1 1 1 . V a n S o e s t , P . J . 1 9 8 2 . Nutritional  e c o l o g y of t h e ruminant.  O & B B o o k s Inc.,  Corvallis, Oregon. V a n S o e s t , P . J . 1 9 9 4 . Nutritional e c o l o g y o f the ruminant. 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 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 w i t h 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 production p a r a m e t e r s a n d V F A a n a l y s e s of c a n o l a m e a l .  Factor  L e v e l of application  Heat  c o n t r o l (0), 1 5 , 3 0 , 6 0 m i n  1  Moisture  0%, 10%, 20%, 3 0 %  2  Protease Xylanase  ,2, 3, 4 :  0%, 0.005%, 0.05%, 0.01%  ,2, 3, 5  0%, 0.001%  :  1  A p p l i e d at 5 5 ° C .  2  A p p l i e d o n a % vol / wt b a s i s .  3  S u p p l i e d by F i n n F e e d s International Ltd., Wiltshire, U.K. Heptex sb-protease. Avizyme 1310.  4  5  152  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 production of c a n o l a m e a l .  Protease  1  (%)  R a t e (ml_*h- ) 1  0  0.077  3  0.005  0.075  3  0.01  0.108  b  0.05  0.089  a b  S.E.M.  3  2  0.004  1  A p p l i e d o n a % vol / wt basis.  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 h e e q u a t i o n Y = A (1 S . E . M . , p o o l e d s t a n d a r d error of the m e a n .  3  a b  - e  M e a n s in a c o l u m n w i t h d i f f e r e n t s u p e r s c r i p t s a r e s i g n i f i c a n t l y d i f f e r e n t  153  - '- ).  <  b(  l>  (P < 0.05).  T a b l e 4 . 3 . T h e effect o f p r o t e a s e a n d x y l a n a s e application o n the length o f the 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 treatment  1  P r o t e a s e (%)  X y l a n a s e (%)  Lag (h)  0  0  0.449  c  0.005  0  0.266  a b  0.01  0  0.140  a  0.05  0  0.273  b  0  0.001  0.187  a b  0.005  0.001  0.294  b  0.01  0.001  0.136  a  0.05  0.001  0.187  a b  S.E.M.  3  2  0.046  1  Applied o n a % vol / wt basis.  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 h e e q u a t i o n Y = A ( 1 - e  3  S . E . M . , p o o l e d standard error of the m e a n .  a c  M e a n s in a c o l u m n w i t h d i f f e r e n t s u p e r s c r i p t s a r e s i g n i f i c a n t l y d i f f e r e n t  154  ( b ( t  ~  l >  ).  (P < 0.05).  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 in vitro V F A production of c a n o l a m e a l .  E n z y m e treatment  1  P r o t e a s e (%)  X y l a n a s e (%)  VFA(mmol/g)  0  0  2.58  0.005  0  3.36^  0.01  0  3.92  d  0.05  0  3.26  a b  0  0.001  3.24  b c  0.005  0.001  2.34  a  0.01  0.001  3.88  d  0.05  0.001  2.66  a b  S.E.M.  2  0.208  1  A p p l i e d o n a % vol / wt b a s i s .  2  S . E . M . , p o o l e d s t a n d a r d error of the m e a n .  a _ d  a  M e a n s in a c o l u m n w i t h d i f f e r e n t s u p e r s c r i p t s a r e s i g n i f i c a n t l y d i f f e r e n t  155  (P < 0.05).  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 to p r o p i o n a t e a n d butyrate to p r o p i o n a t e of c a n o l a m e a l .  Protease  1  (%)  A:P  0  2.789  0.005  3.128  0.01  2.890  0.05  3.736  S.E.M.  3  B:P  2  0.59  a  a  0.68  b  a  0.57  a  0.68  b  a  b  0.154  0.253  1  A p p l i e d o n a % vol / wt b a s i s .  2  M o l a r ratios  3  S . E . M . , p o o l e d s t a n d a r d error of the m e a n .  a b  2  M e a n s in a c o l u m n w i t h d i f f e r e n t s u p e r s c r i p t s a r e s i g n i f i c a n t l y d i f f e r e n t  156  (P < 0.05).  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 to p r o p i o n a t e ( B : P ) of c a n o l a m e a l .  Xylanase  1  (%)  A:P  2  B:P  2  0  2.779  0.57  0.001  3.493  0.69  0.108  0.018  S.E.M.  1  3  A p p l i e d o n a % vol / wt b a s i s .  2  M o l a r ratios  3  S . E . M . , p o o l e d s t a n d a r d error of the m e a n .  157  Figure 4.1. T h e computerized g a s production system (adapted from Pell and Schofield, 1993).  158  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  ab  0.08 0.06 0.04 0.02 0 0  0.005  0.01  0.05  P r o t e a s e l e v e l (% v o l / w t )  F i g u r e 4 . 4 . T h e effect of the addition of p r o t e a s e o n the rate of 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 t h r u m e n fluid.  