Changes in nutrient composition and in vitro ruminal fermentation of total mixed ration silage stored at different temperatures and periods
M a k o t o K o n d o a * , K a z u ma S h i m i z u a , A n u r a g a J a y a n e g a r a b , Ta k a s h i M i s h i m a a , H i r o k i M a t s u i a , S h u i c h i K a r i t a a , M a s a k a z u G o t o a , Ts u t o m u F u j i h a r a a , c .
a
b
F a c u l t y o f B i o r e s o u r c e s , M i e U n i v e r s i t y, Ts u , M i e , 5 1 4 - 8 5 0 7 , J a p a n . F a c u l t y o f A n i m a l S c i e n c e , B o g o r A g r i c u l t u r a l U n i v e r s i t y, B o g o r 1 6 6 8 0 , Indonesia.
c
P h i l i p p i n e C a r a b a o C e n t e r, N u e v a E c i j a , t h e P h i l i p p i n e s
* All correspondence should be addressed to: Makoto Kondo Department of Bioresources, Mie University 1 5 7 7 , K u r i m a m a c h i y a , Ts u , M i e , 5 1 4 - 8 5 0 7 , J a p a n E m a i l : ma k o k @ b i o . mi e - u . a c . j p
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ABSTRACT B A C K G R O U N D : To t a l mi x e d r a t i o n ( T M R ) i s w i d e l y u s e d f o r d a i r y c a t t l e a n d needs to be prepared daily because of rapid deterioration. Ensiling TMR allows p r e s e r v a t i o n a n d s a v e s l a b o u r a t t h e f a r m ; h o w e v e r, s i l a g e f e r m e n t a t i o n ma y i n f l u e n c e v a r i o u s n u t r i t i o n a l c o mp o n e n t s . T h e o b j e c t i v e s o f t h i s s t u d y w e r e t o e v a l u a t e n u t r i t i o n a l c h a n g e s a n d i n v i t ro r u m e n f e r me n t a t i o n o f T M R s i l a g e t h a t w a s s t o r e d a t d i ff e r e n t t e m p e r a t u r e s a n d d u r a t i o n s o n a l a b o r a t o r y s c a l e i n comparison with those of typical TMR before ensiling. R E S U LT S : N o d i s t i n c t c h a n g e s i n c r u d e p r o t e i n ( C P ) , n e u t r a l d e t e r g e n t f i b r e and
non-fibrous
carbohydrate
contents
were
observed
during
silage
f e r m e n t a t i o n . H o w e v e r, c l e a r c h a n g e s w e r e o b s e r v e d i n t h e s o l u b l e C P a n d soluble sugar fractions; solubilisation of the CP fraction in TMR silage was e n h a n c e d b y p r o l o n g e d s t o r a g e a n d h i g h e r s t o r a g e t e mp e r a t u r e s , a n d m o s t soluble sugars were lost during ensiling. Short-chain fatty acid concentrations i n t h e i n v i t ro r u m e n f r o m T M R s b e f o r e a n d a f t e r e n s i l i n g w e r e n o t s i g n i f i c a n t l y d i ff e r e n t ; h o w e v e r, t h r o u g h o u t i n c u b a t i o n , N H 3 - N c o n c e n t r a t i o n s from TMR silages were significantly higher than those from TMR before ensiling. C O N C L U S I O N : A h i g h e r r u m i n a l N H 3 - N c o n c e n t r a t i o n f r o m T M R s i l a g e ma y b e a r e s u l t o f a s h o r t a g e o f f e r m e n t a b l e s u g a r s a n d e n h a n c e d d e a mi n a t i o n o f C P. Feeding TMR ensiled under a high temperature must be investigated to balance p r o t e i n s a n d c a r b o h y d r a t e s f o r r u me n f e r m e n t a t i o n .
K e y w o r d s : t o t a l mi x e d r a t i o n , s i l a g e , s o l u b l e p r o t e i n , i n v i t ro r u m i n a l fermentation
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INTRODUCTION To t a l mi x e d r a t i o n ( T M R ) h a s b e e n w i d e l y u s e d f o r d a i r y c a t t l e b e c a u s e it contains roughage, concentrates, vitamins and minerals in ratios that are w e l l - b a l a n c e d t o me e t t h e a n i m a l ’s r e q u i r e me n t s . H o w e v e r, T M R d e t e r i o r a t e s e a s i l y b e c a u s e i t h a s a h i g h n u t r i e n t c o n t e n t a n d s u f f i c i e n t mo i s t u r e c o n t e n t f o r t h e g r o w t h o f b a c t e r i a , y e a s t a n d mo l d s . 1 T h u s , m o s t f a r m e r s mu s t p r e p a r e T M R o n c e o r t w i c e d a i l y, a n d c o n t r a c t o r s w h o a l s o d o t h i s m u s t d e l i v e r T M R t o f a r m s a l m o s t e v e r y d a y. I n c o n t r a s t , e n s i l e d T M R c a n b e p r e s e r v e d ; t h i s s a v e s labour for TMR preparation, and in some cases, it can be carried for long d i s t a n c e s . 2 L a rg e - s c a l e p r e p a r a t i o n o f T M R s i l a g e b y a c o m p a n y o r c o n t r a c t o r leads to lower feed costs and the use of more high-moisture by-products, which d e t e r i o r a t e e a s i l y. S t u d i e s o n T M R s i l a g e h a v e i n c r e a s e d i n r e c e n t y e a r s , particularly in Asian counties.3-10 Fermentation of TMR silage progresses with h i g h a m o u n t s o f o rg a n i c a c i d s [ a p p r o x i m a t e l y 5 0 t o > 1 0 0 g k g − 1 d r y m a t t e r ( D M ) ] , d e s p i t e t h e h i g h D M c o n t e n t ( a p p r o x i ma t e l y 4 0 0 – 6 0 0 g k g − 1 ) . 2 , 3 , 8 Several studies have shown that TMR silage is stable after opening the silo, e v e n u n d e r h i g h e r t e mp e r a t u r e s , 3 , 8 , 1 1 w h i c h l e a d s t o s t a b l e T M R f e e d i n g without being heated in hot climates. Compared with crop silage, TMR silage typically has high contents of
e n e r g y a n d p r o t e i n s , t h e r e b y e n a b l i n g i t t o m e e t t h e n u t r i e n t r e q u i r e me n t s o f dairy cows. The nutritional value of TMR is based on a mixture of ingredients; t h e r e f o r e , s o me n u t r i e n t s i n T M R s i l a g e a r e e x p e c t e d t o b e l o s t d u r i n g e n s i l i n g , a n d t h e b a l a n c e d i ff e r s b e t w e e n t h e t i m e a t w h i c h i t i s p r e p a r e d a n d t h e t i m e a t w h i c h i t i s f e d t o a n i m a l s . R e s p i r a t i o n b y p l a n t s a n d f e r me n t a t i o n b y m i c r o o rg a n i s m s i n c r o p s i l a g e p r o d u c e s m a l l e r mo l e c u l e s f r o m c a r b o h y d r a t e s
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a n d p r o t e i n s . 1 2 - 1 4 B e c a u s e T M R s i l a g e c a n b e p r e p a r e d t h r o u g h o u t t h e y e a r, w h i c h i s d i ff e r e n t f r o m c r o p s i l a g e , t h e e ff e c t o f t e m p e r a t u r e o n t h e fermentation characteristics and nutrient changes in TMR silages should be u n d e r s t o o d . T h e e n s i l i n g p e r i o d a l s o a ff e c t s t h e s e p a r a me t e r s . I n p a r t i c u l a r, changes in carbohydrates and proteins during ensiling may influence the p r o d u c t i o n o f s h o r t - c h a i n f a t t y a c i d s ( S C FA s ) a n d c o n c e n t r a t i o n s o f N H 3 i n t h e r u m e n , w h i c h a r e h i g h l y r e l a t e d t o e n e rg y a n d p r o t e i n m e t a b o l i s m i n ruminants.15
Therefore,
we
investigated
the
nutritional
changes
and
c h a r a c t e r i s t i c s o f r u m e n f e r m e n t a t i o n i n T M R s i l a g e s s t o r e d a t d i ff e r e n t t e mp e r a t u r e s a n d f o r d i ff e r e n t d u r a t i o n s i n c o mp a r i s o n w i t h t h o s e i n t y p i c a l TMR before ensiling.
M AT E R I A L S A N D M E T H O D S Preparation of TMR and ensiling The ingredients of TMR used in the present study are presented in Ta b l e 1 . I t a l i a n r y e g r a s s s i l a g e , s o rg h u m s i l a g e a n d c o r n s i l a g e , e n s i l e d f o r a p p r o x i m a t e l y 4 , 11 a n d 11 mo n t h s , r e s p e c t i v e l y, w e r e u s e d a s r o u g h a g e . T h e D M c o n t e n t s o f t h e s e s i l a g e s w e r e 4 6 6 , 2 5 7 a n d 2 3 1 g k g − 1 , r e s p e c t i v e l y. T h e other ingredients, including concentrates and minerals, were purchased from feed companies. TMR was designed for dairy cattle, and the total digestible nutrient content was 730 g kg−1 DM, which was calculated from the Standard Ta b l e s o f F e e d C o m p o s i t i o n i n J a p a n . 1 6 T h e s e i n g r e d i e n t s w e r e mi x e d i n a practical scale (total of 2,800 kg), and approximately 100 kg of TMR was used for
the
present
s t u d y.
