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Comparison of methods for predicting digestibility of feeds
Animal Feed Science and Technology, 20 (1988) 203-218 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
203
Comparison of Methods for Predicting Digestibility of Feeds JOCELYNE
AUFRERE
and B R I G I T T E M I C H A L E T - D O R E A U
InstitutNational de la Recherche Agronomique (INRA ), Laboratoire des Aliments, Centre de Recherche Zootechnique et Vdtdrinaire (CRZV), Theix-63122 Ceyrat (France) (Received 29 May 1986; accepted forpublication13 November 1987)
ABSTRACT Aufr~re,J. and Michalet-Doreau, B., 1988. Comparison of methods for predictingdigestibilityof feeds.Anita. Feed Sci. Technol.,20: 203-218. Five techniques were investigated to find a laboratory method for the prediction of the in vivo digestibility of feeds and mixed feeds: two chemical techniques (Weende crude fibre and van Soest methods), one biological technique (in vitro digestibility ) and two enzymatic techniques (digestion by pepsin-cellulase, normal or modified (HCI 0.1 N instead of 1 N) ). The enzymatic degradability (Method B, HC10.1 N) allowed a more accurate prediction of the in vivo organic matter digestibility (OMD) of 24 feeds than the separate data of crude fibre, neutral detergent fibre (NDF), acid detergent fibre (ADF) and acid detergent lignin (ADL) contents or in vitro digestibility. The selected equation obtained with the celhlase method could also be used for feed mixtures.
INTRODUCTION
Many laboratory methods for predicting the digestibility of energy and dry matter of forage exist. They can be classified into three classes: (1) chemical analyses only; (2) fermentation with rumen micro-organisms; (3) hydrolysis by enzymatic preparations. Variable correlations have been established between the indigestible fraction of forages and their crude fibre content Weende method (AFNOR, 1981) or lignocellulose (ADF) of van Soest (1963) content. In contrast with these methods, those involvingrumen micro-organisms or enzymes (such as in vitro or in sacco digestibility and cellulase attack followed or preceded by chemical and/or enzymatic attack) are based on an appreciation of the indigestible residue of forage. These methods for predicting forage digestibility have been the subject of several reviews (Fonnesbeck, 1976; Morrison, 1976; Osbourn and Siddons, 1980; Demarquilly and Jarrige, 1981 ). 0377-8401/88/$03.50
© 1988 Elsevier Science Publishers B.V.
204 These methods, all developed for use on forages, have been much less studied with energy feeds (except Fonnesbeck, 1976). The official method of calculating the energy value of energy feeds or compound feeds in France was proposed by Institut National de la Recherche Agronomique (INRA). It is based on some of the chemical constituents of the feed studied: crude protein, crude fibre and fat (Demarquilly et al., 1978). However, this chemical method has limitations, especially with feeds rich in unusually digestible cell wall constituents such as sugar beet and citrus pulps and soya hulls. For this type of feed, measurement of the lignin content (determined by van Soest's method (van Soest, 1963)) improves the prediction ( Sauvant, 1981 ). The work presented here compares various chemical, biological and enzymatic methods of predicting the digestibility of feeds or feed mixtures. MATERIALSAND METHODS The 24 feeds studied include (Table I): 2 starch-rich cereals; 3 leguminous seeds and protein-rich seed cakes; 13 industrial by-products (citrus pulp, soya hulls) high in easily-digestible cellulose; 6 forages and straw high in poorlydigestible cellulose. The in vivo digestibility (apparent digestibility) of these 24 feeds has been determined earlier. Samples representing 13 diets made up of hay or straw and single concentrates in variable proportions (concentrate feeds ranged from 15 to 60% ) were studied in order to verify the cumulative effect of the enzymatic method. In addition 24 mixture feeds evaluated at the Rowett Research Institute were studied in order to verify the enzymatic method chosen.
