The use of a cellulase technique to predict digestibility, metabolizable and net energy of forages

Animal Feed Science and Technology, 19 (1988) 247-260 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands

247

T h e U s e o f a Cellulase T e c h n i q u e to P r e d i c t D i g e s t i b i l i t y , M e t a b o l i z a b l e and N e t E n e r g y o f Forages* J.L. DE BOEVER, B.G. COTTYN, J.I. ANDRIES, F,X. BUYSSE and J.M. VANACKER

National Institute for Animal Nutrition, Scheldeweg 68, B-9231 Melle-Goatrode (Belgium) ** (Received 14 July 1986; accepted for publication 25 June 1987)

ABSTRACT De Boever, J.L., Cottyn, B.G., Andries, J.I., Buysse, F.X. and Vanacker, J.M., 1988. The use of a cellulase technique to predict digestibility, metabolizable and net energy of forages. Anita. Feed Sci. Technol., 19: 247-260. With samples of 24 mhize silages, 21 grass silages and 18 grass hays the 2N HCl-cellulase-pepsin method and several versions of the pepsin-cellulase method were compared for their accuracy in predicting in vivo organic matter digestibility. The rumen fluid-pepsin in vitro method was used as reference. As none of the tested methods proved to be the best for all forage types, the procedure with the best uniformity was chosen for further study. It was originally developed for concentrates and includes three steps: ( 1 ) pepsin in 0.1 N HC1 at 40 ° C for 24 h; (2) heating the same solution at 80°C for 45 rain to hydrolyse starch; (3) cellulase (from Trichoderma viride) at 40°C for 24 h. With this technique cellulase-digestible organic matter of the dry matter (DOMD) was determined on 50 maize silages, 50 grass silages, 24 grass hays and 16 straws for which composition and digestibility in vitro and in vivo were known. By means of linear regression analysis prediction equations for in vivo DOMD, metabolizable and net energy were derived. The best equations for net energy based on cellulase DOMD showed coefficients of variation of 3.3, 4.2 and 2.6 % for maize silage, grass silage and grass hay, respectively, and of 2.7, 3.5 and 2.6%, respectively, for those based on in vitro DOMD. Prediction of feeding value of straw was very imprecise. In spite of the slightly lower accuracy, the ceUulase technique selected has operational advantages over the tureen-fluid in vitro method for routine feed evaluation.

INTRODUCTION As the nutritive value of forages varies not only between species, but also within one species, a good ration calculation needs an assessment of digesti*Research supported by "Instituut tot Aanmoediging van het Wetenschappelijk Onderzoek in Nijverheid en Landbouw" (I.W.O.N.L.). **Communication No. 649 of the Institutel

0377-8401/88/$03.50

© 1988 Elsevier Science Publishers B.V.