TMR
was
packed
into
1,000-mL
high-density
polyethylene bottles (1.4 mm thickness), fitted with a rubber cap and
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g a s - r e l e a s e b u l b ; t h i s w a s m a i n t a i n e d a t 1 5 ° C o r 3 0 ° C t o mi m i c a n n u a l a v e r a g e a n d s u m m e r c l i ma t e s , r e s p e c t i v e l y. T h e D M d e n s i t y o f t h e s e s i l a g e s w a s adjusted to approximately 330 kg DM m−3, which was close to that of practical TMR silage in Japan. The silos were opened at 2, 5, 10, 30 and 90 days after ensiling. The silos were prepared in five replicates for 30 and 90 days and in three replicates for 2, 5 and 10 days. All bottles were weighed before and after ensiling, and DM loss during fermentation was calculated. An approximately 1 k g T M R s a m p l e w a s r a n d o m l y c o l l e c t e d f i v e t i me s d u r i n g p r e p a r a t i o n b e f o r e ensiling.
I n v i t ro r u m i n a l f e r m e n t a t i o n s t u d y A n i n v i t ro r u mi n a l s t u d y w a s c o n d u c t e d a c c o r d i n g t o t h e m e t h o d s described
by
Uddin
et
al.17
TMRs
before
and
after
ensiling
(n
=
5
r e p l i c a t e s / t r e a t me n t ) w e r e d r i e d a t 6 0 ° C f o r 4 8 h a n d g r o u n d t o p a s s t h r o u g h a 1 - m m s c r e e n . A p p r o x i m a t e l y 1 g o f s a mp l e w a s w e i g h e d i n t o a 1 0 0 - m L g l a s s v i a l . R u m e n f l u i d w a s c o l l e c t e d v i a a r u me n c a n n u l a b e f o r e t h e m o r n i n g feeding from two Holstein cows that were fed TMR of the same nutrient value a s t h a t i n t h e p r e s e n t s t u d y. T h e f l u i d s a m p l e s w e r e p o o l e d a n d f i l t e r e d t h r o u g h f o u r l a y e r s o f c h e e s e c l o t h . G l a s s v i a l s w e r e f i l l e d w i t h 5 0 mL o f m e d i u m c o n s i s t i n g o f o n e p a r t o f r u m e n f l u i d a n d t w o p a r t s o f M c D o u g a l l b u ff e r, i n c u b a t e d i n a 3 9 ° C w a t e r b a t h a n d s h a k e n a t 1 6 0 r p m. A f t e r 4 , 8 a n d 2 4 h o f i n c u b a t i o n , 0 . 5 mL o f t h e l i q u i d me d i u m i n t h e v i a l s w a s w i t h d r a w n w i t h a s t e r i l i s e d s y r i n g e f o r S C FA a n d N H 3 - N a n a l y s e s . T h e i n v i t ro r u mi n a l s t u d y w a s r e p l i c a t e d t w i c e , a n d t h e me a n v a l u e s a r e p r e s e n t e d .
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C h e mi c a l a n a l y s e s DM contents of TMRs before and after ensiling were determined by oven-drying, as described above, without correcting for loss of volatiles. F e r me n t e d p r o d u c t s ( l a c t i c a c i d , S C FA a n d N H 3 - N ) a n d t h e p H o f T M R s w e r e determined from water extracts. TMRs (30 g) were macerated with 210 mL distilled water and filtered to obtain a water extract. The pH values of the e x t r a c t s w e r e m e a s u r e d u s i n g a p H me t e r. L a c t i c a c i d a n d S C FA c o n c e n t r a t i o n s w e r e a n a l y s e d w i t h a h i g h - p e r f o r ma n c e l i q u i d c h r o m a t o g r a p h y ( H P L C ) s y s t e m e q u i p p e d w i t h a n i o n - e x c l u s i o n c o l u m n u s i n g a p o s t - c o l u m n p H - b u ff e r e d electron conductivity detection method (Shimadzu Kyoto, Japan). NH3-N contents in the extracts were determined using the steam distillation method. The compositions of the following nutrients were analysed in dried and ground s a m p l e s , a s d e s c r i b e d a b o v e . O rg a n i c m a t t e r ( O M ) , c r u d e p r o t e i n ( C P, t o t a l nitrogen × 6.25) and ether extract (EE) contents were measured according to t h e o f f i c i a l me t h o d s o f t h e A s s o c i a t i o n o f O f f i c i a l A n a l y t i c a l C h e m i s t s . 1 8 O M w a s c a l c u l a t e d b y s u b t r a c t i n g t h e a s h c o n t e n t ( me t h o d n o . 9 4 2 . 0 5 ) . C P w a s d e t e r m i n e d u s i n g t h e K j e l d a h l me t h o d ( 9 8 4 . 1 3 ) , a n d t h e E E c o n t e n t w a s d e t e r m i n e d b y S o x h l e t e x t r a c t i o n ( me t h o d n o . 9 2 0 . 3 9 ) . N e u t r a l d e t e r g e n t f i b r e ( N D F ) w a s d e t e r mi n e d a c c o r d i n g t o t h e m e t h o d s o f Va n S o e s t e t a l . 1 9 u s i n g b o t h s o d i u m s u l p h i t e a n d h e a t - s t a b l e a l p h a - a my l a s e a n d e x p r e s s e d a s t h e a s h - f r e e f o r m . T h e a m o u n t o f s o l u b l e C P e x t r a c t e d w i t h n e u t r a l b u ff e r w a s determined using the method of Licitra et al.20 Non-fibrous carbohydrate (NFC) contents were calculated using the following equation: NFC = OM − CP − N D F − E E . 1 5 S o l u b l e s u g a r s e x t r a c t e d w i t h a q u e o u s e t h a n o l ( 8 0 0 mL L − 1 ) were
analysed
using
the
phenol–sulphuric
acid
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reaction.20
The
S C FA
c o n c e n t r a t i o n i n l i q u i d m e d i u m o f t h e i n v i t ro r u m e n w a s a n a l y s e d b y H P L C , a s d e s c r i b e d a b o v e . T h e N H 3 - N c o n c e n t r a t i o n i n t h e me d i u m w a s d e t e r mi n e d using the indophenol reaction.22
Statistical analyses A completely randomised design was employed to allocate treatments into e x p e r i me n t a l u n i t s . F e r m e n t a t i o n c h a r a c t er i s t i c s , n u t r i e n t c o m p o s i t i o n a n d t h e i n v i t ro r u m i n a l f e r m e n t a t i o n p a r a m e t e r s o f T M R s b e f o r e a n d a f t e r e n s i l i n g w e r e a n a l y s e d b y o n e - w a y a n a l y s i s o f v a r i a n c e ( A N O VA ) a n d f u r t h e r t e s t e d u s i n g Tu k e y ’s t e s t t o c o mp a r e t r e a t me n t m e a n s . T h e T M R d a t a b e f o r e a n d a f t e r ensiling were compared using an orthogonal contrast test. The TMR data after e n s i l i n g w e r e s u b s e q u e n t l y a n a l y s e d b y f a c t o r i a l A N O VA , i n w h i c h t h e t w o f i x e d f a c t o r s w e r e t h e n u mb e r o f s t o r a g e d a y s a n d t e mp e r a t u r e . A l l s t a t i s t i c a l a n a l y s e s w e r e p e r f o r m e d u s i n g S A S s o f t w a r e v e r. 9 . 3 ( S A S I n s t i t u t e , C a r y, N C , USA).
R E S U LT S A N D D I S C U S S I O N The pH value of TMR before ensiling was 5.8, while the pH values of the s i l a g e s a f t e r e n s i l i n g r a n g e d f r o m 4 . 2 t o 4 . 4 ( Ta b l e 2 ) ( P < 0 . 0 5 ) . T h e p H rapidly dropped during the ensiling period until 10 days after ensiling in TMR s t o r e d a t 3 0 ° C , a n d t h e s e v a l u e s w e r e ma i n t a i n e d f o r 9 0 d a y s ( F i g u r e 1 ) . T h e pH of TMR silage stored at 15°C gradually changed and decreased from 30 to 9 0 d a y s . L a c t i c a c i d w a s t h e d o mi n a n t f e r me n t e d p r o d u c t i n T M R s i l a g e a n d was significantly higher in silages stored at 30°C than in those stored at 15°C ( P < 0 . 0 0 1 ) ( Ta b l e 2 ) . W h e n t h e e n s i l i n g p e r i o d w a s p r o l o n g e d f r o m 3 0 t o 9 0
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days, the changes in the lactic acid content were small at both storage t e mp e r a t u r e s . T h e a c e t i c a c i d c o n t 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 w i t h e n s i l i n g ( P < 0 . 0 5 ) , a n d a s i g n i f i c a n t i n t e r a c t i o n e ff e c t w a s d e t e c t e d b e t w e e n t h e e n s i l i n g p e r i o d a n d t e mp e r a t u r e ( P < 0 . 0 5 ) . A h i g h o rg a n i c a c i d c o n t e n t w a s a characteristic of TMR silage, although it also contains high DM.2,9 According to previous studies using practical scales, lactic acid in TMR silage can be as high as 30–100 g kg−1DM and it varies depending on the feed ingredients, s e a s o n a n d e n s i l i n g m e t h o d . 2 , 6 , 9 , 2 3 A h i g h D M c o n t e n t s u p p r e s s e s f e r me n t a t i o n in
silage13
because
bacterial
growth
and
metabolism
are
lower
in
a
l o w - m o i s t u r e e n v i r o n me n t . S p e c i f i c b a c t e r i a a r e r e p o r t e d l y f o u n d i n T M R silage but have not been isolated from crop silage.23 Interactions among these s p e c i f i c b a c t e r i a a n d h i g h l y f e r me n t a b l e s u b s t r a t e s i n T M R c o u l d e n h a n c e organic acid production in TMR silages. Butyric acid, an indicator of undesirable
fermentation,
was
found
in
low
amounts.
