The use of an enzymatic technique to predict digestibility, metabolizable and net energy of compound feedstuffs for ruminants

Animal Feed Science and Technology, 14 (1986} 203--214 Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands

203

THE USE OF AN ENZYMATIC TECHNIQUE TO PREDICT DIGESTIBILITY, METABOLIZABLE AND NET ENERGY OF COMPOUND FEEDSTUFFS FOR RUMINANTS 1

J.L. DE BOEVER, B.G. COTTYN, F.X. BUYSSE, F.W. WAINMAN 2 and J.M. VANACKER National Institute for Animal Nutrition, Scheldeweg 68, B-9231 Melle-Gontrode (Belgium) The Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen AB2 9SB (Gt. Britain) (Received 18 May 1985; accepted for publication 12 December 1985)

ABSTRACT De Boever, J.L., Cottyn, B.G., Buysse, F.X., Wainman, F.W. and Vanacker, J.M., 1986. The use of an enzymatic technique to predict digestibility, metabolizable and net energy of compound feedstuffs for ruminants. Anita. Feed Sci. Technol., 14: 203--214. The enzymatic procedure investigated comprises 3 steps: (1) pepsin in 0.1 M HC1 at 40°C for 24 h; (2) starch hydrolysis in the same solution at 80°C for 45 min; (3) cellulase (from Trichoderma viride) at 40°C for 24 h. Cellulase digestible organic matter of the dry matter (CDOMD), measured by this technique was better correlated with the in vivo DOMD of 40 compound feeds, val~ying between 0.59 and 0.90, than was the in vitro IVDOMD following Tilley and Terry. Multiple linear regression analysis with the Weende components, gross energy, CDOMD and IVDOMD as parameters was used to establish equations for the prediction of metabolizable and net energy of the 40 compounds. Selected equations were tested on two independent data bases of compounds. The results of these tests showed that CDOMD is competitive with IVDOMD in predictive ability. The cellulase method was more repeatable than the in vitro method. As a result of its many advantages, the cellulase technique is the method of choice.

INTRODUCTION T h e possibility of assessing or c o n t r o l l i n g the energy c o n t e n t of c o n c e n t r a t e s is i m p o r t a n t t o t h e m a n u f a c t u r e r , t h e p u r c h a s e r a n d , i n t h e case o f dispute, to the competent authorities. The energetic value of raw materials as well as c o m p o u n d f e e d s d e p e n d s m a i n l y o n t h e i r d i g e s t i b i l i t y . S i n c e dig e s t i b i l i t y e x p e r i m e n t s w i t h a n i m a l s are e x p e n s i v e , t i m e - c o n s u m i n g a n d req u i r e l a r g e q u a n t i t i e s o f f e e d , t h e y are u n s u i t a b l e f o r large scale f e e d e v a l u Research supported by " I n s t i t u u t tot Aanmoediging van het Wetenschappelijk Onderzoek in Nijverheid en Landbouw" (I.W.O.N.L.). Communication No. 595 of the Institute.

0377-8401/86/$03.50

© 1986 Elsevier Science Publishers B.V.

204 ation. Therefore, many attempts have been made to predict the energy cont e n t of concentrated feeds from relatively simple parameters (Kirchgessner and Kellner, 1981; Sauvant, 1981; Sch6ner and Pfeffer, 1981; Wainman et al., 1981; C o t t y n et al., 1984). Prediction equations using only chemical parameters were less accurate (RSD ~ 4%) than regressions based on incubations using rumen liquor (RSD : 2.2--2.8%). However, the low reproducibility between laboratories (Wainman et al., 1981; Van Der Meer, 1 9 8 3 ) a n d the need for fistulated animals are points against these latter techniques. These disadvantages can be avoided by the use of commercially available enzymatic preparations. The aim of the present work was to investigate the reliability of an enzymatic m e t h o d in predicting the digestibility and energy content of c o m p o u n d feeds and to compare it with the in vitro m e t h o d of Tilley and Terry (1963). MATERIALS AND METHODS The 40 feedingstuffs used in the investigations comprised 33 Belgian commercial dairy cattle concentrates, 5 compounds of lower energy c o n t e n t and 2 maize grains. In vivo feed evaluation The energy content of these 40 feeds was calculated using the m e t h o d of Van Es (1978), which was introduced to Belgium by Buysse et al. (1977). The steps in the calculation are as follows: (1) Gross energy, calculated Schiemann et al. (1971)