248 bility of each forage batch. Digestibility experiments with animals are expensive, time-consuming and require large quantities of feed, so they are unsuitable for practical feed evaluation. Among the existing laboratory methods, those using rumen fluid appear the most precise for predicting the nutritive value of forages (Cottyn et al., 1986). However low reproducibility between laboratories (Wainman et al., 1981; van der Meer, 1983) and the need for fistulated animals are disadvantages with these techniques. Thus, many attempts during the last 15 years have been made to predict the nutritive value of forages by using enzymatic preparations (reviews by Marten and Barnes, 1980; Osbourn and Siddons, 1980; Jones, 1986). Our aim was to find a quick and reliable enzymatic method for the prediction of digestibility and energy value of forages. MATERIALAND METHODS The forages investigated included 50 maize silages, 50 grass silages, 24 grass hays and 16 straws. All these roughages originated from crops grown at our institute (sandy-loam soil; 700 m m rain/year) in the course of the years 1968-1984. Samples of the feeds, ground to pass a 1-mm screen, were chemically analysed for crude protein, oil, crude fibre, neutral detergent fibre and ash. Crude protein ( N × 6.25) was determined with an automatic Kjel-foss apparatus. For determination of oil, samples were extracted with diethyl ether for a minimum of 6 hours. Crude fibre was analysed with an automatic fibertec apparatus (Tecator). Neutral detergent fibre was determined by the method of Goering and van Soest (1970). Ash content was obtained after incineration at 600 ° C. For 35 maize silages, 31 grass silages, 20 grass hays and 2 straws digestibility was determined with 5 non-lactating cows, while 5 adult wethers were used for the remaining forages. The animals were fed near the maintenance level with forage as the sole ration, except the straw which was supplemented with soyabean meal. After an adaptation period of 2 to 3 weeks, total faeces were collected and sampled once a day for an experimental period of 10 days. As sheep and cows showed no systematic difference in digesting forages of moderate and good quality (de Boever et al., 1984), no distinction was made between digestion coefficients of the two species. All analyses were carried out on oven-dried ( ~ 60°C) samples; no corrections were made for loss of volatile components. Gross energy was calculated from chemical composition by using the equation of Schiemann et al. (1971). Metabolizable and net energy were both derived from chemical composition and in vivo digestion coefficients by means of formulae proposed by van Es (1978). In the system of van Es, introduced to Belgium by Buysse et al. (1977), net energy lactation is expressed in V E M

249

V E M :-

N E L X 1000 4.184 X 1.65

where N E L is the net energy lactation expressed in MJ. Concerning the enzymatic digestibility, two basic procedures were investigated, the 2N HCl-cellulase-pepsin technique of Kellner and Kirchgessner (1976) (Method A) and the pepsin-cellulase method of Jones and Hayward (1975) (Method B ). The first procedure was carried out exactly as prescribed, using Cellulase C from an Aspergillus mould and supplied by Carl Roth, Germany (Roth). The second procedure, of which 10 versions were tested (B1-B10), was somewhat modified. Thus, the 2 (or 3) stages took place in the same sintered grass crucible (porosity 1 ), with shaking after about 5 h of incubation. At the beginning of the comparative test 500-mg samples were incubated, subsequently 300 mg were used, respecting however the proportions of the solutions. Two commercially-available cellulase preparations, produced by Trichoderma viride, were compared: Onozuka R-10 from Maruzen Chem: Co., Japan (Ono); Prep. No. 39074 from BDH, England ( B D H ) . Other factors studied were the length of the incubation period, the concentration of hydrochloric acid solution, the ratio of cellulase to sample and the use of a starchgelatinizing step. The enzyme preparations, stored in a refrigerator, were used within a year of supply. For convenience, the test of methods was limited to a series of 24 maize silages, with in vivo organic matter digestibility (OMD) ranging from 66.0 to 79.7%, a series of 21 grass silages (in vivo OMD: 55.2-78.6% ) and a series of 18 grass hays (in vivo OMD: 54.6-81.1% ). More versions were tested on the grass silage, because of its importance and higher variability. All determinations were carried out in duplicate, and the mean cellulase OMD results used to calculate correlations with in vivo OMD. These relationships were compared with that using in vitro OMD, the reference method. In vitro OMD (Method C) was determined following the classical 2-stage technique of Tilley and Terry (1963). Prediction equations for digestibility and energy value were derived from the whole data base. Linear regressions were calculated between the in vivo digestible organic matter of the dry matter (DOMD) and crude fibre (CF), neutral detergent fibre (NDF), ash, cellulase DOMD or in vitro DOMD and the same parameters in combination with ash. Calculated metabolizable energy (ME) and n e t energy lactation ( N E L ) were estimated by means of multiple regression analysis using the following variables: crude protein (CP); CF; NDF; diethyl ether extract (EE); ash; cellulase DOMD (CDOMD) or in vitro DOMD ( IVDOMD ) .