Although
some
d i ff e r e n c e s w e r e o b s e r v e d i n t h e b u t y r i c a c i d c o n t e n t b e f o r e a n d a f t e r e n s i l i n g , the changes were small. Fermentation by butyric acid bacteria such as Clostridium does not actively occur in high DM TMR silages because their water requirement is high.12 The butyric acid in TMR silages was from crop silages, which are used in TMR as ingredients, and was not produced during the fermentation of TMR silages, as shown by Cao et al.24 The storage temperature a n d d u r a t i o n a ff e c t e d t h e N H 3 - N : t o t a l N ( T N ) r a t i o i n T M R s i l a g e s ( P < 0 . 0 5 ) ; h o w e v e r, t h e r a t i o w a s 3 2 . 5 – 4 9 . 6 g k g − 1 T N , w h i c h w a s l o w e r t h a n t h a t o f c r o p silage (100–200 g kg−1TN).12 These data indicate that compared with crop s i l a g e , d e a m i n a t i o n ma y b e s t r o n g l y s u p p r e s s e d i n T M R s i l a g e . D M l o s s e s d u r i n g e n s i l i n g w e r e < 5 % i n a l l T M R s i l a g e s , a n d n o e ff e c t o f t h e s t o r a g e
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t e mp e r a t u r e o r d u r a t i o n w a s o b s e r v e d . D M l o s s e s w h i l e e n s i l i n g c r o p s i l a g e o c c u r a s a r e s u l t o f m i c r o b i a l f e r m e n t a t i o n , e ff l u e n t a n d p l a n t r e s p i r a t i o n , a n d l o s s e s h a v e b e e n e s t i ma t e d t o b e 7 % t o > 4 0 % . 1 2 R e l a t i v e l y l o w e r l o s s o f D M from TMR silage than those from crop silage can be explained by several f a c t o r s . N o e f f l u e n t w a s o b s e r v e d f r o m a n y o f t h e T M R s i l a g e s , w h i c h ma y have been because of their high DM content. Losses due to plant respiration may also have been lower because the crops incorporated into TMRs had been e n s i l e d ; t h u s , mo s t p l a n t c e l l s w o u l d n o t h a v e b e e n p h y s i o l o g i c a l l y a c t i v e . C o m p a r e d w i t h f e r m e n t a t i o n p r o d u c t s i n T M R s i l a g e s , i t i s mo r e i m p o r t a n t t o d e t e r mi n e t h e n u t r i e n t c o m p o s i t i o n b e c a u s e n u t r i e n t s d i r e c t l y a ff e c t m i l k p r o d u c t i o n b y d a i r y c o w s . N o d i s t i n c t c h a n g e s i n O M , C P, E E o r N D F c o n t e n t s w e r e o b s e r v e d d u r i n g s i l a g e f e r m e n t a t i o n ( Ta b l e 3 ) . T h e r e w e r e no clear changes in the NFC content as a result of the ensiling period or t e mp e r a t u r e , w h e r e a s t h e s o l u b l e s u g a r c o n t e n t s i g n i f i c a n t l y d e c r e a s e d t o < 1 0 g kg−1 DM (P < 0.05). Starch, sugars, pectin and organic acids are included in the NFC fraction.15 Soluble sugars are converted to lactic acid and acetic acid during silage fermentation; therefore, the NFC content did not change much; h o w e v e r, t h e c o mp o n e n t s c l e a r l y c h a n g e d . B a c t e r i a i n t h e r u me n r a p i d l y o b t a i n e n e r g y a s AT P f r o m t h e b r e a k d o w n o f s u g a r s b u t n o t f r o m o rg a n i c a c i d s ; t h u s , the energy supply from rapidly fermentable substrates may have been lower in TMR silages after ensiling than in those before ensiling. Hall et al.25 reported on the neutral detergent soluble carbohydrate fraction in detail and showed that t h e c o mp o n e n t s i n t h e N F C f r a c t i o n v a r y a m o n g f e e d s t u ff s . D e t a i l e d a n a l y s e s of the NFC fractions in TMR silages are required to reveal rumen-fermentable carbohydrates.
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Clear changes were observed in the soluble CP fraction, which increased from 302 g kg−1 before ensiling to 338–455 g kg−1 after ensiling on a CP basis. This result indicates that proteins in TMR were broken down to p e p t i d e s , a mi n o a c i d s a n d N H 3 d u r i n g e n s i l i n g . A h i g h s o l u b l e C P i n t a k e b y cattle causes excess NH3-N in the rumen, leading to a higher urinary N loss.15,26 The soluble CP fractions were significantly higher in TMR silage stored at 30°C than in that stored at 15°C after ensiling for 90 days (P < 0.05); h o w e v e r, t h e r e s u l t w a s n o t s i g n i f i c a n t a f t e r e n s i l i n g f o r a s h o r t e r d u r a t i o n . Prolonging the ensiling period from 30 to 90 days also increased the soluble CP content (P < 0.05). Compared with the fermentation process shown in Figure 1, changes in the protein fractions clearly occurred after acid was produced and the pH of TMR silage was stabilised. Protein degradation during ensiling of crop silage is thought to be caused by proteases from fresh plant material and m i c r o o rg a n i s m s . 1 2 A l t h o u g h w e d i d n o t d e t e r m i n e p r o t e a s e a c t i v i t i e s i n t h e ingredients, the contribution of these activities to protein degradation during ensiling would be low because these activities are much lower in silage than in fresh materials.27 The proteolytic activity of grains during ensiling remains unknown. It has been reported that protease activities in grains gradually decline
during
ma t u r a t i o n , 2 8
whereas
they
drastically
increase
after
germination.29 These findings suggest that protease activities in grains used as T M R i n g r e d i e n t s a r e l o w b e c a u s e t h e g r a i n s a r e w e l l ma t u r e d a n d h a v e n o t germinated.
Heat
processing
grains
by
s t e a mi n g
and
rolling
partially
d e a c t i v a t e s e n z y m e s ; h o w e v e r, t h e e f f e c t o f p r o c e s s i n g o n t h e s e a c t i v i t i e s remains unknown. Protein breakdown caused by Clostridium and enterobacteria that produce proteases occurs in silage.12 As described above, the possibility
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t h a t C l o s t r i d i u m g r e w i n T M R s i l a g e s w a s l o w b e c a u s e o f h i g h D M . M o r e o v e r, b u t y r i c a c i d w a s n o t p r o d u c e d d u r i n g e n s i l i n g i n t h e p r e s e n t s t u d y. C a o e t a l . 9 reported that the number of enterobacteria are consistently low during TMR fermentation. Therefore, it seems that protein degradation by these bacteria is n o t a ma j o r f a c t o r d u r i n g T M R e n s i l i n g . I n c o n t r a s t , l a c t i c a c i d b a c t e r i a m a y have been predominant in TMR silages because of the high lactic acid content and low pH. Lactic acid bacteria are virtually non-proteolytic12; thus, their c o n t r i b u t i o n t o p r o t e i n d e g r a d a t i o n i n T M R s i l a g e s w o u l d b e e x t r e me l y l o w. H o w e v e r, t h e s o l u b l e p r o t e i n c o n t e n t i n c r e a s e d f r o m 3 0 t o 9 0 d a y s i n t h e p r e s e n t s t u d y, s u g g e s t i n g t h a t l a c t i c a c i d b a c t e r i a ma y h a v e h a d s o me e f f e c t o n p r o t e i n b r e a k d o w n . Wi n t e r s e t a l . 3 0 d e m o n s t r a t e d t h a t s o me l a c t i c a c i d b a c t e r i a e n h a n c e p r o t e i n d e g r a d a t i o n d u r i n g e n s i l i n g . H o w e v e r, f u r t h e r s t u d i e s o n proteases of lactic acid bacteria are required to understand protein degradation in TMR silage. I n t h e i n v i t ro r u m e n s t u d y, t h e S C FA c o n c e n t r a t i o n f r o m T M R a f t e r a 4-h incubation before ensiling was slightly higher than that after ensiling; h o w e v e r, n o s i g n i f i c a n t d i ff e r e n c e w a s o b s e r v e d a mo n g t h e t r e a t me n t s a f t e r 8 a n d 2 4 - h i n c u b a t i o n s ( Ta b l e 4 ) . T h e s e r e s u l t s i n d i c a t e t h a t t h e e n e rg y s u p p l y for ruminants from TMR silage after ensiling was not lower than that from TMR before ensiling. Lactic acid in the rumen is mainly metabolised to propionic acid as well as acetic and butyric acids.31 The high lactic acid c o n c e n t r a t i o n i n T M R a f t e r e n s i l i n g m a y h a v e b e e n me t a b o l i s e d t o p r o p i o n i c a c i d i n t h e i n v i t ro r u m e n , a s s h o w n b y t h e l o w e r r a t i o s o f a c e t i c a n d p r o p i o n i c acids from TMR silages. Throughout incubation, NH3-N concentrations from T M R s i l a g e s i n t h e i n v i t ro r u m e n w e r e 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 o s e f r o m
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TMR silages before ensiling. These results are consistent with those of Cao et al.24 who showed higher NH3-N concentrations in the rumen of sheep fed a TMR silage than in that of sheep fed TMR before ensiling. No NH3 absorption b y t h e r u m e n w a l l o r f l o w t o t h e o m a s u m o c c u r r e d i n t h e c l o s e d i n v i t ro r u m e n system; thus, lowering the NH3 content was highly dependent on bacterial uptake. NH3 production in this system was mainly derived from the degradation of feed protein. The higher NH3-N concentration from TMR silages in the in v i t ro r u me n ma y h a v e b e e n a r e s u l t o f t h e r a p i d r e l e a s e o f N H 3 f r o m p r o t e i n s and lower NH3 uptake by bacteria. As described above, TMR after ensiling contained a higher soluble CP content than that before ensiling. Soluble p r o t e i n s c o n t a i n s m a l l N c o m p o u n d s s u c h a s p e p t i d e s a n d a mi n o a c i d s , w h i c h r a p i d l y r e l e a s e N H 3 i n t o t h e r u m e n . 1 5 S i m i l a r l y, f e w e r s u g a r s r e ma i n e d i n T M R s i l a g e , w h i c h l e d t o a s h o r t a g e o f e n e rg y f o r r u m e n b a c t e r i a t o t a k e u p N H 3 . The NH3-N concentration at the beginning of the incubation was 3.38 mg dL−1 ( d a t a n o t s h o w n ) ; t h u s , N H 3 ma y h a v e b e e n t a k e n u p b y b a c t e r i a u n t i l 4 h o f incubation when TMR before ensiling was used as the substrate. In contrast, the release of NH3 from CP was higher than its uptake by rumen bacteria when T M R s i l a g e s w e r e u s e d . T h e s e r e s u l t s s u g g e s t t h a t N m e t a b o l i s m i n t h e r u me n o f c o w s f e d T M R s i l a g e a f t e r e n s i l i n g w a s a l t e r e d i n c o mp a r i s o n w i t h t h a t i n t h e r u me n o f c o w s f e d T M R b e f o r e e n s i l i n g .