Determining in vivo digestibility Each in vivo digestibility measurement was realized on 6 mature castrated wethers, 3 or 4 years old. Digestibility measures were done with total collection of faeces for 6 days after a preliminary period of 15 days. Feeds involved in this study were associated with hay and represented 60% of the diet dry matter, a level established to avoid disturbing rumen functions. Some diets were supplemented with urea to obtain a nitrogen content (about 2% ) necessary to meet the requirements of rumen micro-organisms. Diets were fed twice a day (23 g digestible organic matter kg-1 po.75) at near maintenance level. This method reduced or minimized associative digestibility between feed and hay, so feed digestibility was calculated using the difference method. In vivo digestibility coefficients of the organic matter varied from 46.3 for straw to 91.0 for soya bean meal for the separate feedstuffs and from 53.1 to 78.9 for the complete rations. The accuracy of prediction of these digestibility measurements was on average + 2.5 (variation coefficient 3% ).
205
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Methods for estimating feed digestibility Chemical methods The 24 feeds were analysed for ash, total protein (N. Kjeldahl X 6.25), lipid (ether extract) and crude fibre (Weende). In addition, cell-wall constituents: neutral detergent fibre (NDF), acid detergent fibre (ADF) and acid detergent lignin (ADL), were determined according to van Soest (1963) and van Soest and Wine {1967), adapted by Giger et al. (1979).
In vitro digestibility In vitro digestibility was determined according to the method of Tilley and Terry (1963). The feeds with protein contents below 10% were completed with urea, glucose and yeast (Wilkins, 1967).
Enzymatic degradability The technique used was that generally employed in the laboratory for forages (Aufr~re, 1982), modified due to the presence of starch in certain feeds (acid hydrolysis of starch at 80 ° C ). This technique involves three stages: (1) pre-treatment with pepsin (pepsin Merck no. 7190; 1:10 000) in hydrochloric acid (0.2% pepsin in 1 N HC1 (method A) or 0.1 N HC1 (method B)) in a water bath at 40 ° C for 24 h; (2) starch hydrolysis in a water bath in the same mixture for exactly 30 min at 80 ° C; (3) attack by cellulase (cellulase Onozuka R 10 extracted from Trichoderma viride, Yakult Honsha Co. Ltd, Japan) after filtration and rinsing, for 24 h in a water bath at 40 ° C. RESULTS
Predicting digestibility from chemical composition The chemical compositions of the feeds studied are given in Table I. Average total protein content (TNC) is 16.3%, but certain feeds have contents up to 52%. Crude fibre content also varies considerably, from 2 to 51%. As for forage, the organic matter digestibility ( OMD, in % ) was estimated from the crude fibre content (CBO expressed in g kg - 1 0 M ) but with poor accuracy (eqn. (1)). OMD = 84.5 - 0.54 CBO R = 0.575, RSD = 12.5, N= 24
(1)
Introduction in the above regression equation of other chemical constituents in addition to crude fibre (total crude protein, ash or starch) does not much improve accuracy in predicting digestibility. Differences between measurements and estimated values are large, on av-
207 in vtvo organic matter d i g e s t i b i l i t y (Oil))
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I
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Fig. 1.The relationbetween organic matter digestibilitymeasured in vivo ( O M D ) and that estimated from the crude fibre content (CBO) and/or the cell-wallcontent (NDFO). • = C B O ; /x = N D F O . For key to abbreviationssee Table I.
erage 11.4 points, 16% in relative value. For a given crude fibre content, organic matter digestibility varies considerably according to the basic material. Wheat straw and citrus pulp both have crude fibre contents of about 40% but their digestibilities are 46.3 and 75.4, respectively. Similarly, rapeseed hulls and sugar beet pulp have, for the same crude fibre content (approximately 19% ), very different digestibilities (58.9 and 85.3, respectively). Further, this predicting equation largely underestimates the digestibility of feeds rich in highly digestible cell walls, particularly pea and soya hulls, sugar beet and citrus pulps (Fig. 1 ). If these 4 feeds are excluded, the average difference between measurements and estimates is reduced, from 11.4 to 7.7; the variation coefficient drops from 16 to 13%, which is still rather high. Since Weende's crude fibre does not properly represent cell wall in a feed, other analytical methods were tried. The results of cell-wall constituents according to van Soest's method, shown in Table I, provide an improved appreciation of the importance of cell walls and their composition. The cell-wall fraction is better represented by NDF (expressed in g k g - 1 0 M ) than crude fibre content, giving an improved prediction of digestibility (RSD=9.2).