161  0  0.01  0.02  0.03  0.04  0.05  P r o t e a s e l e v e l (% v o l / w t )  F i g u r e 4 . 5 . T h e effect of the addition of 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 the length of the lag p h a s e of c a n o l a m e a l  i n c u b a t e d in vitro w i t h r u m e n f l u i d .  162  F i g u r e 4 . 6 . T h e effect of the addition of 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 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 t h r u m e n f l u i d .  163  CD CD  -+—' CD O  2.5  <  + 0  0.01  + 0.02  0.03  0.04  0.05  P r o t e a s e l e v e l (% v o l / w t )  Figure  4.7.  Linear  relationship  between  level  of  protease  a p p l i c a t i o n a n d t h e ratio 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 vitro 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 = 1 7 . 5 4 6 * p r o t e a s e level + 2.851 : n = 4, R  2  164  = 0.839, S . E . = 0.17).  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 B r i t i s h C o l u m b i a ( B . C . ) , m a x i m i z i n g f e e d utilization  is a primary  goal a s feed  costs increase. Greater a w a r e n e s s of the  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 d a i r y a n d a n i m a l p r o d u c t i o n in t h i s 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 nutrient w a s t e s . B a r l e y g r a i n is the 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 major alternative protein s o u r c e to s o y b e a n m e a l u s e d in r a t i o n s f o r d a i r y c a t t l e . C a n o l a m e a l i s r e s t r i c t e d in d a i r y c a t t l e d i e t s b e c a u s e o f it's h i g h f i b e 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 r a i n i s r e l a t i v e l y h i g h in  starch,  however  research  for improving  it's n u t r i t i o n a l  value  is a l s o  as a  c o n s e q u e n c e o f it's r e l a t i v e l y h i g h f i b e r c o n t e n t . Current p r o c e s s i n g technology for ruminant feedstuffs entails  conventional  m e t h o d s . T h e p o s s i b 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 t h p r o c e s s i n g t e c h n o l o g y h a s yet to b e realized. T h e objective of t h e current study w a s to investigate t h e feasibility  of applying  e n z y m e s a n d mild  hydrothermal  treatment to barley a n d  c a n o l a m e a l in o r d e r t o i m p r o v e t h e i r n u t r i t i o n a l v a l u e f o r d a i r y c a t t l e .  5.1 B A R L E Y In vitro a n a l y s e s w e r e u s e d t o e v a l u a t e t h e e f f e c t s o f b a r l e y t r e a t e d w i t h pg 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 i g e s t i b i l i t i e s o f b a r l e y 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 , d a i r y p r o d u c e r s  165  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 . C . f a c e a u n i q u e c h a l l e n g e t o m a t c h t h e h i g h s o l u b l e n i t r o g e n 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 ( v o n K e y s e r l i n g k et a l . , 1 9 9 6 ) w i t h a  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 o u r c e in o r d e r t o  maximize rumen  microbial  p r o t e i n 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 region  also require  intensive agricultural an  active  role  by  p r a c t i c e s in t h i s e n v i r o n m e n t a l l y p r o d u c e r s to  reduce  fragile  nutrient 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 of n i t r o g e n 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 of f e e d utilization. M i l d h e a t a p p e a r e d not to h a v e a n effect, w h e r e a s the addition of m o i s t u r e h a d a q u a d r a t i c relationship with digestibility. F r o m t h i s 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 i g e s t i b i l i t y o f b a r l e y . H o w e v e r , p o t e n t i a l a p p l i c a t i o n s e x i s t for 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 level (0.01%)  o r a c o m b i n a t i o n of p r o t e a s e a n d p - g l u c a n a s e to  improve the  feeding  quality of barley.  