CONCLUSIONS TMR after ensiling had lower soluble sugar and higher soluble CP contents than that before ensiling. CP solubilisation was enhanced in TMR silage by both prolonged storage and higher storage temperatures. The
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presence of a higher ruminal NH3 concentration from TMR silage during the in v i t ro r u m i n a l i n c u b a t i o n m a y h a v e b e e n c a u s e d b y a s h o r t a g e o f f e r me n t a b l e s u g a r s a n d e n h a n c e d d e a mi n a t i o n o f s o l u b l e C P c o m p o u n d s . F u r t h e r s t u d i e s a r e required to investigate the nutritional changes in TMR silage on a practical s c a l e a n d t o i d e n t i f y N b a l a n c e i n r u mi n a n t s f e d T M R b e f o r e a n d a f t e r e n s i l i n g .
ACKNOWLEDGEMENTS A u t h o r s a l s o t h a n k t o D r Ya m a mo t o a n d me m b e r s i n M i e P r e f e c t u r a l Livestock Institute for supply of TMR and rumen fluid. This research is supported by a grant from the ‘Research for production of valuable livestock by feeding self-sufficient forage crops’ project of the Ministry of Agriculture, Forestry and Fisheries of Japan.
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Vo d k i n L O a n d S c a n d a l i o s J G , D e v e l o p m e n t a l e x p r e s s i o n o f g e n e t i c a l l y d e f i n e d p e p t i d a s e s i n m a i z e . P l a n t P h y s i o l 6 3 : 11 9 8 - 2 0 4 ( 1 9 7 9 ) .
29
F e l l e r U , S o o n g T S T a n d H a g e ma n R H , P a t t e r n s o f p r o t e o l y t i c e n z y m e a c t i v i t i e s i n d i ff e r e n t t i s s u e s o f g e r mi n a t i n g c o r n ( Z e a m a y s L . ) . P l a n t a 140:155-162 (1978).
30
Wi n t e r s A L , C o c k b u r n J E , D h a n o a M S a n d M e r r y R J , E ff e c t s o f l a c t i c a c i d bacteria in inoculants on changes in amino acid composition during e n s i l a g e o f s t e r i l e a n d n o n s t e r i l e r y e g r a s s . J A p p l M i c ro b i o l 8 9 : 4 4 2 - 4 5 1 (2000).
31
J a a k k o l a S a n d H u h t a n e n P, R u m e n f e r me n t a t i o n a n d m i c r o b i a l p r o t e i n s y n t h e s i s i n c a t t l e g i v e n i n t r a r u mi n a l i n f u s i o n s o f l a c t i c a c i d w i t h a g r a s s s i l a g e b a s e d d i e t . J A g r i c S c i 11 9 : 4 11 - 4 1 8 ( 1 9 9 2 ) .
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Ta b l e 1 . I n g r e d i e n t s o f T M R u s e d i n t h i s s t u d y Ingredients
kg-1
g DM
Italian ryegrass silage
138
Sorghum silage
78
Maize silage
70
Beet pulp
68
Steam rolled maize
208
Steam rolled barley
45
Wheat barn
44
Roasted soybean
48
Soybean meal
123
Corn gluten feed
45
Mineral mixture
12
Calcium carbonate
8
Sodium chloride
4
Magnesium oxide
4
Others
2
To t a l
1000
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Tab l e 2 . F er m en t at i o n ch ar a ct er i st i c s o f T M R b ef o r e an d af t er en si l i n g B ef o r e en si l i n g -1
551
Dr y m at t er ( g k g )
5.79a
30 days 1 5 °C
2
547
P r o b ab i l i t y
90 days
3 0 °C
2
539
1 5 °C
2
536
4.44b
4.28c
4.26c
1
3 0 °C
SEM 2
548 4.20d
Bef o r e v s Af t er
Da y #
Temp . #
D ay × Temp . #
***
***
***
4.01 0.006
***
-1
20.1c
89.3b
108.9a
92.5b
104.7a
1.90
***
-1
12.6b
32.8ab
34.2ab
39.3a
31.0b
1.73
***
2.3a
2.0a
2.0a
1.4b
1.4b
0 . 11
***
***
21.0d
32.6c
46.0ab
42.6b
49.6a
1.29
***
***
pH La c t i c aci d ( g k g D M) Ac et i c a ci d ( g k g DM ) -1
B u t y r i c a ci d ( g k g DM ) -1
Af t er en si l i n g 1
NH 3 - N ( g k g t o t al N)
3.39 3.88 4.57 4.57 DM l o s s e s ( % ) 0.571 1 S t o r ag e p er i o d o f T MR si l a g e. 2 S t o r ag e t e mp er at u r e o f T M R si l a g e. a,b,c,d Val u e s wi t h d i ff er en t l et t er s i n a r o w ar e si g n i f i c an t l y d i ff e r en t ( P < 0 . 0 5 ) . * P < 0.05, ** P < 0.01, *** P < 0.001. # C o n d u ct ed o n l y t o s am p l e s af t er e n si l i n g .
*** * ***
*
Tab l e 3 . N u t r i en t co m p o si t i o n o f T M R b ef o r e an d af t er en s i l i n g B ef o r e en s i l i n g -1
Or g an i c m at t er ( g k g DM ) C P ( g k g - 1 D M) Soluble CP (g kg-1 CP) N F C 3 ( g k g - 1 DM )
A f t er en s i l i n g 30 days 15°C
2
1
P r o b ab i l i t y
90 days
3 0 °C
2
1 5 °C
2
1
3 0 °C
SEM 2
B ef o r e v s A f t er
918a
915bc
913c
916b
0.5
155c
163ab
166a
153c
158bc
1.4
**
***
*
302d
338cd
354c
401b
455a
8.8
***
***
***
364a
349ab
335b
321b
345ab
6.5
**
***
52.6a
7.1b
7.2b
7.0b
7.2b
0.72
***
Ether ex tract ( g k g-1DM)
30.4b
30.1b
32.9ab
34.3a
34.4a
0.91
*
381 376 380 404 378 7.7 S t o r ag e p er i o d o f T MR si l a g e. 2 S t o r ag e t e mp er at u r e o f T M R si l a g e. 3 No n f i b r o u s car b o h y d r at e. a,b,c,d Val u e s wi t h d i ff er en t l et t er s i n a r o w ar e si g n i f i c an t l y d i ff e r en t ( P < 0 . 0 5 ) . * P < 0 .05, ** P < 0.01, *** P < 0.001 # C o n d u ct ed o n l y t o s am p l e s af t er e n si l i n g . 1
Temp . #
916ab
S o l u b l e s u g a r s ( g k g - 1 DM ) NDF (g k g-1D M)
Day#
**
Da y × Temp . # *** *
Tab l e 4 . Co n c en t r at i o n s o f s h o r t - ch ai n f at t y a ci d an d a m mo n i a n i t r o gen i n i n vi t ro r u me n i n cu b at ed w i t h T M R b ef o r e an d af t er en si l i n g B ef o r e en s i l i n g
A f t er en s i l i n g 30 days 1 5 °C
2
1
P r o b ab i l i t y
90 days
3 0 °C
2
1 5 °C
2
1
30°C
SEM 2
B ef o r e v s Af t er
D ay #
Temp . #
Da y × Temp . #
4 h af t er i n c u b at i o n To t al S C FA 3 ( m M ) A P r at i o
4 -1
NH3-N (mg dL )
39.8a
37.7bc
3.37
a
1.42
c
3.00
bc
3.31
b
37.5c
39.3ab
38.5abc
2.67
d
3.09
b
2.88
c
4.89
a
4.23
a
4.93
a
0.40
**
**
0.048
***
**
***
0.173
***
*
***
*
8 h af t er i n c u b at i o n To t al S C FA ( m M ) A P r at i o -1
NH3-N (mg dL ) 2 4 h af t er i n To cu bt al at iSoC n FA ( m M ) A P r at i o
5
64.1 2.65 0.15
d
102 2.62
-1
65.9 a
67.0
2.21
b
1.93
c
98 a b
2.28
66.0
1.95
c
2.67
b
103 b a
2.08
67.1
2.19
b
3.61
a
102 c a
2.30
0.62
2.07
bc
0.033
***
3.17
ab
0.141
***
103 b a
2.07
***
**
1.5 c a
0.019
NH3-N (mg dL ) 4.65 6.67 7.75 7.41 7.09 0.466 S t o r ag e p er i o d o f T M R si l ag e. 2 S t o r ag e t e mp er at u r e o f T M R s i l a g e. 3 S h o r t -ch ai n f at t y a c i d . 4 R at i o o f a c et i c a ci d t o p r o p i o n i c aci d a,b,c,d Val u e s wi t h d i ff er en t l et t er s i n a r o w ar e si g n i f i c an t l y d i ff e r en t (P < 0 . 0 5 ) . * P < 0 .05, ** P < 0.01, *** P < 0.001 # C o n d u ct ed o n l y t o s am p l e s af t er e n s i l i n g. 1
***
*** ***
***
6 5.5
pH
5 4.5 4 3.5 0
10
20
30
40
50
60
70
80
90
30 40 50 60 70 Ensiling period (day)
80
90
Ensiling period (day) 120
Lactic acid (g kg-1 DM)
100 80 60 40 20 0 0
10
20
Figure 1. Changes in pH and lactic acid concentration of TMR silages stored at 15°C and 30°C.○; stored at 15°C, ■; stored at 30°C.