from

chemical

composition

following

GE (MJ kg DM- ~) = 0.0241 CP + 0.0366 EE + 0.0209 CF + 0.0170 NFE [0.0006 sugars] * (g kg DM -1) • the sugar con'ection is to be applied for contents > 80 g kg DM(2) Metabolizable energy, calculated from chemical composition and the digestion coefficients with a formula proposed by Van Es (1978), derived from data from Rostock and Wageningen. ME (MJ kg DM-1) = 0.0172 DCP + 0.0377 DEE + 0.0138 DCF + 0.0146 DNFE - [0.0006 sugars] * (g kg DM -1) (3) N e t energy lactation NEL (MJ kg DM -1) = 0.6 [1 + 0.0004 (q - 57)] X ME X 0.9752 X 4.184 with q =

ME GE

X 100

The feed-unit-lactation " V E M " , as used in Belgium, can be calculated as:

205 VEM kg DM- 1 = NEL in MJ kg DM- 1 X

1000 4.184 X 1.65

The chemical composition was determined using the Weende scheme of analysis. The digestion coefficients o f the compounds were obtained using 5 wethers, fed at maintenance level, with total faeces collection over 10 days. In 34 experiments the concentrate was combined with hay in a 80 : 20 ratio, whereas in the others it was given alone.

Simulation techniques The digestibility in vitro was determined following the classical 2 stage technique (2 times 48 h) o f Tilley and Terry (1963), as described by Aerts et al. (1975). As in the previous work o f C o t t y n et al. (1984) the 2 N HCl--cellulasepepsin technique of Kellner and Kirchgessner (1976) gave a poor estimate of the net energy o f fibre-rich concentrates, the search at our institute for a useful enzymatic m e t h o d was continued. From a screening test with 18 versions o f enzymatic methods, the slightly modified technique of Aufr'ere and Michalet-Doreau (1983) proved to be the quickest and most accurate in predicting the in vivo digestibility of dairy concentrates (De Boever et al., 1986). The working m e t h o d is described below:

(a ) Principle To facilitate cellulolytic breakdown, the feed sample is treated with a pepsin solution. As concentrates contain starch, the latter is removed by hydrolysing at 80°C. Finally, cell walls are attacked by cellulase.

(b ) Reagents (1) Pepsin-- hydrochloric acid solution Dissolve 2 g pepsin (Merck no. 7190, 1 : 10.000) in 1 litre 0.1 M HC1. (2) Cellulase--buffer solution (2.1) Acetate buffer pH 4.8 (0.1 M) -- Dilute 5.9 ml acetic acid (CH3 COOH, 96%) with water to 1 litre (Solution A). -- Dissolve 13.6 g sodium acetate (CH3 COONa 3H20) in water and make the volume up to 1 litre (Solution B). Mix 400 ml of Solution A with 600 ml o f Solution B and control pH. Increase in pH results from the addition o f Solution B, decrease from the addition o f Solution A. (2.2) To avoid the risk that the cellulase used may be no longer available, two preparations, both extracted from Trichoderma viride, were tested: (i) Onozuka R--10, from Maruzen Chem. Co. (Japan}; price 1984: 22 US$ per 10 g; dissolve 3.3 g in 1 litre buffer (ii) No. 39074, from BDH Chem. (England); price 1984: £ 6.90 per 10 g; dissolve 13.5 g in 1 litre buffer

206

(c) Procedure (1) An air
Relationship between in vivo and e n z y m e digestibility Less is known about the use of enzyme preparations to simulate ruminal digestion of c o m p o u n d and straight feeds than o f forages. Available information from the literature and our findings are shown in Table II. Where possible, a comparison is made with the two-stage in vitro digestibility of Tilley and Terry (1963). The different enzymatic methods, shown in Table II, could be classified in 4 basic procedures: (a) Pepsin--cellulase: developed by Jones and Hayward (1975) for predicting the digestibility o f grasses. To adapt the technique for concentrates,

207 TABLE I Range in chemical composition, digestibility and energy content of the 40 dairy cattle concentrates x-

Sx

Min.