5.2 CANOLA MEAL R e s u l t s o f t h e in vitro d i g e s t i b i l i t i e s 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 z e d 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 o b t a i n information of the effects of the e n z y m e a n d mild hydrothermal treatments o n the fermentation characteristics of c a n o l a m e a l . T h e results of the 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 t w o 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 elicit their e f f e c 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  166  differences between treatments  t h a n the digestibility  experiment, although  similar  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 n c i p 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 enzyme  activity.  The  results  of the  gas  production  experiment  indicated  these  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 elicited a q u a d r a t i c effect o n the digestibility of 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 t h x y l a n a s e w a s detrimental  to digestibility,  s i m i l a r to t h e r e s u l t s p r e v i o u s l y o b s e r v e d with  barley,  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 e v e l a p p e a r e d t o b e s u c c e s s f u l in i n c r e a s i n g the fermentability of 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 t h a t 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 n u t r i t i v e v a l u e o f 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 explained  by  performance  the trials  addition be  of  protease.  conducted  on  However,  it  is  selected treatments  important to  that  clearly  primarily animal  define  the  p o t e n t i a 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 competitive market for both dairy p r o d u c e r s a n d the f e e d a l i k e , will  promote  r e s e a r c h into i n n o v a t i v e  processing technology  industry  s u c h a s that  i n v e s t i g a t e d in t h e e x p e r i m e n t s d i s c u s s e d in t h i s 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 " o f i m p l e m e n t i n g e n z y m e s i n t o r u m i n a n t f e e d s h a v e n o t b e e n d i s c u s s e d in t h i s t h e s i s , enzymes  are  an  expensive  commodity  and  167  their  practical  applications  remain  d e p e n d e n t u p o n t h e r e t u r n o n i n v e s t m e n t into m o s t l i k e l y , m o r e e x p e n s i v e , " v a l u e a d d e d " product.  168  5.4 REFERENCES Huntington, J . A . 1995. R u m e n Compounder, M a y issue.  fermentation  dynamics.  Pages  17-19  in  Feed  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 . Nutr., 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 z a t i o n 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 protein. J . Dairy S c i . , 7 5 : 3 4 5 - 3 5 7 . von  K e y s e r l i n g k , M . A . G . , Swift,  M. L ,  P u c h a l a , R. a n d J . A . S h e l f o r d .  Degradability characteristics of dry matter a n d c r u d e protein ruminants. A n i m . F e e d S c i . Technol., 57:291-311.  169  1996.  of f o r a g e s  in  6.0 APPENDICES  170  1 °  9  C  ^"  LO I s  'CD CO CD  CO  o to  co >, 0 _CD  1  E o  T—  XI  C  O  I - CD  00  00 O  LO CM  S  co oo  CM 00 TtCD  E  Oj| <M  >% CD "O SZ 0) CD O i : CD  N  I S  C  CD  co co  CD  £ -!= <  SZ  CD  XI  CD  CD  O  c CD  T-  O  t1  CD  CD  CM CD CN  CD CO CD  CD XI  o  CQ. CD  T3  •c CD  T3  O  '  1—  00  X>  LO  CD"  x  CO  I S  TO  1 -  CN  o g o  05  CO  3  CD  a)  CD CT  XI  To co  CO CD "CD  -C -*—» LL  O  0 CD  CO  CO  I -  T—  CD  S  CM  CD  O  C  -n CJ  O  CD  X  C  CL  CO  x  CO  CO  o  CD  ct* CD CO O X CD CD >,<6 -t—» N  X  < LU Z T-  w  O  CD  CQ.CL  CD W  11  CD  _ t — • CD -=•  3  Q.  CD  r  ro  T3  co $  =  T3  <D  to  °  =3  N LU  0 3  CO CD ^ ~ CM ~"  CD CO CD  C  •° >,  O CD  c  3  O CD CO  LU  E  to  >.u.  