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M a k o t o K o n d o a * , K a z u ma S h i m i z u a , A n u r a g a J a y a n e g a r a b , Ta k a s h i M i s h i m a a , H i r o k i M a t s u i a , S h u i c h i K a r i t a a , M a s a k a z u G o t o a , Ts u t o m u F u j i h a r a a , c .
a
b
F a c u l t y o f B i o r e s o u r c e s , M i e U n i v e r s i t y, Ts u , M i e , 5 1 4 - 8 5 0 7 , J a p a n . F a c u l t y o f A n i m a l S c i e n c e , B o g o r A g r i c u l t u r a l U n i v e r s i t y, B o g o r 1 6 6 8 0 , Indonesia.
c
P h i l i p p i n e C a r a b a o C e n t e r, N u e v a E c i j a , t h e P h i l i p p i n e s
* All correspondence should be addressed to: Makoto Kondo Department of Bioresources, Mie University 1 5 7 7 , K u r i m a m a c h i y a , Ts u , M i e , 5 1 4 - 8 5 0 7 , J a p a n E m a i l : ma k o k @ b i o . mi e - u . a c . j p
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ABSTRACT B A C K G R O U N D : To t a l mi x e d r a t i o n ( T M R ) i s w i d e l y u s e d f o r d a i r y c a t t l e a n d needs to be prepared daily because of rapid deterioration. Ensiling TMR allows p r e s e r v a t i o n a n d s a v e s l a b o u r a t t h e f a r m ; h o w e v e r, s i l a g e f e r m e n t a t i o n ma y i n f l u e n c e v a r i o u s n u t r i t i o n a l c o mp o n e n t s . T h e o b j e c t i v e s o f t h i s s t u d y w e r e t o e v a l u a t e n u t r i t i o n a l c h a n g e s a n d i n v i t ro r u m e n f e r me n t a t i o n o f T M R s i l a g e t h a t w a s s t o r e d a t d i ff e r e n t t e m p e r a t u r e s a n d d u r a t i o n s o n a l a b o r a t o r y s c a l e i n comparison with those of typical TMR before ensiling. R E S U LT S : N o d i s t i n c t c h a n g e s i n c r u d e p r o t e i n ( C P ) , n e u t r a l d e t e r g e n t f i b r e and
non-fibrous
carbohydrate
contents
were
observed
during
silage
f e r m e n t a t i o n . H o w e v e r, c l e a r c h a n g e s w e r e o b s e r v e d i n t h e s o l u b l e C P a n d soluble sugar fractions; solubilisation of the CP fraction in TMR silage was e n h a n c e d b y p r o l o n g e d s t o r a g e a n d h i g h e r s t o r a g e t e mp e r a t u r e s , a n d m o s t soluble sugars were lost during ensiling. Short-chain fatty acid concentrations i n t h e i n v i t ro r u m e n f r o m T M R s b e f o r e a n d a f t e r e n s i l i n g w e r e n o t s i g n i f i c a n t l y d i ff e r e n t ; h o w e v e r, t h r o u g h o u t i n c u b a t i o n , N H 3 - N c o n c e n t r a t i o n s from TMR silages were significantly higher than those from TMR before ensiling. C O N C L U S I O N : A h i g h e r r u m i n a l N H 3 - N c o n c e n t r a t i o n f r o m T M R s i l a g e ma y b e a r e s u l t o f a s h o r t a g e o f f e r m e n t a b l e s u g a r s a n d e n h a n c e d d e a mi n a t i o n o f C P. Feeding TMR ensiled under a high temperature must be investigated to balance p r o t e i n s a n d c a r b o h y d r a t e s f o r r u me n f e r m e n t a t i o n .
K e y w o r d s : t o t a l mi x e d r a t i o n , s i l a g e , s o l u b l e p r o t e i n , i n v i t ro r u m i n a l fermentation
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INTRODUCTION To t a l mi x e d r a t i o n ( T M R ) h a s b e e n w i d e l y u s e d f o r d a i r y c a t t l e b e c a u s e it contains roughage, concentrates, vitamins and minerals in ratios that are w e l l - b a l a n c e d t o me e t t h e a n i m a l ’s r e q u i r e me n t s . H o w e v e r, T M R d e t e r i o r a t e s e a s i l y b e c a u s e i t h a s a h i g h n u t r i e n t c o n t e n t a n d s u f f i c i e n t mo i s t u r e c o n t e n t f o r t h e g r o w t h o f b a c t e r i a , y e a s t a n d mo l d s . 1 T h u s , m o s t f a r m e r s mu s t p r e p a r e T M R o n c e o r t w i c e d a i l y, a n d c o n t r a c t o r s w h o a l s o d o t h i s m u s t d e l i v e r T M R t o f a r m s a l m o s t e v e r y d a y. I n c o n t r a s t , e n s i l e d T M R c a n b e p r e s e r v e d ; t h i s s a v e s labour for TMR preparation, and in some cases, it can be carried for long d i s t a n c e s . 2 L a rg e - s c a l e p r e p a r a t i o n o f T M R s i l a g e b y a c o m p a n y o r c o n t r a c t o r leads to lower feed costs and the use of more high-moisture by-products, which d e t e r i o r a t e e a s i l y. S t u d i e s o n T M R s i l a g e h a v e i n c r e a s e d i n r e c e n t y e a r s , particularly in Asian counties.3-10 Fermentation of TMR silage progresses with h i g h a m o u n t s o f o rg a n i c a c i d s [ a p p r o x i m a t e l y 5 0 t o > 1 0 0 g k g − 1 d r y m a t t e r ( D M ) ] , d e s p i t e t h e h i g h D M c o n t e n t ( a p p r o x i ma t e l y 4 0 0 – 6 0 0 g k g − 1 ) . 2 , 3 , 8 Several studies have shown that TMR silage is stable after opening the silo, e v e n u n d e r h i g h e r t e mp e r a t u r e s , 3 , 8 , 1 1 w h i c h l e a d s t o s t a b l e T M R f e e d i n g without being heated in hot climates. Compared with crop silage, TMR silage typically has high contents of
e n e r g y a n d p r o t e i n s , t h e r e b y e n a b l i n g i t t o m e e t t h e n u t r i e n t r e q u i r e me n t s o f dairy cows. The nutritional value of TMR is based on a mixture of ingredients; t h e r e f o r e , s o me n u t r i e n t s i n T M R s i l a g e a r e e x p e c t e d t o b e l o s t d u r i n g e n s i l i n g , a n d t h e b a l a n c e d i ff e r s b e t w e e n t h e t i m e a t w h i c h i t i s p r e p a r e d a n d t h e t i m e a t w h i c h i t i s f e d t o a n i m a l s . R e s p i r a t i o n b y p l a n t s a n d f e r me n t a t i o n b y m i c r o o rg a n i s m s i n c r o p s i l a g e p r o d u c e s m a l l e r mo l e c u l e s f r o m c a r b o h y d r a t e s
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a n d p r o t e i n s . 1 2 - 1 4 B e c a u s e T M R s i l a g e c a n b e p r e p a r e d t h r o u g h o u t t h e y e a r, w h i c h i s d i ff e r e n t f r o m c r o p s i l a g e , t h e e ff e c t o f t e m p e r a t u r e o n t h e fermentation characteristics and nutrient changes in TMR silages should be u n d e r s t o o d . T h e e n s i l i n g p e r i o d a l s o a ff e c t s t h e s e p a r a me t e r s . I n p a r t i c u l a r, changes in carbohydrates and proteins during ensiling may influence the p r o d u c t i o n o f s h o r t - c h a i n f a t t y a c i d s ( S C FA s ) a n d c o n c e n t r a t i o n s o f N H 3 i n t h e r u m e n , w h i c h a r e h i g h l y r e l a t e d t o e n e rg y a n d p r o t e i n m e t a b o l i s m i n ruminants.15
Therefore,
we
investigated
the
nutritional
changes
and
c h a r a c t e r i s t i c s o f r u m e n f e r m e n t a t i o n i n T M R s i l a g e s s t o r e d a t d i ff e r e n t t e mp e r a t u r e s a n d f o r d i ff e r e n t d u r a t i o n s i n c o mp a r i s o n w i t h t h o s e i n t y p i c a l TMR before ensiling.
M AT E R I A L S A N D M E T H O D S Preparation of TMR and ensiling The ingredients of TMR used in the present study are presented in Ta b l e 1 . I t a l i a n r y e g r a s s s i l a g e , s o rg h u m s i l a g e a n d c o r n s i l a g e , e n s i l e d f o r a p p r o x i m a t e l y 4 , 11 a n d 11 mo n t h s , r e s p e c t i v e l y, w e r e u s e d a s r o u g h a g e . T h e D M c o n t e n t s o f t h e s e s i l a g e s w e r e 4 6 6 , 2 5 7 a n d 2 3 1 g k g − 1 , r e s p e c t i v e l y. T h e other ingredients, including concentrates and minerals, were purchased from feed companies. TMR was designed for dairy cattle, and the total digestible nutrient content was 730 g kg−1 DM, which was calculated from the Standard Ta b l e s o f F e e d C o m p o s i t i o n i n J a p a n . 1 6 T h e s e i n g r e d i e n t s w e r e mi x e d i n a practical scale (total of 2,800 kg), and approximately 100 kg of TMR was used for
the
present
s t u d y.