Max.

Dry matter (g kg -1)

869

14

842

901

Chemical composition (g kg DM-1) Crude protein (CP) Crude fibre (CF) Ether extract (EE) N-free extractives (NFE) Ash Starch

2O0 98 43 567 93 208

68 50 17 87 23 126

78 17 15 410 15 35

383 236 91 813 131 646

Digestible organic matter of the dry matter (%) In vivo DOMD Cellulase Ono (CDOMD Ono) Cellulase BDH (CDOMD BDH) In vitro (IVDOMD) Energy content (MJ kg DM-~) GE (calculated) GE (determined): ME (calculated) NEL (calculated) VEM kg DM-I (calculated)

73.7 78.3 79.7 72.4 18.01 17.40 11.76 7.12 1032

6.0 5.9 6.0 5.5

58.7 61.1 61.9 59.4

0.61 16.20 0.59 15.80 1.21 8.97 0.85 5.36 123 776

90.3 92.9 94.6 87.8 18.98 18.80 14.55 9.24 1338

' Starch content was polarimetrically determined following the C.E.C. : GE was determined by adiabatic bomb calorimetry. D o w m a n and Collins ( 1 9 8 2 ) and Aufr~re and M i c h a l e t - D o r e a u ( 1 9 8 3 ) used a m y l o g l u c o s i d a s e at 6 0 ° C and pepsin at 8 0 ° C , respectively, t o gelatinize starch. (b) 2 N H C l - c e l l u l a s e - - p e p s i n : described b y Kellner and Kirchgessner ( 1 9 7 6 ) f o r use with forages. (c) Neutral detergent--cellulase, p r e c e d e d b y a m y l o g l u c o s i d a s e ( D o w m a n and Collins, 1 9 8 2 ) (d) H 2 0 - - e n z y m e m i x t u r e - - p e p s i n : originally m e n t i o n e d b y Sauvant ( 1 9 8 0 ) , b u t d e s c r i b e d b y Castagna et al. ( 1 9 8 4 ) . T h e d a t a in Table II, s h o w t h a t cellulase t e c h n i q u e s c o m p r i s i n g a pre-treatm e n t w i t h pepsin o r n e u t r a l d e t e r g e n t and starch h y d r o l y s i s are highly correlated with in vivo digestibility o f c o n c e n t r a t e s . C o n s i d e r i n g o u r results and t h o s e o f D o w m a n and Collins ( 1 9 8 2 ) , t h e relationship with p e p s i n - cellulase was even b e t t e r t h a n with r u m e n fluid--pepsin. I n a E u r o p e a n in vitro ringtest with 52 p a r t i c i p a t i n g l a b o r a t o r i e s c o m p a r i n g in vitro (Tilley and T e r r y ) , pepsin--cellulase, N D F - - c e l l u l a s e and e n z y m e m i x t u r e - - p e p s i n (Van D e r Meer, 1 9 8 3 ) , all 3 e n z y m a t i c m e t h o d s p r e d i c t e d t h e in vivo digestibility o f 6 c o n c e n t r a t e s with a h i g h e r a c c u r a c y t h a n t h e in vitro, d u e m a i n l y t o t h e l o w e r r e p r o d u c i b i l i t y o f t h e latter.

24 rations with 25--100% grain

Clark and Beard (1977)

Range in Y

Enzymatic procedure

' Digestibility of the dry m a t t e r (DMD). 2 Digestible organic m a t t e r in t h e d r y m a t t e r (DOMD).