ZS  £  XI  C C  -*-»  L—  CO  CD CD  CD —I  O  CD  -CD i—»  CD  -*—<  _co  co"  — CO  T3 T3 •C  CM  to 'c =3 CD > — t - <  o CD  < C CD CN  T3 co o  CO JD .Q CD  C£  TO  D)  oo  CD  0-  CD-  S'  CL X CD CO  S  CD >  1  CD  |  o  I -  LO  CN  T3 L-  |  co CN CM  E o  CD CO CO CD  or CD  X  CT  CL Q. CO CD  L—  o I— CD CD CO CD  CM  oo d  c  CO  c O  CD -•—»  CO x:  CO CD CO  E  Z)  "-»—»  CO  CD  M—  c o CD  C  CT  CD  _l  •4-»  N  CD CO o o  CO CD  o  E  CD  CO CN O CD D_  CD  0)  CM  2  CO  171  2  I. " 0  APPENDIX 6.2 COMPOSITION OF THE CONCENTRATE RATION FED TO DONOR COWS. T a b l e 6.2. Nutrient c o m p o s i t i o n of the dairy c o n c e n t r a t e ration. Ingredient  C o n t e n t (% a s f e d b a s i s )  Barley (11.5% C P )  58.6  W h e a t millrun  14.7  Canola meal  11.2  Dairy fines  3.5  Beet pulp  3.0  Molasses/cane/LI  2.7  Limestone  1.75  C o r n (8.5%)  1.0  Salt a n d trace mineral mix  0.88  Distillers grain  0.87  S o y a oil  0.75  Raypell binder  0.7  Meat and bone meal  0.35  172  APPENDIX 6.3. SAS PROGRAM USED TO EVALUATE B A R L E Y DATA. options nocentre linesize=75; f i l e n a m e invitro ' f i l e n a m e ' ; data; infile i n v i t r o ; i n p u t f e e d r e p i v d m d ivtd a d f n d f h e a t m o i s t b e t a p r o t x y l ; title 'in v i t r o r e s u l t s a n d f i b e r a n a l y s e s o f b a r l e y ' ; proc glm; c l a s s h e a t moist b e t a prot x y l ; m o d e l i v d m d ivtd a d f n d f = h e a t | m o i s t | b e t a | p r o t | x y l / n o u n i s s 3 ; r a n d o m heat*moist*beta*prot*xyl / test; Ismeans heat moist*beta  moist  moist*prot  beta  prot  moist*xyl  xyl  heat*moist  beta*prot  heat*beta  beta*xyl  prot*xyl  heat*moist*prot  heat*moist* xyl  heat*prot*xyl  heat*beta*xyl  moist*beta*prot  moist*beta*xyl  moist*prot*xyl  beta*prot*xyl  heat*moist*beta*prot  heat*moist*prot*xyl  heat*prot  heat*xyl  heat*moist*beta heat*beta*prot  heat*beta*prot*xyl heat*moist*beta*xyl  moist*beta*prot*xyl / stderr e = heat*moist*beta*prot*xyl; 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 r a c t i o n s 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 t o t h e l a r g e n u m b e r o f f a c t o r s in t h i s e x p e r i m e n t , t h e e r r o r t e r m i n c l u d e s the h i g h e s t o r d e r interaction a s a n indication of e x p e r i m e n t a l error. T h e  173  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 statement.  174  APPENDIX 6.4. SAS PROGRAM USED TO EVALUATE CANOLA MEAL DATA. options nocentre linesize=75; f i l e n a m e invitro ' f i l e n a m e ' ; data; infile i n v i t r o ; i n p u t f e e d r e p i v d m d ivtd a d f n d f h e a t m o i s t p r o t x y l ; title 'in v i t r o r e s u l t s a n d f i b e r a n a l y s e s o f c a n o l a m e a l ; proc glm; c l a s s h e a t moist prot x y l ; m o d e l i v d m d ivtd a d f n d f = h e a t | m o i s t | p r o t | x y l / n o u n i s s 3 ; r a n d o m heat*moist *prot*xyl / test; Ismeans heat moist*xyl  moist  prot*xyl  moist*prot*xyl  prot  xyl  h e a f m o i s t heat*prot  heat*moist*prot  heat*moist*prot*xyl  heat*moist* xyl  heat*xyl  moist*prot  heat*prot*xyl  / stderr e = heat*moist*prot*xyl;  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 r a c t i o n s 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 t o t h e l a r g e n u m b e r o f f a c t o r s in t h i s e x p e r i m e n t , t h e e r r o r t e r m i n c l u d e s the h i g h e s t o r d e r interaction a s a n indication of e x p e r i m e n t a l error. 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 h e s u m o f all 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 statement.  175  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 t o e s t a b l i s h t h e 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 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 this relationship a n d to convert voltage r e s p o n s e to t h e resulting g a s v o l u m e (Pell a n d S c h o f i e l d , 1993). Interpretation of g a s production results requires a c c o m m o d a t i o n for highly soluble g a s e s s u c h a s C 0  2  (Pell a n d Schofield, 1995).  