TMR
was
packed
into
1,000-mL
high-density
polyethylene bottles (1.4 mm thickness), fitted with a rubber cap and
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g a s - r e l e a s e b u l b ; t h i s w a s m a i n t a i n e d a t 1 5 ° C o r 3 0 ° C t o mi m i c a n n u a l a v e r a g e a n d s u m m e r c l i ma t e s , r e s p e c t i v e l y. T h e D M d e n s i t y o f t h e s e s i l a g e s w a s adjusted to approximately 330 kg DM m−3, which was close to that of practical TMR silage in Japan. The silos were opened at 2, 5, 10, 30 and 90 days after ensiling. The silos were prepared in five replicates for 30 and 90 days and in three replicates for 2, 5 and 10 days. All bottles were weighed before and after ensiling, and DM loss during fermentation was calculated. An approximately 1 k g T M R s a m p l e w a s r a n d o m l y c o l l e c t e d f i v e t i me s d u r i n g p r e p a r a t i o n b e f o r e ensiling.
I n v i t ro r u m i n a l f e r m e n t a t i o n s t u d y A n i n v i t ro r u mi n a l s t u d y w a s c o n d u c t e d a c c o r d i n g t o t h e m e t h o d s described
by
Uddin
et
al.17
TMRs
before
and
after
ensiling
(n
=
5
r e p l i c a t e s / t r e a t me n t ) w e r e d r i e d a t 6 0 ° C f o r 4 8 h a n d g r o u n d t o p a s s t h r o u g h a 1 - m m s c r e e n . A p p r o x i m a t e l y 1 g o f s a mp l e w a s w e i g h e d i n t o a 1 0 0 - m L g l a s s v i a l . R u m e n f l u i d w a s c o l l e c t e d v i a a r u me n c a n n u l a b e f o r e t h e m o r n i n g feeding from two Holstein cows that were fed TMR of the same nutrient value a s t h a t i n t h e p r e s e n t s t u d y. T h e f l u i d s a m p l e s w e r e p o o l e d a n d f i l t e r e d t h r o u g h f o u r l a y e r s o f c h e e s e c l o t h . G l a s s v i a l s w e r e f i l l e d w i t h 5 0 mL o f m e d i u m c o n s i s t i n g o f o n e p a r t o f r u m e n f l u i d a n d t w o p a r t s o f M c D o u g a l l b u ff e r, i n c u b a t e d i n a 3 9 ° C w a t e r b a t h a n d s h a k e n a t 1 6 0 r p m. A f t e r 4 , 8 a n d 2 4 h o f i n c u b a t i o n , 0 . 5 mL o f t h e l i q u i d me d i u m i n t h e v i a l s w a s w i t h d r a w n w i t h a s t e r i l i s e d s y r i n g e f o r S C FA a n d N H 3 - N a n a l y s e s . T h e i n v i t ro r u mi n a l s t u d y w a s r e p l i c a t e d t w i c e , a n d t h e me a n v a l u e s a r e p r e s e n t e d .
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C h e mi c a l a n a l y s e s DM contents of TMRs before and after ensiling were determined by oven-drying, as described above, without correcting for loss of volatiles. F e r me n t e d p r o d u c t s ( l a c t i c a c i d , S C FA a n d N H 3 - N ) a n d t h e p H o f T M R s w e r e determined from water extracts. TMRs (30 g) were macerated with 210 mL distilled water and filtered to obtain a water extract. The pH values of the e x t r a c t s w e r e m e a s u r e d u s i n g a p H me t e r. L a c t i c a c i d a n d S C FA c o n c e n t r a t i o n s w e r e a n a l y s e d w i t h a h i g h - p e r f o r ma n c e l i q u i d c h r o m a t o g r a p h y ( H P L C ) s y s t e m e q u i p p e d w i t h a n i o n - e x c l u s i o n c o l u m n u s i n g a p o s t - c o l u m n p H - b u ff e r e d electron conductivity detection method (Shimadzu Kyoto, Japan). NH3-N contents in the extracts were determined using the steam distillation method. The compositions of the following nutrients were analysed in dried and ground s a m p l e s , a s d e s c r i b e d a b o v e . O rg a n i c m a t t e r ( O M ) , c r u d e p r o t e i n ( C P, t o t a l nitrogen × 6.25) and ether extract (EE) contents were measured according to t h e o f f i c i a l me t h o d s o f t h e A s s o c i a t i o n o f O f f i c i a l A n a l y t i c a l C h e m i s t s . 1 8 O M w a s c a l c u l a t e d b y s u b t r a c t i n g t h e a s h c o n t e n t ( me t h o d n o . 9 4 2 . 0 5 ) . C P w a s d e t e r m i n e d u s i n g t h e K j e l d a h l me t h o d ( 9 8 4 . 1 3 ) , a n d t h e E E c o n t e n t w a s d e t e r m i n e d b y S o x h l e t e x t r a c t i o n ( me t h o d n o . 9 2 0 . 3 9 ) . N e u t r a l d e t e r g e n t f i b r e ( N D F ) w a s d e t e r mi n e d a c c o r d i n g t o t h e m e t h o d s o f Va n S o e s t e t a l . 1 9 u s i n g b o t h s o d i u m s u l p h i t e a n d h e a t - s t a b l e a l p h a - a my l a s e a n d e x p r e s s e d a s t h e a s h - f r e e f o r m . T h e a m o u n t o f s o l u b l e C P e x t r a c t e d w i t h n e u t r a l b u ff e r w a s determined using the method of Licitra et al.20 Non-fibrous carbohydrate (NFC) contents were calculated using the following equation: NFC = OM − CP − N D F − E E . 1 5 S o l u b l e s u g a r s e x t r a c t e d w i t h a q u e o u s e t h a n o l ( 8 0 0 mL L − 1 ) were
analysed
using
the
phenol–sulphuric
acid
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reaction.20
The
S C FA
c o n c e n t r a t i o n i n l i q u i d m e d i u m o f t h e i n v i t ro r u m e n w a s a n a l y s e d b y H P L C , a s d e s c r i b e d a b o v e . T h e N H 3 - N c o n c e n t r a t i o n i n t h e me d i u m w a s d e t e r mi n e d using the indophenol reaction.22
Statistical analyses A completely randomised design was employed to allocate treatments into e x p e r i me n t a l u n i t s . F e r m e n t a t i o n c h a r a c t er i s t i c s , n u t r i e n t c o m p o s i t i o n a n d t h e i n v i t ro r u m i n a l f e r m e n t a t i o n p a r a m e t e r s o f T M R s b e f o r e a n d a f t e r e n s i l i n g w e r e a n a l y s e d b y o n e - w a y a n a l y s i s o f v a r i a n c e ( A N O VA ) a n d f u r t h e r t e s t e d u s i n g Tu k e y ’s t e s t t o c o mp a r e t r e a t me n t m e a n s . T h e T M R d a t a b e f o r e a n d a f t e r ensiling were compared using an orthogonal contrast test. The TMR data after e n s i l i n g w e r e s u b s e q u e n t l y a n a l y s e d b y f a c t o r i a l A N O VA , i n w h i c h t h e t w o f i x e d f a c t o r s w e r e t h e n u mb e r o f s t o r a g e d a y s a n d t e mp e r a t u r e . A l l s t a t i s t i c a l a n a l y s e s w e r e p e r f o r m e d u s i n g S A S s o f t w a r e v e r. 9 . 3 ( S A S I n s t i t u t e , C a r y, N C , USA).
R E S U LT S A N D D I S C U S S I O N The pH value of TMR before ensiling was 5.8, while the pH values of the s i l a g e s a f t e r e n s i l i n g r a n g e d f r o m 4 . 2 t o 4 . 4 ( Ta b l e 2 ) ( P < 0 . 0 5 ) . T h e p H rapidly dropped during the ensiling period until 10 days after ensiling in TMR s t o r e d a t 3 0 ° C , a n d t h e s e v a l u e s w e r e ma i n t a i n e d f o r 9 0 d a y s ( F i g u r e 1 ) . T h e pH of TMR silage stored at 15°C gradually changed and decreased from 30 to 9 0 d a y s . L a c t i c a c i d w a s t h e d o mi n a n t f e r me n t e d p r o d u c t i n T M R s i l a g e a n d was significantly higher in silages stored at 30°C than in those stored at 15°C ( P < 0 . 0 0 1 ) ( Ta b l e 2 ) . W h e n t h e e n s i l i n g p e r i o d w a s p r o l o n g e d f r o m 3 0 t o 9 0
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days, the changes in the lactic acid content were small at both storage t e mp e r a t u r e s . T h e a c e t i c a c i d c o n t 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 w i t h e n s i l i n g ( P < 0 . 0 5 ) , a n d a s i g n i f i c a n t i n t e r a c t i o n e ff e c t w a s d e t e c t e d b e t w e e n t h e e n s i l i n g p e r i o d a n d t e mp e r a t u r e ( P < 0 . 0 5 ) . A h i g h o rg a n i c a c i d c o n t e n t w a s a characteristic of TMR silage, although it also contains high DM.2,9 According to previous studies using practical scales, lactic acid in TMR silage can be as high as 30–100 g kg−1DM and it varies depending on the feed ingredients, s e a s o n a n d e n s i l i n g m e t h o d . 2 , 6 , 9 , 2 3 A h i g h D M c o n t e n t s u p p r e s s e s f e r me n t a t i o n in
silage13
because
bacterial
growth
and
metabolism
are
lower
in
a
l o w - m o i s t u r e e n v i r o n me n t . S p e c i f i c b a c t e r i a a r e r e p o r t e d l y f o u n d i n T M R silage but have not been isolated from crop silage.23 Interactions among these s p e c i f i c b a c t e r i a a n d h i g h l y f e r me n t a b l e s u b s t r a t e s i n T M R c o u l d e n h a n c e organic acid production in TMR silages. Butyric acid, an indicator of undesirable
fermentation,
was
found
in
low
amounts.