208 TABLE II Equations predicting organic matter digestibility from chemical composition (expressed in % of OM) Constant
Crudefibre (CBO)
84.50 94.13 81.03 80.68 93:88 96.01 85.37 95.50
-0.54*
Cell walls (NDFO)
Lignocellulose (ADFO)
Lignin (ADLO)
-0.49* -0.29* - 1.70" -0.51" - 0.40*
0.03 - 1.13" -1.49" -1.16"
-0.18 +0.08
-0.44*
R
RSD
0.58 0.79 0.44 0.66 0.80 0.89 0.71 0.90
12.5 9.2 13.7 11.5 9.4 6.9 11.0 6.9
*P < 0.05. U s e of the N D F c o n t e n t instead of crude fibre c o n t e n t in digestibility-predicting e q u a t i o n s results in a reduction in the difference b e t w e e n calculated values and m e a s u r e m e n t s , generally 6.2 c o m p a r e d w i t h 11.4. Precision of prediction from N D F values ( R S D = 13.7) is slightly better t h a n that o b t a i n e d in vivo organic matter digestibility
BEET /
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IRAWQcORN'BT
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r : 0.936
in vitro digestibility (TILLEY et TERRY)
Fig. 2. The relationbetween in vivo organic matterdigestibilityand in vitro digestibility of Tilley and Terry. For keytoabbreviations see Table I.
209
from ADF contents (RSD = 13.7) or ADL contents ( RSD = 11.5) but this difference is not significant (Table II ). Two-by-two combinations of parameters give a further slight increase in predicting accuracy, the residual standard deviation being reduced from 9.2 to 6.9 when both lignin and NDF contents are taken into account, but again this improvement is not significant and the standard deviation remains high. Using the summative model of Goering and van Soest (1970), the predicting equation results in a difference between calculated values and measurements (7.0) which is slightly higher than that between predicting values from NDF content and measurements (6.2). However it must be taken into account that the summative model was developed upon forages and is applied here on concentrate feeds.
Predicting digestibility from in vitro digestibility ( Tilley and Terry, 1963) For the 24 feeds studied, the relation between in vivo organic matter digestibility ( OMD ) and in vitro dry matter digestibility (IVMD, in % ) is given by eqn. (2) OMD = 13.0 + 0.807 IVMD R = 0.936, RSD -- 5.4, N-- 24
(2)
The mean difference between measured and calculated values is 4.2, a considerable improvement in comparison with that obtained by chemical methods, although straw treated with sodium hydroxide deviates greatly from the regression line, at a distance of 13.7 (Fig. 2). Many studies (Ololade et al., 1970; Rexen and Vestergaard Thomsen, 1976; Bergen et al., 1979; Istasse et al., 1981 ) have shown that digestibility of cereal straw or maize cobs treated with sodium hydroxide is largely overestimated by this method. A revised equation linking in vitro digestibility with in vivo digestibility, after excluding the straw data, was calculated (eqn, ( 3 ) ) O M D = 14.6+0.789 IVMD R = 0.955, RSD = 4.4, N = 23
(3)
Exclusion of the straw data improved precision. The residual standard deviation was reduced from 5.4 for 24 samples to 4.4 for the 23 remaining.
Estimating digestibility from enzymatic degradability The enzymatic degradability method used for forage (Aufr~re, 1982) was applied to the 24 feeds studied. With forage, cellulase attack was preceded by hydrolysis with pepsin in an acid medium (1 N HC1, Method A). However, this method had to be slightly modified to analyse energy feeds of high starch contents. It was necessary to hydrolyse the starch before enzymatic attack.
210 in vivo organic matter digestibihty (OMD)
BEET SOYB.M O ~ PEA,H "
COR N LURP" %~m~GLF
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Pepsin-cellulase d i g e s t i b i l i t y (DCO) HCl 0 . I N
Fig. 3. The relation between in vivo organic matter digestibility and pepsin-cellulase digestibility with 1 N hydrochloric acid and 0.1 N hydrochloric acid. For key to abbreviations see Table I.