6 1 - - 8 6 ' pepsin--0.1 N HCI cellulase B D H pepsin--0.] N HC] cellulase Asp. cellulase Asp. -amylase Aerts (1982) 32 commercial 75--89 2 N HCI compounds cellulase Asp. pepsin--0.1 N HCI 65--86 amyloglucosidase Wainman et al. 24 c o m p o u n d s neutral d e t e r g e n t (1981) cellulase BDH D o w m a n and 12 c o m p l e t e feeds 5 9 - 8 1 2 pepsin--0.1 N HCI amyloglucosidase Collins (1982) cellulase BDH amyloglucosidase neutral detergent cellulase BDH 44--91 pepsin--0.1 N HCI Aufrbre and 25 b y - p r o d u c t s pepsin--O. 1 N HCI Michalet-Doreau cellulase O n o (1983) 66--85 H~O Castagna et al. ] 1 c o m p o u n d s m i x t u r e of amylase, (1984) hemicellulase 31--92 + cellulase Novo 18 ingredients pepsin--0.1 N HCI 68--92 pepsin--0.1 N HC1 De Boever et al. 40 c o n c e n t r a t e s pepsin--0.1 N HCI (this report) cellulase O n o or cellulase BDH

N u m b e r and nature o f samples

Author(s)

4.1 2.2

3.3 1.7 1.7

0.85 0.85 0.79

0.91

0.98

0.81

0.91 0.95 0.95

42.7+0.44x 43.1+0.44x

10.7+0.90x

-9.3+1.06x,

20.2+0.72x,

33.5+0.62x,

Y= Y=-19.8+l.13x,

1.1+0.89x

Y=

Y=

Y=

Y=

Y=

Y=

2 4 h - - 4 0 ° C Y = 36.4+0.59x, 4 h--40°C 24h--40°C Y= 0.8+0.93x~ 45'--80°C 24 b - - 4 0 ° C Y = - ] . 4 + 0 . 9 4 x ,

3.3

3.2

0.93 --

0.96 --

2.0

4.1

0.60

6.2

RSD

41.7+0.46x

r

Y=

24 h - - 4 0 ° C 48 h - - 4 0 " C 24 h - - 4 0 ° C 48 h - - 4 0 ° C 48 h - - 4 0 ° C 24 h - - 2 5 ° C 30'--] 00°C 24 h--40~C 48 h - - 4 0 ° C 16 h 60°C lh 24 h - - 4 0 " C 24 h - - 4 0 ° C 16 h - - 6 0 ° C 24 h - - 4 0 ° C 16 h - - 6 0 ° C lh 24 h - - 4 0 ° C 24 h - - 4 0 " C 30'--80~C 24 h - - 4 0 ° C i0' - - 7 0 ° C

Y = a+bx I

r

Y=

1.9+0.99x2 0.92

2.1

0.95 --

1.7

Y = 12.7+0.85x2 0.94

Y=-5.8+l.13x2

2.0

3.6

RSD

Y = 1 2 . 8 + 0 . 8 6 x 2 0.83

Y = 15.7+0.78x2 0.89

Y = a+bx 2

Linear relationship b e t w e e n in vivo OM digestibility (Y) and digestibility in e n z y m e s (x,) or in r u m e n fluid (x2)

T A B L E II

b~ o

209 E q u a t i o n s t o p r e d i c t t h e in vivo digestible organic m a t t e r in t h e d r y m a t t e r are given in T a b l e III. T h e t w o t e s t e d cellulase p r e p a r a t i o n s s h o w e d a c o m p a r a b l e a c t i v i t y , t a k i n g into a c c o u n t t h e p r e s c r i b e d dosage. TABLE III Equations to predict in vivo-DOMD (%)

in vivo DOMD = 0.973 CDOMDon o -- 2.49 = 0.969 CDOMDBD g - 3.55 = 1.027 IVDOMD - 0.70

R2

RSD

CV (%)

0.93 0.94 0.90

1.6 1.5 1.9

2.11 2.04 2.63

E q u a t i o n s to p r e d i c t t h e e n e r g y value ( M E , N E L , V E M )