250 RESULTS AND DISCUSSION

Test of enzymatic methods I n Table I some details of the investigated methods are summarized. The main results for the extent of OM digestion, the correlation with in vivo OMD and the repeatability are given in Table II. For maize silage, all tested enzymatic methods (coefficient of variation ( C V) ranging from 2.4 to 2.9% ) were as accurate as the in vitro method. Although not the only differing factor, the absence of a heating step to gelatinize starch was probably the main cause for the low repeatability of the pepsin/0.1N HC1-BDH cellulase procedure (Method B6). The 2N HCl-cellulase-pepsin method of Kellner a n d Kirchgessner (1976) ( Method A) appeared little suited for grass silage and hay. Pace et al. (1984), working with grasses and legumes, also found a poorer correlation to in vitro OMD with 2N HCt-cellulase-pepsin than with pepsin-cellulase. For grass hay, the lowest variation with in vivo OMD was obtained with pepsin in 1N H C L - B D H celtulase (Method B 1, CV= 3.0% ). This version, however, which was also proposed by Aufr~re (1982) for use with forages, gave high variability (CV> 7.4% ) with grass silage. Searching for the cause, we studied the influence of the HC1 concentration used in the pepsin treatment. For 0.050, 0.075, 0.100, 0.250, 0.500 and 1.000N HCI the CV's of the relation between Ono cellulase OMD and in vivo OMD were 5.9, 6.3, 5.3, 6.0, 6.6 and 7.4%. But, even with the optimal HCI concentration of 0.1N, the pepsin-cellulase method ( CV= 5.3% for Ono (Method B4) and 5.9 for BDH (Method B3 ) ) remained less accurate than the in vitro method (Method C, CV= 4.1% ). With cellulase BDH, organic matter digestion and accuracy further increased either by increasing the ratio of cellulase to sample from 0.80 to 1.33 (Method B5), or extending the incubation from 24 to 48 h (Method B6). Doubling the pepsin treatment time, however, brought no improvement (Method B 8 ) . The two ceIlulase preparations showed comparable results, taking into account the need of a 4 times higher dose for BDH t h a n for Ono. While adding a starch gelatinizing step (Methods B9 and B10) for ensiled and dried grasses resulted in more organic matter being digested and a slightly better repeatability, it gave a small reduction in accuracy. This study showed that :none of the tested enzymatic methods was best for all 3 forage types. Because confusion from using different methods would be more disadvantageous t h a n using a slightly less precise method, we chose one method for further evaluation. It comprises three steps: (1) pepsin in 0.1N HC1 (24 h, 40°C); (2) pepsin in 0.1N HC1 (45 min, 80°C); (3) cellulase from Trichoderma uiride (24 h, 40 ° C ). It was also proposed for concentrates, and is described in detail by de Boever et al. (1986b). With this procedure (Methods

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253 B9 + B10 in Table II), the CV's of the cellulase-in vivo OMD relationship were 2.5, 5.9 a n d 4.7%, respectively, for maize silage, grass silage and grass hay, compared with 2.8, 4.1 and 3.1% for in vitro-in vivo OMD. The repeatability of the chosen method was comparable with that of the in vitro method. The enzymatic procedure with neutral detergent reagent as a pretreatment (Dowman and Collins, 1982) was not involved in the comparative study, because this method proved more difficult to manipulate than those employing pepsin, and this resulted in a lower repeatability (Dowman and Collins, 1982; de Boever et al., 1986a). The reliability of the selected method, with cellulase BDH (Method B9), was further investigated with 50 maize silages, 50 grass silages, 24 grass hays and 16 straws. Dabble Data for chemical composition, digestibility and energy value of the forages are shown in Table III. The maize silages were harvested at several stages of maturity, from the early milk to the late dough-dent stage. Similarly the grass silages and grass hays covered the range in quality prevailing in a temperate climate. Of the 16 straws (8 barley and 8 wheat), 7 were untreated, while 6 were treated with ammonia and 3 with urea. The precision with which the digestibility experiments were carried out was satisfactory. The coefficient of variation of the in vivo OMD obtained from the data of 5 animals averaged 1.55, 1.34, 1.16 and 3.08% for the 50 maize silages, 50 grass silages, 24 grass hays and 16 straws respectively. Regardless of feed type, in vitro DOMD values agreed fairly well with in vivo values. While cellulase DOMD values for good quality forages and concentrates (de Boever et al., 1986b) were higher t h a n the in vivo values, the opposite was true for forages with in vivo DOMD less than about 70%, and the difference between the two values increased with decreasing quality. In spite of having the same in vivo digestibility, pepsin-cellulase solubilized more dry matter from legumes t h a n from grasses, perhaps because the latter contain more cell walls (Aufr~re, 1982). According to McQueen and van Soest (1975) a major difference between rumen fluid and enzyme procedures is that in vitro fermentation permits adaptation of microorganisms, or selection of species capable of degrading a certain association of cell-wall constituents, while modification of enzymes is not possible. Therefore, each forage group was considered separately when deriving prediction equations. Prediction of digestibility and energy value Table IV shows the error (coefficient of variation) with which different parameters predict in vivo DOMD. Prediction of in vivo DOMD by means of