211 TABLE III Enzymaticdegradabilityof diet (DCO) Rations (constituentsas % DM)
DCO DCO additive= global DCOi×li%
Straw (84) +soyabean meal (15) NaOH straw (84) +soya bean meal (15) Straw (84)+maize (5)+soya bean (11) NaOH straw (84) +maize (5) +soya bean meal Barley (59.9) +hay (40.1) Barley (29.7) +hay (70.3) Maize (62)+hay (38) Lupin (60.6) +hay (39.4) Beet pulp (30) +hay (70) Beet pulp (39.9) + hay (70) Rapeseedmeal (60.3)+hay (39.7) Soyabean meal (30) +hay (70) Soyabean meal (60) +hay (40)
39.1 51.5 42.6 49.9 75.4 64.0 75.5 74.2 65.2 74.9 69.8 65.1 79.6
39.5 53.1 40.2 53.9 74.2 64.5 76.5 76.0 64.5 76.1 73.4 65.3 78.6
The equation associating organic matter digestibility (OMD) and enzymatic degradability as a proportion of the organic matter (DCO, in % ) is shown in eqn. (4), Method A OMD = 12.3 + 0.781 DCO
(4)
R = 0.905, RSD = 6.5, N = 24 Study of the deviations from the regression suggest that enzymatic degradability underestimates the digestibility of feeds rich in easily digestible cellulose such as pea, horse bean and soya hulls and citrus pulp (Fig. 3). In contrast, Method A overestimates digestibility when the basic product has a high hemicellulose content (hemicellulose calculated by the difference N D F - ADF) as is the case for maize cobs and whole plant residues and bran. For these feeds, the difference between the values measured and calculated is high (11.2, 9.7 and 11.8, respectively). However, their high hemicellulose contents do not appear to be the direct cause of their deviation from the model: straw, which also contains a considerable proportion of hemicellulose does not deviate from the regression line. The factor involved appears rather to be the pentosans which constitute most of the hemicellulose fraction of maize cobs and wheat bran (Cerning and Guilbot, 1974; Riquet, 1979). The explanation for this may be that pentosans, considered as poorly digestible by ruminants (Hungate, 1966), are hydrolysed by hydrochloric acid (Salo, 1957; van Soest, 1967; Schaller, 1978; McQueen and Nicholson, 1979). The hydrochloric acid attack (1 N HC1) which precedes cellulase action could be the cause of overestimation of digestibility for pentosan-rich feeds.
212 in vivo organic matter d i g e s t i b i l i t y (OMD)
90
Y= X
85
80
~O
75
i
70
!
75
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80
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85
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90
-b
OMDestimated by pepsin-cellulase digestibility
Fig. 4. T h e relation between in vivo organic m a t t e r digestibility a n d organic m a t t e r digestibility estimated by pepsin-cellulase m e t h o d for 24 concentrate mixtures of the Rowett Institute.
Following this hypothesis, the action of acid hydrolysis was reduced before the enzyme attack with a weaker concentration of hydrochloric acid (0.1 N, Method B). Applied to the 24 feeds, degradability with 0.1 N acid is on average 5.9 lower TABLE IV Comparison of m e t h o d of predicting organic m a t t e r digestibility Variables
NDF +ADL
Residual s t a n d a r d deviation
7.4
Correlation between deviations measured OMD - calculated OMD with NDF+ADL DIV DCO
1.000 ---
DIV 5.4
- 0.098 1.000 --
DCO 3.2
0.233 0.252 1.000
213 than that obtained with I N acid. However, the reduction varies with the feed considered, 26.6 and 11.9, respectively, for maize cobs and plant residues and 15.2 for bran, compared with 4.2 for the other feeds. The equation relating organic matter digestibility (OMD) and enzymatic degradability, measured with the weaker hydrochloric analysis (0.1 N HC1) and compared, as before, with the organic matter content (DCO, in % ) is eqn. (5), Method B OMD -- 22.6 + 0.699 DCO R = 0.978, RSD -- 3.2, N= 24
(5)
As might be expected, the difference between measured and calculated values is reduced, 2.5 compared with 5.0 for Method A (Fig. 3b), particularly as a result of reduced deviations for maize cobs, and plant residues and bran ( 3.8 against a previous 10.9). To determine the cumulative effect of this enzymatic method on mixed feeds, measurements of degradability by cellulase (with pre-treatment in 0.1 N acid medium) were made on representative samples of 13 rations of forage (hay or straw) and various concentrates. First, we calculated the difference between degradability by cellulase either measured on the rations or calculated from the degradabilities of each of the basic feeds (Table III). The measurements and calculations are not significantly different, deviations varying from - 4.0 to + 2.4 according to the feeds considered; enzymatic degradability is thus an additive measurement. Secondly, for 24 mixed feeds of the Rowett Institute we calculated the variations between digestibilities measured and those estimated from eqn. (5). They ranged from - 2 . 5 to + 3.5 (Fig. 4). Enzymatic degradability can thus be used also to predict digestibility of concentrate mixtures. The average accuracy of the estimation judged by the mean difference between measured and calculated values is better than that obtained for feeds, respectively 1.!0 compared with 2.5 found for the 24 feeds.