P r e d i c t i o n o f t h e e n e r g y value was b a s e d o n m u l t i p l e regression analysis using t h e a l l - c o m b i n a t i o n p r o c e d u r e w i t h t h e f o l l o w i n g variables Y=a

+ b l X l + b2X2 + . . . + b6X6

w h e r e Y = " c a l c u l a t e d " ME or N E L , b o t h in MJ kg DM- 1, o r V E M kg DMand Xz = " d e t e r m i n e d " gross e n e r g y ( G E in MJ kg D M - I ) , X2 = c r u d e p r o t e i n (CP), X3 = c r u d e fibre ( C F ) , X4 = d i e t h y l e t h e r e x t r a c t ( E E ) and X s = ash. V a r i a b l e s X2-X.~ w e r e e x p r e s s e d in % on d r y m a t t e r and X6 = o n e o f t h e f o l l o w i n g p a r a m e t e r s : p e r c e n t a g e digestible organic m a t t e r o f t h e d r y m a t t e r in cellulase ( C D O M D O n o o r C D O M D B D H ) o r in vitro ( 1 V D O M D ) . T h e results o f t h e regression analysis are s h o w n in T a b l e IV. O n l y t h o s e regression e q u a t i o n s are r e p o r t e d in w h i c h t h e c o n t r i b u t i o n o f each variable was significant f o r P = 0 . 0 0 1 , and w h i c h r e d u c e d t h e residual s t a n d a r d deviat i o n to less t h a n 3.0%. D u e to t h e n e e d f o r high a c c u r a c y , regressions based exclusively o n c h e m ical c o m p o s i t i o n did n o t satisfy, c o n f i r m i n g t h e research and l i t e r a t u r e review o f C o t t y n et al. ( 1 9 8 4 ) . Selected e q u a t i o n s always containc
As t h e e q u a t i o n s in T a b l e I I l and IV were g e n e r a t e d f r o m t h e basic d a t a , t h e i r R S D - v a l u e s i m p l y r e f l e c t t h e e r r o r o f fitting t h e c o n s t a n t s ( c a l i b r a t i o n

210 TABLE IV Equations to predict the metabolizable and net energy value (n = 40)' Regression equation ME

R2

RSD

C V (%)