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256 TABLE V Equations to predict in vivo DOMD and energy value of forages by the in vitro DOMD method ( IVDOMD ) Rl

RSD

CV %

Maize silage DOMD ME NEL

= 0.798 IVDOMD + 15.1 = 0.125 IVDOMD + 0.247 EE + 1.41 = 0.089 IVDOMD + 0.167 E E - 0.25

0.81 0.82 0.83

1.5 0.26 0.18

2.2 2.4 2.7

Grass silage DOMD ME NEL

= 0.686 I V D O M D - 0.207 Ash + 24.2 = 0.122 IVDOMD + 0.170 EE + 1.82 = 0.077 IVDOMD + 0.116 EE + 0,66

0.95 0.92 0.92

1.6 0.32 0.20

2.7 3.3 3.5

Grass hay DOMD ME NEL

= 0.867 IVDOMD ÷ 10.4 = 0.110 I V D O M D - 0.045 N D F + 5,65 = 0.074 IVDOMD - 0.033 N D F + 3.10

0.92 0.93 0.94

1.3 0.22 0.14

2.1 2.3 2.6

DOMD, IVDOMD, EE, Ash, N D F as % of DM; M E and N E L in MJ kg- 1 DM. RSD = Residual standard deviation; R l = determination coefficient; CV= coefficients of variation. C D O M D was w o r s e t h a n by I V D O M D , b u t as g o o d as w i t h t h e c o m b i n a t i o n s CF or NDF+ash, and better than by single chemical parameters. The precision with which CDOMD estimated in vivo DOMD of grass silages and hays TABLE VI Equations to predict in vivo DOMD and energy value of forages by the cellulase DOMD method ( CDOMD ) Rl

RSD

CV %

Maize silage DOMD ME NEL

=0.610 CDOMD + 28.2 = 0.094 CDOMD ÷ 0.237 EE + 3.58 =0.067 CDOMD+O.160 EE + 1.26

0.71 0.73 0.75

1.8 0.32 0.22

2.6 2.9 3.3

Grass silage DOMD ME NEL

=0.499 CDOMD--0.432 A s h + 37.7 =0.110 CDOMD+O.077 C F + 0.112 E E + 0 .7 1 =0.071 CDOMD+O.038 CF+0,075 E E ÷ 0 . 1 4

0.91 0.91 0.89

2.1 0.34 0.24

3.7 3.6 4.2

Grass hay DOMD ME NEL

=0.685 CDOMD-0.740 A s h + 28.3 =0.090 CDOMD-O.042 NDF-O.091 A s h + 7.57 =0.063 CDOMD-O.029 N D F - 0 . 5 6 Ash-l-4.05

0.92 0.93 0.94

1.3 0.22 0.15

2.1 2.3 2.6

DOMD, CDOMD, EE, CF, Ash, N D F as % of DM; M E and N E L in MJ kg- 1DM. RSD = Residual standard deviation; R ~= determination coefficient; CV= coefficients of variation.