Comparison of different methods of predicting the OMD The enzymatic method used (pepsin-cellulase) predicts organic matter digestibility of feeds with greater accuracy than the chemical methods, when cellwall constituents are considered separately or for the combination NDF and ADL (Table IV). In addition, the differences between the observed and calculated values were compared for each of the three models used (principal component analysis). The differences calculated from NDF and ADL contents vary in opposite direction to in vitro digestibility especially for feeds rich in highly digestible cell walls. The digestibility of these feeds is underestimated with the cell-wall contents and overestimated by in vitro digestibility. Never-
214 theless, overall there is no significant correlation between the deviations calculated by any one of the three methods used. DISCUSSION In France, to predict energy feed digestibility and thus energy value, equations involving the chemical composition of these feeds were proposed by Demarquilly et al. (1978) from a series of data concerning the composition and digestibility of 61 common basic feeds given in the 1978 INRA Tables. However, the precision of prediction obtained with crude fibre content remains very poor (RSD -- 12.5, N = 24). As shown by Sauvant (1981), deviations from predictions may be very large for certain feeds. This is particularly true for feeds with easily digestible cellulose (pea and soya hulls, sugar beet and citrus pulp ), for which digestibility is considerably underestimated. The regression equation involving cell-wall contents determined by the method of van Soest improves prediction accuracy (RSD = 9.2 with NDF content), especially for feeds with a large amount of easily digestible cellulose. Sauvant (1981), Wainman et al. (1981) and Castagna et al. (1984) found that the residual standard deviation from the OMD estimate of different feeds from NDF contents is further improved if the ADL is introduced into the model. Nevertheless, the precision obtained remains lower (RSD -- 6.9) than that of Sauvant (1981) ( RSD = 3.2, N = 45) and Castagna et al. (1984) (RSD = 3.4, N - 18). Use of the summative model of Goering and van Soest (1970) does not improve prediction accuracy; however such comparison is not totally possible, since the summative model is applied upon data from which the equations were not developed. Biological or enzymatic methods have also been proposed for predicting concentrate digestibility. The in vitro digestibility of Tilley and Terry (1963) has been used: (1) to compare basic materials by Dowman and Collins (1982) for barley ( RSD = 2.1, N = 28 ) and oats (RSD = 3.8, N = 16 ) and by Schmid et al. (1975) and Collins et al. (1980) for cereal silages; (2) to compare mixed rations by Kumeno et al. (1967), Nik-Khah and Beard (1977), Giger et al. (1979) and Mathiesen and Moller (1983). For the 24 feeds used in the present study, in vitro digestibility gives a better prediction of the feed in vivo digestibility than the combination NDF and ADL ( RSD -- 4.4 and 6.9, respectively). In contrast, Marten et al. (1975) obtained, for cereals and sorghum silage, equally good digestibility estimates with ADF residues and in vitro digestibility. The use of methods measuring feed degradability under enzyme action has recently been developed for concentrates (Abe et al., 1974; Clark and Beard, 1977; Ilsraelsen et al., 1978; Dowman and Collins, 1982; Castagna et al., 1984). They vary considerably,but all are based on two steps: (1) a pre- or post-treatment by chemicals (HCI, detergent) and/or enzymes (amylase, pepsin, pron-
215
TABLE V Comparison of differentmethods of enzymatic degradation (de Boever et al.,1984) References
Measurement method
Type of cellulaseused
Kellner and Kirchgessner (1976)