= 0.150 CDOMDon o =0.161CDOMDBD H =0.149CDOMDBD H =0.162CDOMDon o =0.146IVDOMD

+ 0.241 E E +0.605GE+0.233EE+0.580GE+0.335EE-

0.99 11.60 1.10 11.02 0.23

0.96 0.95 0.95 0.95 0.94

0.24 2.04 0.26 2.21 0.26 2.23 0.28 2.38 0.30 2.55

NEL = 0 . 1 1 2 C D O M D o n o =0.112CDOMDBD H =0.118CDOMDon o =0.121CDOMDBD H =0.111IVDOMD

+0.159EE+0.152EE+0.500GE+0.369GE+0.228EE-

2.37 2.49 0 . 0 2 0 C P 2 - 10.41 8.96 1.88

0.96 0.95 0.95 0.95 0.94

0.18 2.54 0.19 2.64 0.19 2.73 0.20 2.85 0.21 2.91

VEM = 16.28 CDOMDon o =16.28CDOMDBD H = 17.09CDOMDon o = 17.56 CDOMDBD H = 16.04 IVDOMD

+ 23.05 EE +21.98EE+72.38GE+ 53.44 G E + 33.09 EE -

343 361 2.90CP 2 - 1508 1298 273

0.96 0.95 0.95 0.95 0.94

26 27 28 29 30

2.54 2.64 2.73 2.85 2.91

' GE, ME and NEL are expressed in MJ kg DM-', VEM kg DM - ' , CDOMD, IVDOMD (cellulase and in vitro digestible organic matter), EE and CP are measured in % of the DM. 2 Significant at P < 0.01; all other parameters are significant at P < 0.001. error). The real validity of the selected equations can only be proved by testing the best fit using independent data (prediction error). Two such data bases were available (a) 8 2 c o n c e n t r a t e m i x t u r e s , c o m p o u n d e d a t o u r I n s t i t u t e in t h e y e a r s 1967-1983, a n d s u b j e c t e d t o t h e s i m u l a t i o n t e c h n i q u e s a n d in vivo f e e d e v a l u a t i o n u s i n g " c a l c u l a t e d " g r o s s a n d m e t a b o l i z a b l e e n e r g y , as d e s c r i b e d above. (b) the 24 compound feedingstuffs evaluated at the Rowett Research I n s t i t u t e , w i t h all d a t a d e r i v e d f r o m t h e T h i r d R e p o r t ( W a i n m a n e t al., 1 9 8 1 ) , e x c e p t t h e c e l l u l a s e a n d in v i t r o d i g e s t i b i l i t y , w h i c h w e r e d e t e r m i n e d in o u r l a b o r a t o r y . N E L w a s c a l c u l a t e d f r o m " d e t e r m i n e d " g r o s s a n d m e t a bolizable energy. B a r l e y , s u g a r b e e t p u l p a n d s o y a b e a n o i l m e a l w e r e f r e q u e n t l y u s e d in the compounds o f G o n t r o d e , w h e r e a s in t h o s e o f t h e R o w e t t I n s t i t u t e grains, grain by-products and some low grade ingredients predominated. The chemical compositions, digestibilities and energy values of these two data b a s e s a r e s u m m a r i z e d in T a b l e V. C o n s i d e r a t i o n o f T a b l e s I a n d V, s h o w s that the range of the basic data includes most of the independent data, thus fulfilling the statistical condition for testing the equations from Table III and IV. T h e r e s u l t s in T a b l e V I s h o w t h a t t h e e q u a t i o n s w i t h e i t h e r C D O M D o r IVDOMD are robust enough to predict the in vivo DOMD of independent

211 TABLE

V

Range in chemical composition,

Chemical Crude Crude Ether Ash

digestibility and energy content

Gontrode

n = 82

x

sx

Rowett

c o m p o s i t i o n I (g kg D M -1) protein 161 40 fibre 126 35 extract 2 31 22 86 12

Digestible organic matter of the In vivo 77.7 C elIulase Ono 82.5 C ellulase BDH 83.6 In vitro 76.8 Energy content (MJ kg DM q) G E 1'3 17.77 ME 3 12.03 NEL 7.34 VEM kg DM-' 1064

Min.

Max.

94 41 6 64

365 189 84 120

dry matter (%) 4.1 61.3 4.2 67.9 3.9 68.1 4.5 57.8

0.59 0.72 0.52 76

of independent

16.24 10.49 6.23 902

n = 24

x

sx

168 88 42 93

85.6 87.4 89.3 83.2

18.12 11.55 6.95 1006

Min.

20 35 13 22

71.2 75.0 76.2 70.4

19.23 13.41 8.35 1210

data bases

5.2 5.8 6.0 5.8 0.65 0.99 0.71 102

Max.

127 38 25 54

209 167 73 149

58.5 61.5 62.7 55,8

81.4 85.5 87.2 81.8

16.30 9.75 5.63 815

The chemical composition and gross energy of the Rowett feeds are means calculated laboratories, respectively. 2 In Gontrode and in Rowett diethyl ether and petroleum spirit were used, respectively. 3GE and ME were calculated at Gontrode, while determined at the Rowett.