Although
some
d i ff e r e n c e s w e r e o b s e r v e d i n t h e b u t y r i c a c i d c o n t e n t b e f o r e a n d a f t e r e n s i l i n g , the changes were small. Fermentation by butyric acid bacteria such as Clostridium does not actively occur in high DM TMR silages because their water requirement is high.12 The butyric acid in TMR silages was from crop silages, which are used in TMR as ingredients, and was not produced during the fermentation of TMR silages, as shown by Cao et al.24 The storage temperature a n d d u r a t i o n a ff e c t e d t h e N H 3 - N : t o t a l N ( T N ) r a t i o i n T M R s i l a g e s ( P < 0 . 0 5 ) ; h o w e v e r, t h e r a t i o w a s 3 2 . 5 – 4 9 . 6 g k g − 1 T N , w h i c h w a s l o w e r t h a n t h a t o f c r o p silage (100–200 g kg−1TN).12 These data indicate that compared with crop s i l a g e , d e a m i n a t i o n ma y b e s t r o n g l y s u p p r e s s e d i n T M R s i l a g e . D M l o s s e s d u r i n g e n s i l i n g w e r e < 5 % i n a l l T M R s i l a g e s , a n d n o e ff e c t o f t h e s t o r a g e
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t e mp e r a t u r e o r d u r a t i o n w a s o b s e r v e d . D M l o s s e s w h i l e e n s i l i n g c r o p s i l a g e o c c u r a s a r e s u l t o f m i c r o b i a l f e r m e n t a t i o n , e ff l u e n t a n d p l a n t r e s p i r a t i o n , a n d l o s s e s h a v e b e e n e s t i ma t e d t o b e 7 % t o > 4 0 % . 1 2 R e l a t i v e l y l o w e r l o s s o f D M from TMR silage than those from crop silage can be explained by several f a c t o r s . N o e f f l u e n t w a s o b s e r v e d f r o m a n y o f t h e T M R s i l a g e s , w h i c h ma y have been because of their high DM content. Losses due to plant respiration may also have been lower because the crops incorporated into TMRs had been e n s i l e d ; t h u s , mo s t p l a n t c e l l s w o u l d n o t h a v e b e e n p h y s i o l o g i c a l l y a c t i v e . C o m p a r e d w i t h f e r m e n t a t i o n p r o d u c t s i n T M R s i l a g e s , i t i s mo r e i m p o r t a n t t o d e t e r mi n e t h e n u t r i e n t c o m p o s i t i o n b e c a u s e n u t r i e n t s d i r e c t l y a ff e c t m i l k p r o d u c t i o n b y d a i r y c o w s . N o d i s t i n c t c h a n g e s i n O M , C P, E E o r N D F c o n t e n t s w e r e o b s e r v e d d u r i n g s i l a g e f e r m e n t a t i o n ( Ta b l e 3 ) . T h e r e w e r e no clear changes in the NFC content as a result of the ensiling period or t e mp e r a t u r e , w h e r e a s t h e s o l u b l e s u g a r c o n t e n t s i g n i f i c a n t l y d e c r e a s e d t o < 1 0 g kg−1 DM (P < 0.05). Starch, sugars, pectin and organic acids are included in the NFC fraction.15 Soluble sugars are converted to lactic acid and acetic acid during silage fermentation; therefore, the NFC content did not change much; h o w e v e r, t h e c o mp o n e n t s c l e a r l y c h a n g e d . B a c t e r i a i n t h e r u me n r a p i d l y o b t a i n e n e r g y a s AT P f r o m t h e b r e a k d o w n o f s u g a r s b u t n o t f r o m o rg a n i c a c i d s ; t h u s , the energy supply from rapidly fermentable substrates may have been lower in TMR silages after ensiling than in those before ensiling. Hall et al.25 reported on the neutral detergent soluble carbohydrate fraction in detail and showed that t h e c o mp o n e n t s i n t h e N F C f r a c t i o n v a r y a m o n g f e e d s t u ff s . D e t a i l e d a n a l y s e s of the NFC fractions in TMR silages are required to reveal rumen-fermentable carbohydrates.
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Clear changes were observed in the soluble CP fraction, which increased from 302 g kg−1 before ensiling to 338–455 g kg−1 after ensiling on a CP basis. This result indicates that proteins in TMR were broken down to p e p t i d e s , a mi n o a c i d s a n d N H 3 d u r i n g e n s i l i n g . A h i g h s o l u b l e C P i n t a k e b y cattle causes excess NH3-N in the rumen, leading to a higher urinary N loss.15,26 The soluble CP fractions were significantly higher in TMR silage stored at 30°C than in that stored at 15°C after ensiling for 90 days (P < 0.05); h o w e v e r, t h e r e s u l t w a s n o t s i g n i f i c a n t a f t e r e n s i l i n g f o r a s h o r t e r d u r a t i o n . Prolonging the ensiling period from 30 to 90 days also increased the soluble CP content (P < 0.05). Compared with the fermentation process shown in Figure 1, changes in the protein fractions clearly occurred after acid was produced and the pH of TMR silage was stabilised. Protein degradation during ensiling of crop silage is thought to be caused by proteases from fresh plant material and m i c r o o rg a n i s m s . 1 2 A l t h o u g h w e d i d n o t d e t e r m i n e p r o t e a s e a c t i v i t i e s i n t h e ingredients, the contribution of these activities to protein degradation during ensiling would be low because these activities are much lower in silage than in fresh materials.27 The proteolytic activity of grains during ensiling remains unknown. It has been reported that protease activities in grains gradually decline
during
ma t u r a t i o n , 2 8
whereas
they
drastically
increase
after
germination.29 These findings suggest that protease activities in grains used as T M R i n g r e d i e n t s a r e l o w b e c a u s e t h e g r a i n s a r e w e l l ma t u r e d a n d h a v e n o t germinated.
Heat
processing
grains
by
s t e a mi n g
and
rolling
partially
d e a c t i v a t e s e n z y m e s ; h o w e v e r, t h e e f f e c t o f p r o c e s s i n g o n t h e s e a c t i v i t i e s remains unknown. Protein breakdown caused by Clostridium and enterobacteria that produce proteases occurs in silage.12 As described above, the possibility
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t h a t C l o s t r i d i u m g r e w i n T M R s i l a g e s w a s l o w b e c a u s e o f h i g h D M . M o r e o v e r, b u t y r i c a c i d w a s n o t p r o d u c e d d u r i n g e n s i l i n g i n t h e p r e s e n t s t u d y. C a o e t a l . 9 reported that the number of enterobacteria are consistently low during TMR fermentation. Therefore, it seems that protein degradation by these bacteria is n o t a ma j o r f a c t o r d u r i n g T M R e n s i l i n g . I n c o n t r a s t , l a c t i c a c i d b a c t e r i a m a y have been predominant in TMR silages because of the high lactic acid content and low pH. Lactic acid bacteria are virtually non-proteolytic12; thus, their c o n t r i b u t i o n t o p r o t e i n d e g r a d a t i o n i n T M R s i l a g e s w o u l d b e e x t r e me l y l o w. H o w e v e r, t h e s o l u b l e p r o t e i n c o n t e n t i n c r e a s e d f r o m 3 0 t o 9 0 d a y s i n t h e p r e s e n t s t u d y, s u g g e s t i n g t h a t l a c t i c a c i d b a c t e r i a ma y h a v e h a d s o me e f f e c t o n p r o t e i n b r e a k d o w n . Wi n t e r s e t a l . 3 0 d e m o n s t r a t e d t h a t s o me l a c t i c a c i d b a c t e r i a e n h a n c e p r o t e i n d e g r a d a t i o n d u r i n g e n s i l i n g . H o w e v e r, f u r t h e r s t u d i e s o n proteases of lactic acid bacteria are required to understand protein degradation in TMR silage. I n t h e i n v i t ro r u m e n s t u d y, t h e S C FA c o n c e n t r a t i o n f r o m T M R a f t e r a 4-h incubation before ensiling was slightly higher than that after ensiling; h o w e v e r, n o s i g n i f i c a n t d i ff e r e n c e w a s o b s e r v e d a mo n g t h e t r e a t me n t s a f t e r 8 a n d 2 4 - h i n c u b a t i o n s ( Ta b l e 4 ) . T h e s e r e s u l t s i n d i c a t e t h a t t h e e n e rg y s u p p l y for ruminants from TMR silage after ensiling was not lower than that from TMR before ensiling. Lactic acid in the rumen is mainly metabolised to propionic acid as well as acetic and butyric acids.31 The high lactic acid c o n c e n t r a t i o n i n T M R a f t e r e n s i l i n g m a y h a v e b e e n me t a b o l i s e d t o p r o p i o n i c a c i d i n t h e i n v i t ro r u m e n , a s s h o w n b y t h e l o w e r r a t i o s o f a c e t i c a n d p r o p i o n i c acids from TMR silages. Throughout incubation, NH3-N concentrations from T M R s i l a g e s i n t h e i n v i t ro r u m e n w e r e 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 o s e f r o m
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TMR silages before ensiling. These results are consistent with those of Cao et al.24 who showed higher NH3-N concentrations in the rumen of sheep fed a TMR silage than in that of sheep fed TMR before ensiling. No NH3 absorption b y t h e r u m e n w a l l o r f l o w t o t h e o m a s u m o c c u r r e d i n t h e c l o s e d i n v i t ro r u m e n system; thus, lowering the NH3 content was highly dependent on bacterial uptake. NH3 production in this system was mainly derived from the degradation of feed protein. The higher NH3-N concentration from TMR silages in the in v i t ro r u me n ma y h a v e b e e n a r e s u l t o f t h e r a p i d r e l e a s e o f N H 3 f r o m p r o t e i n s and lower NH3 uptake by bacteria. As described above, TMR after ensiling contained a higher soluble CP content than that before ensiling. Soluble p r o t e i n s c o n t a i n s m a l l N c o m p o u n d s s u c h a s p e p t i d e s a n d a mi n o a c i d s , w h i c h r a p i d l y r e l e a s e N H 3 i n t o t h e r u m e n . 1 5 S i m i l a r l y, f e w e r s u g a r s r e ma i n e d i n T M R s i l a g e , w h i c h l e d t o a s h o r t a g e o f e n e rg y f o r r u m e n b a c t e r i a t o t a k e u p N H 3 . The NH3-N concentration at the beginning of the incubation was 3.38 mg dL−1 ( d a t a n o t s h o w n ) ; t h u s , N H 3 ma y h a v e b e e n t a k e n u p b y b a c t e r i a u n t i l 4 h o f incubation when TMR before ensiling was used as the substrate. In contrast, the release of NH3 from CP was higher than its uptake by rumen bacteria when T M R s i l a g e s w e r e u s e d . T h e s e r e s u l t s s u g g e s t t h a t N m e t a b o l i s m i n t h e r u me n o f c o w s f e d T M R s i l a g e a f t e r e n s i l i n g w a s a l t e r e d i n c o mp a r i s o n w i t h t h a t i n t h e r u me n o f c o w s f e d T M R b e f o r e e n s i l i n g .