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258 was improved by accounting for the ash content, while IVDOMD + ash was better than IVDOMD alone for grass silages. In the case of straw, all the parameters studied, or combinations of them, caused large prediction errors. Barber et aI. (1984) also did not find significant relationships between fibre fractions, cellulase or in vitro determinations and in vivo DOMD for untreated straw. However, Steg and den Boer (1982) reported a satisfactory in vitro-in vivo OMD regression for a group of 16 straws, of which 6 were untreated, 8 treated with NH3 and 2 with NaOH. The selected equations for prediction of in vivo DOMD based on in vitro and cellulase DOMD are shown in Table V and VI respectively. The RSD values for grass silages and hays were approximately one percentage unit lower than those obtained by Barber et al. (1984), who reported 2,7 and 2.0, respectively, with in vitro DOMD, and 3.2 and 2.3, respectively, with cellulase DOMD. Because M E served to calculate N E L by a simple mathematical function, the multiple regression analyses for predicting the respective energy values resulted in similar equations concerning the significant parameters, but with a somewhat lower accuracy for the N E L equations. The residual errors of the latter, associated with either IVDOMD or CDOMD as sole predictor and with these parameters in combination with one and two chemical variables, are given in Table VII. The contribution of a third chemical parameter was never significant ( P > 0.05). As with digestibility, prediction of energy values based on CDOMD had greater error than using IVDOMD, especially in the case of maize a n d grass silages. For maize silage the prediction by IVDOMD as well as CDOMD was improved by accounting for the ether extract. These combinations, with respective errors of 2.7 and 3.3%, could not be improved significantly by accounting for an additional parameter. In the case of grass silage, the lowest errors were obtained with I V D O M D + E E (3.5%) and C D O M D + C F + E E (4.2%). With grass hay, IVDOMD + N D F + E E and CDOMD + N D F + ash gave the best predictions, with respective errors of 2.4 and 2.6%. Although the biological relevance of the ether extract is questionable, its inclusion is justified both by statistics and because determination of this usually routinely analysed parameter entails no supplemental work. The best equations to predict calculated metabolizable and net energy of forages by in vitro and cellulase DOMD and their precision are shown in Tables V and VI, respectively. The RSD values of the M E equations for grass silages and hays were, respectively, three and two times smaller than those of the regressions based on the single parameters in vitro or cellulase DOMD given by Barber et al. (1984). For practical feed evaluation, the reduced accuracy of the cellulase technique is more than offset by its simplicity, and by its higher reproducibility (Wainman et al., 1981; van der Meer, 1983). When prediction equations based on cellulase DOMD are to be routinely used in other laboratories, problems may arise from differences in cellulase activity (in our experience it is never greater

259 t h a n 1.5 d i g e s t i b i l i t y u n i t s ) o r f r o m s m a l l 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 procedure. To correct for such differences, two or preferably more s t a n d a r d s a m p l e s s h o u l d b e i n c l u d e d in e a c h cellulase run, a n d a n a l y s e d in t r i p l i c a t e . T h e w i t h i n - r u n r e g r e s s i o n o f t h e celtuIase r e s u l t s o f t h e s e s t a n d a r d s a m p l e s o n t h e i r c e l I ~ a s e values, as o c c u r r e d d u r i n g t h e d e v e l o p m e n t o f t h e p r e d i c t i o n e q u a t i o n s , is t h e n u s e d t o a d j u s t t h e c e l l u l a s e digestibility v a l u e s of the test samples.

ACKNOWLEDGEMENTS .

T h e a u t h o r s t h a n k Ir. 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 o f A g r i c u l t u r e R e s e a r c h , G h e n t , for s t a t i s t i c a l a n a l y s i s . W e a r e i n d e b t e d t o M r s . L. de W u l f for h e r skilled t e c h n i c a l a s s i s t a n c e .

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