19.15 13.66 8.45 1224 from 5 and 4

TABLE VI Test of equations to predict in vivo-DOMD (%) on two independent data bases of compounds Gontrode (n = 82) ± sa '

CDOMDono CDOMDBD H IVDOMD

+ 0.1 -+ 2.1 - 0.3 t 1.8 + 0.5 -+ 2.4

Rowett (n = 24)

P E (%)2

~ +- s,~

P E (%)

2.69 2.41 3.24

- 0.7 -~ 1.8 - 0.9 -+ 2.0 + 0.4 + 2.1

2.82 3.17 3.10

' Mean difference (predicted -- real value) .~ standard deviation. ~/e(predicted - real value) 2 2Prediction error:

× n-2

100 _ ; Y = mean of real values. Y

compounds. Application of the prediction equations for metabolizable and n e t energy, s h o w n in Tables VII and VIII, respectively, a p p e a r e d successful o n l y w h e n u s i n g t h e c o r r e c t p a r a m e t e r s , n a m e l y " d e t e r m i n e d " gross e n e r g y a n d " d i e t h y l " e t h e r e x t r a c t . I n t h e case o f G o n t r o d e c o m p o u n d s , t h e u s e o f " c a l c u l a t e d " i n s t e a d o f " d e t e r m i n e d " gross e n e r g y , t h e f o r m e r o n a v e r a g e , a m o u n t i n g t o g r e a t e r v a l u e s (see a b o v e ) , l e a d s t o a s e r i o u s o v e r e s t i m a t i o n o f t h e e n e r g y v a l u e . F o r R o w e t t feeds, t h e u s e o f " p e t r o l e u m " s p i r i t f o r f a t e x t r a c t i o n , w h i c h gives l o w e r v a l u e s t h a n d o e s t h e u s e o f " d i e t h y l " e t h e r

212 (Bassler and Putzka, 1984), resulted in an u n d e r e s t i m a t i o n of the energy value. Despite the fact that prediction equations based on CDOMD had a lower residual standard deviation than those with IVDOMD, they were somewhat less a c c u r a t e in p r e d i c t i n g t h e e n e r g y v a l u e o f i n d e p e n d e n t c o m p o u n d s . TABLE VII Test of equations to predict ME (MJ kg DM -1) on two independent data bases of compounds Components

CDOMDono, CDOMDBDH, CDOMDBDH, CDOMDono, IVDOMD,

Gontrode (n = 82)

Rowett (n = 24)

-E = SA '

P E (%)2

~ ± sA

P E (%)

+ 0.07 +- 0.34 + 0 . 5 7 ± 0.47 + 0 . 0 2 ~ 0.33 + 0 . 6 2 ± 0.50 0.03 = 0.32

2.93 6.24 2.74 6.70 2.66

0.32 ~: 0.30 +0.07-" 0.40 - 0.35± 0.31 + 0 . 0 9 ± 0.35 - 0.14 ± 0.27

3.89 3.58 4.15 3.19 2.71

EE GE EE GE EE

-

1,2 See Table VI.

TABLE VIII Test of equations to predict NEL (MJ kg DM -1) on two independent data bases of compounds Components

CDOMDono, CDOMDBDH, CDOMDono, CDOMDBDH, IVDOMD,

Gontrode (n = 82)

EE EE GE, CP GE EE

Rowett (n = 24)

X ~ sA 1

P E (%)2

~ ± sA

+ 0 . 0 6 ~ 0.26 + 0 . 0 2 ± 0.25 + 0,54 ± 0.35 + 0.39 ~- 0.37 0.01 ± 0.24

3.61 3.44 8.89 7.36 3.34

+ + -

-

0.22~ 0.22 0.22± 0.26 0.22 ± 0.29 0.02 ± 0.31 0.09 ± 0.20

P E (%)

4.65 5.07 5.41 4.51 3.22

1,2 See Table VI.

Repeatability

and

reproducibility

From the determination of organic matter digestibility of the 146 (40 + 8 2 + 2 4 ) c o n c e n t r a t e s , t h e r e p e a t a b i l i t y w i t h i n a n d b e t w e e n series o f t h e inv e s t i g a t e d t e c h n i q u e s was c a l c u l a t e d . T h e r e p e a t a b i l i t y is t h e v a l u e b e l o w which t h e a b s o l u t e d i f f e r e n c e b e t w e e n t w o test results o b t a i n e d in t h e same l a b o r a t o r y m a y b e e x p e c t e d t o lie, w i t h a p r o b a b i l i t y o f 9 5 % . R e s u l t s i n T a b l e I X s h o w t h a t t h e c e l l u l a s e t e c h n i q u e is m o r e r e p e a t a b l e t h a n t h e i n v i t r o m e t h o d . T h e h i g h e r r e p e a t a b i l i t y w i t h i n series is m a i n l y d u e t o t h e f a c t t h a t all o p e r a t i o n s t a k e p l a c e i n t h e s a m e c r u c i b l e , w h i l e t h e u s e o f t h e s a m e