CONCLUSIONS TMR after ensiling had lower soluble sugar and higher soluble CP contents than that before ensiling. CP solubilisation was enhanced in TMR silage by both prolonged storage and higher storage temperatures. The
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presence of a higher ruminal NH3 concentration from TMR silage during the in v i t ro r u m i n a l i n c u b a t i o n m a y h a v e b e e n c a u s e d b y a s h o r t a g e o f f e r me n t a b l e s u g a r s a n d e n h a n c e d d e a mi n a t i o n o f s o l u b l e C P c o m p o u n d s . F u r t h e r s t u d i e s a r e required to investigate the nutritional changes in TMR silage on a practical s c a l e a n d t o i d e n t i f y N b a l a n c e i n r u mi n a n t s f e d T M R b e f o r e a n d a f t e r e n s i l i n g .
ACKNOWLEDGEMENTS A u t h o r s a l s o t h a n k t o D r Ya m a mo t o a n d me m b e r s i n M i e P r e f e c t u r a l Livestock Institute for supply of TMR and rumen fluid. This research is supported by a grant from the ‘Research for production of valuable livestock by feeding self-sufficient forage crops’ project of the Ministry of Agriculture, Forestry and Fisheries of Japan.
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casei
or
4
N k o s i B D , a n d M e e s k e R , E f f e c t s o f e n s i l i n g t o t a l l y mi x e d p o t a t o h a s h r a t i o n with
or
without
a
heterofermentative
bacterial
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on
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Ta b l e 1 . I n g r e d i e n t s o f T M R u s e d i n t h i s s t u d y Ingredients
kg-1
g DM
Italian ryegrass silage
138
Sorghum silage
78
Maize silage
70
Beet pulp
68
Steam rolled maize
208
Steam rolled barley
45
Wheat barn
44
Roasted soybean
48
Soybean meal
123
Corn gluten feed
45
Mineral mixture
12
Calcium carbonate
8
Sodium chloride
4
Magnesium oxide
4
Others
2
To t a l
1000
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Tab l e 2 . F er m en t at i o n ch ar a ct er i st i c s o f T M R b ef o r e an d af t er en si l i n g B ef o r e en si l i n g -1
551
Dr y m at t er ( g k g )
5.79a
30 days 1 5 °C
2
547
P r o b ab i l i t y
90 days
3 0 °C
2
539
1 5 °C
2
536
4.44b
4.28c
4.26c
1
3 0 °C
SEM 2
548 4.20d
Bef o r e v s Af t er
Da y #
Temp . #
D ay × Temp . #
***
***
***
4.01 0.006
***
-1
20.1c
89.3b
108.9a
92.5b
104.7a
1.90
***
-1
12.6b
32.8ab
34.2ab
39.3a
31.0b
1.73
***
2.3a
2.0a
2.0a
1.4b
1.4b
0 . 11
***
***
21.0d
32.6c
46.0ab
42.6b
49.6a
1.29
***
***
pH La c t i c aci d ( g k g D M) Ac et i c a ci d ( g k g DM ) -1
B u t y r i c a ci d ( g k g DM ) -1
Af t er en si l i n g 1
NH 3 - N ( g k g t o t al N)
3.39 3.88 4.57 4.57 DM l o s s e s ( % ) 0.571 1 S t o r ag e p er i o d o f T MR si l a g e. 2 S t o r ag e t e mp er at u r e o f T M R si l a g e. a,b,c,d Val u e s wi t h d i ff er en t l et t er s i n a r o w ar e si g n i f i c an t l y d i ff e r en t ( P < 0 . 0 5 ) . * P < 0.05, ** P < 0.01, *** P < 0.001. # C o n d u ct ed o n l y t o s am p l e s af t er e n si l i n g .
*** * ***
*
Tab l e 3 . N u t r i en t co m p o si t i o n o f T M R b ef o r e an d af t er en s i l i n g B ef o r e en s i l i n g -1
Or g an i c m at t er ( g k g DM ) C P ( g k g - 1 D M) Soluble CP (g kg-1 CP) N F C 3 ( g k g - 1 DM )
A f t er en s i l i n g 30 days 15°C
2
1
P r o b ab i l i t y
90 days
3 0 °C
2
1 5 °C
2
1
3 0 °C
SEM 2
B ef o r e v s A f t er
918a
915bc
913c
916b
0.5
155c
163ab
166a
153c
158bc
1.4
**
***
*
302d
338cd
354c
401b
455a
8.8
***
***
***
364a
349ab
335b
321b
345ab
6.5
**
***
52.6a
7.1b
7.2b
7.0b
7.2b
0.72
***
Ether ex tract ( g k g-1DM)
30.4b
30.1b
32.9ab
34.3a
34.4a
0.91
*
381 376 380 404 378 7.7 S t o r ag e p er i o d o f T MR si l a g e. 2 S t o r ag e t e mp er at u r e o f T M R si l a g e. 3 No n f i b r o u s car b o h y d r at e. a,b,c,d Val u e s wi t h d i ff er en t l et t er s i n a r o w ar e si g n i f i c an t l y d i ff e r en t ( P < 0 . 0 5 ) . * P < 0 .05, ** P < 0.01, *** P < 0.001 # C o n d u ct ed o n l y t o s am p l e s af t er e n si l i n g . 1
Temp . #
916ab
S o l u b l e s u g a r s ( g k g - 1 DM ) NDF (g k g-1D M)
Day#
**
Da y × Temp . # *** *
Tab l e 4 . Co n c en t r at i o n s o f s h o r t - ch ai n f at t y a ci d an d a m mo n i a n i t r o gen i n i n vi t ro r u me n i n cu b at ed w i t h T M R b ef o r e an d af t er en si l i n g B ef o r e en s i l i n g
A f t er en s i l i n g 30 days 1 5 °C
2
1
P r o b ab i l i t y
90 days
3 0 °C
2
1 5 °C
2
1
30°C
SEM 2
B ef o r e v s Af t er
D ay #
Temp . #
Da y × Temp . #
4 h af t er i n c u b at i o n To t al S C FA 3 ( m M ) A P r at i o
4 -1
NH3-N (mg dL )
39.8a
37.7bc
3.37
a
1.42
c
3.00
bc
3.31
b
37.5c
39.3ab
38.5abc
2.67
d
3.09
b
2.88
c
4.89
a
4.23
a
4.93
a
0.40
**
**
0.048
***
**
***
0.173
***
*
***
*
8 h af t er i n c u b at i o n To t al S C FA ( m M ) A P r at i o -1
NH3-N (mg dL ) 2 4 h af t er i n To cu bt al at iSoC n FA ( m M ) A P r at i o
5
64.1 2.65 0.15
d
102 2.62
-1
65.9 a
67.0
2.21
b
1.93
c
98 a b
2.28
66.0
1.95
c
2.67
b
103 b a
2.08
67.1
2.19
b
3.61
a
102 c a
2.30
0.62
2.07
bc
0.033
***
3.17
ab
0.141
***
103 b a
2.07
***
**
1.5 c a
0.019
NH3-N (mg dL ) 4.65 6.67 7.75 7.41 7.09 0.466 S t o r ag e p er i o d o f T M R si l ag e. 2 S t o r ag e t e mp er at u r e o f T M R s i l a g e. 3 S h o r t -ch ai n f at t y a c i d . 4 R at i o o f a c et i c a ci d t o p r o p i o n i c aci d a,b,c,d Val u e s wi t h d i ff er en t l et t er s i n a r o w ar e si g n i f i c an t l y d i ff e r en t (P < 0 . 0 5 ) . * P < 0 .05, ** P < 0.01, *** P < 0.001 # C o n d u ct ed o n l y t o s am p l e s af t er e n s i l i n g. 1
***
*** ***
***
6 5.5
pH
5 4.5 4 3.5 0
10
20
30
40
50
60
70
80
90
30 40 50 60 70 Ensiling period (day)
80
90
Ensiling period (day) 120
Lactic acid (g kg-1 DM)
100 80 60 40 20 0 0
10
20
Figure 1. Changes in pH and lactic acid concentration of TMR silages stored at 15°C and 30°C.○; stored at 15°C, ■; stored at 30°C.
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