G a s c o m p o s i t i o n is a l s o important b e c a u s e of differing solubilities. V o l u m e c h a n g e s related to c a l i b r a t i o n c o r r e c t i o n s involving 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 i n s i g n i f i c a n t in t h i s 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 t h i n t h e f l a s k r e s u l t s in s o m e o f t h e g a s t o 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 t o 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 h e r e l a t i o n s h i p b e t w e e n t h e 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 t o it's p a r t i a l p r e s s u r e within a fixed v o l u m e container (Schofield a n d Pell, 1995). vol. C0  2  (aqueous) / vol. C0  2  (gas) = 0.0246 R T (VaA/g),  ( E q u a t i o n 1)  w h e r e R i s 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 * K ) , T i s t h e t e m p e r a t u r e in 0  d e g r e e s K e l v i n , Va i s t h e l i q u i d v o l u m e in t h e f l a s k a n d Vg i s t h e a v a i l a b l e g a s v o l u m e within t h e flask. Calibration of the g a s production system w a s conducted under the 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  176  e q u i p m e n t w e r e p r e w a r m e d t o 3 9 ° C ± 1 ° C a n d 1 5 m L o f buffer 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 mL). T h e f l a s k s w e r e repeatedly injected with either 5, 10, 1 5 or 2 0 m L of C 0  2  t h r o u g h t h e port a n d a l l o w e d t o equilibrate f o r 1 0 m i n . T h e d a t a in  T a b l e 6.3 illustrates t h e effect o f p r e s s u r e o n t h e solubility o f C 0 . S e n s o r r e s p o n s e 2  w a s o b t a i n e d b y subtracting t h e difference b e t w e e n t h e v o l t a g e r e c o r d e d at b a s e l i n e from t h evoltage recorded at 10 min. A calibration correction factor w a s obtained for C0  2  s o l u b i l i t y i n t h i s 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) f o r C 0 [COJ  aq/[C0 ]gas  = 0.0246 R T *15 mL/41.5  2  2  at 39°C:  mL = 0.227  T h e g a s production data collected forthe present study w a s calculated using the 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 = 0 . 9 9 4 ) w h i c h d e s c r i b e s t h e relationship 2  between g a s production and subsequent voltage response: Y = 64.60 ±3.58  (mL /V)X-1.27±0.47  mL,  (Equation 2)  w h e r e Y represents the g a s v o l u m e a n d X is the voltage r e s p o n s e p r o d u c e d by the sensors. T a b l e 6.3. T h e effect o f C 0  co  2  Injected 5 mL  2  Amount of C 0  solubility o f g a s o n it's ability t o i n d u c e a p r e s s u r e  2  C0  2  available to induce  Voltage  d i s s o l v e d in buffer  pressure change  R e s p o n s e (V)  1.14 m L  3.86 m L  0.0747  10 m L  2.27 m L  7.73 m L  0.1450  15 m L  3.40 m L  11.60 m L  20 m L  4.54 m L  15. 4 6 m L  0.2039 0.2534  177  6.5.1 REFERENCES Pell, A . N . a n d P . Schofield. 1993. C o m p u t e r i z e d monitoring of g a s production 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 . Schofield, P . a n d A . N . Pell. 1995. Validity of using accumulated g a s pressure r e a d i n g s t o 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 n v o l v i n g t h r e e forages. J . Dairy S c i . , 78:2230-2238.  178  < LL  >- O _l  CO  _l a:  < o  LU  o  o  X CO  V  < O  o  co  • « •  Od  h-  CO  < 11  °  d  II  II  CO LU  > O o  o  °° E  •  AMPL  co"  •  •  o lCO  + X  •  1-  X  CD O) CD  •  o _i zQ> o < LU O 0£ LL. Q. CO  3  o o  < 1-  •• %  • •  CD o < O 3 o UJ Q LL Q O o o a. z O CO o  1-  GA EC  LU  a _j  CO UJ LU i - CO X Q  Z LU  5  Q. O _l 0. < < o  O  a z  < r2  o o  o o  CN  o d o  o co  o  o d  CD  (6/-|iu) uouonpojd se6 papjooa^j  179  o d  CM  o d  APPENDIX 6.7. S A S PROGRAM USED TO DETERMINE G A S PRODUCTION PARAMETERS FOR CANOLA MEAL. options nocentre linesize=75; filename g a s production 'filename'; data; infile g a s p r o d u c t i o n ; input time f e e d r e p l f e e d r e p 2 f e e d r e p 3 ; title ' t e s t o f fitting s i m p l e e x p o n e n t i a l w i t h l a g ' ; p r o c nlin m e t h o d = d u d ; p a r m s a=1 b=0.1 l a g = 0 ; m o d e l y = a*(1 - e x p ( - b * ( t - l a g ) ) ) ; output out=b predicted=yhat residual=yres ; proc print; proc plot; p l o t y*t='o' y h a t * t = " p " / o v e r l a y ; run ;  180  CD  Q CO  CD  q o  > wo ^  '•4-»  LO CN  |£« CT i< T—  2 Q. CO CD CD  2  3 "  O  O  H—  o CD  Q CO  -f—<  CO  r C  o c  o  CO CD  -4—'  c  o o "co E 1_  CD  2  c CO CO CD  . c  Q E o >^  D  -a  c o  I* *i >  0C  T3 _,-  BCO CO T -  2  CD  n >  —  o " ca c O O -I  CO CD CO  o co  c  CD Ui (0 CD  •*~  d  d  CN  Tl-  5 0  c  :  O  _CD £ _Q CO  i— a?  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