213 b a t c h o f cellulase r e d u c e s t h e d i f f e r e n c e b e t w e e n weeks. F u r t h e r m o r e , it a p p e a r s f r o m t h e studies o f W a i n m a n et al. ( 1 9 8 1 ) and V a n D e r Meer ( 1 9 8 3 ) t h a t cellulase t e c h n i q u e s are m o r e r e p r o d u c i b l e t h a n m e t h o d s using r u m e n fluid. H o w e v e r , w h e n p r e d i c t i o n e q u a t i o n s b a s e d on ceUulase digestibility are to b e used r o u t i n e l y in o t h e r l a b o r a t o r i e s , p r o b l e m s m a y arise f r o m possible d i f f e r e n c e s in a c t i v i t y b e t w e e n b a t c h e s o f t h e cellulase p r e p a r a t i o n , o r f r o m small d i f f e r e n c e s in t h e e x e c u t i o n o f t h e prescribed p r o c e d u r e . T o allow f o r c o r r e c t i o n o f such d i f f e r e n c e s , 4 or m o r e s t a n d a r d s a m p l e s o f k n o w n digestibility should be i n c l u d e d w i t h each set o f u n k n o w n s a m p l e s ; w i t h t h e c a l c u l a t e d s i m p l e regression, results s h o u l d b e a d j u s t e d t o t h e s a m e level o f cellulase digestibility as o c c u r r e d d u r i n g t h e development of prediction equations. TABLE IX Repeatability (r) of the organic matter digestibility determination Within series

Cellulase Ono Cellulase BDH In vitro

Between series

nI

Sd2

r3

r (%)~

n

Sd

r

r (%)

82 24 124

0.46 0.53 0.86

0.92 1.06 1.72

1.05 1.19 2.10

70 122 81

0.69 0.84 1.11

1.38 1.68 2.22

1.57 1.88 2.71

i n = number of duplicate determinations. 2Sd = standard deviation of the difference between duplicates. 3r=2×

s d.

~r (%) = r in percent of the mean organic matter digestibility of 146 concentrates; 87.9, 89.4 and 81.8% for cellulase Ono, BDH and in vitro respectively. CONCLUSIONS

T h e cellulase t e c h n i q u e , a c c o r d i n g t o t h e p r e s c r i b e d p r o c e d u r e , has a c o m p a r a b l e ability t o p r e d i c t t h e in vivo digestibility a n d e n e r g y value o f c o n c e n t r a t e d feeds as t h e in vitro m e t h o d o f Tilley a n d T e r r y . E n z y m a t i c t e c h n i q u e s n o t o n l y save m o n e y , w o r k a n d t i m e c o m p a r e d w i t h m e t h o d s using r u m e n liquor, b u t also h a v e i m p o r t a n c e f o r a n i m a l w e l f a r e since t h e r e is no n e e d f o r t h e use o f f i s t u l a t e d animals. Finally, b u t o f e q u a l i m p o r t a n c e , e n z y m a t i c m e t h o d s are s i m p l e r in t h e i r e x e c u t i o n a n d h a v e b e t t e r r e p r o d u c i b i l i t y . This m a k e s t h e m highly suitable f o r r o u t i n e use. ACKNOWLEDGEMENTS T h e a u t h o r s t h a n k It. R. M o e r m a n s o f t h e B i o m e t r i c U n i t o f t h e C e n t r e f o r A g r i c u l t u r e R e s e a r c h , G h e n t , f o r statistical analysis. T h e y are i n d e b t e d t o Mrs. L. De W u l f f o r h e r skilled t e c h n i c a l assistance.

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