2 N HCI Cellulase Pepsin
30 rain,I000C 24 h, 40°C 48 h, 40°C
D o w m a n and Collins (1982) Standard method ADAS, U.K. (Method chain CEE)
NDS Cellulase With amyloglucosidase A (pre-treatment)
I h, 100°C 24 h, 40°C
Castagna et al. (1984)
{Method chain CEE)
H20 Cocktail enzyme I Pepsin Cocktail enzyme II
I0 min, 700C 24 h, 40°C 4 h, 40°C 24 h, 40 °C
Jones and Hayward {1975) GotoandMinson (1977) Aufrbre (1982) (Method chain CEE)
AmyloglucosidaseB 16h, 55°C Pepsin 24h, 40°C Cellulase 24 h, 40°C Reducing volume
Sauvant (1980)
Pepsin H20 Cellulase
16 h, 60°C
24h, 40°C 48 h, 40°C
Aufrbre and Doreau (1983)
Pepsin Pepsin Cellulase
24 h, 40°C 45 rain, 80°C 24h, 40°C
Prediction of OMD from celluiase digestibility r2
RSD
Roth
0.64
2.2
BDH Roth Onozuka Roth Onozuka
0.46 0.59 0.60 0.64 0.66
2.7 2.4 2.3 2.2 2.2
Novoenzymes
0.57
2.4
Novoenzymes
0.57
2.6
BDH Onozuka BDH + Sigma BDH Onozuka
0.75 0.79 0.73 0.71 0.74
1.8 1.7 1.9 2.O 1.9
BDH Onozuka BDH Onozuka
0.75 0.77 0.72 0.77
1.8 1.8 1.9 1.8
Onozuka
0.79
1.7
R S D = residualstandard deviation.
ase) and (2) attack by cellulase alone or mixed with other enzymes (amylase, hemicellulase). The enzymatic method presented here estimates digestibility of feeds with a better accuracy (RSD = 3.2, N = 24). When applied to concentrate mixtures, our method also gives acceptable results, the mean deviation between measured values and those estimated by eqn. (5) is smaller for the 13 rations (1.1) than for the 24 feeds studied (2.5). This improved accuracy for concentrate mixtures probably results from the fact that the estimation errors for each of the components usually compensate for each other. The repeatability of the method is satisfactory (1.2), and can be divided into within-series or within-
216
days (0.8) and between-series or between-days standard deviations (0.9). The simplicity of the method, all the operations being carried out in the same container, largely explains the good intra-series repeatability obtained. A series of analyses was organized within the European Economic Communities by Van Der Meet (1982, 1983) to evaluate the repeatability and reproducibility of an enzymatic degradation procedure: 34 laboratories were involved and 3 methods of enzyme degradation were tested. One of the latter, pepsin + cellulase, was very similar to the method proposed in this paper. The European study indicated that variation within a laboratory remains small, although a little higher than found previously, 1.8 compared with 1.2, but the variation between laboratories was large ( 5.3 ) whatever the method of enzyme degradation used. The use of control samples in each series is thus necessary to permit comparison between laboratories. In 1984 de Boever et al. compared 18 methods of enzymatic degradability on 31 concentrates with known digestibilities. The accuracy of estimation varied from 1.7 to 2.7 according to the method used: the best predictions were obtained with pepsin-cellulase methods (RSD = 1.7-2.0) (Table V ). The best method (RSD = 1.7 ) is an adaptation of the one proposed in this paper (pepsin hydrolysis in acid conditions and attack by cellulase) but differs by the duration of acid hydrolysis (45 min compared with 30 min), and the relative proportions of cellulase and substrate (30% compared with 10% expressed as a percentage). This study indicates that enzymatic degradability provides an estimation of the digestibility of energy feeds used in the manufacture of concentrates with greater precision than those obtained by other chemical or biological methods. Systematic comparisons of several methods for estimating feed digestibility have also shown this superiority of enzymatic degradability over in vitro digestibility (van der Meet, 1982; Mathiesen and Moller, 1983; de Boever et al., 1984). Nevertheless, it is difficult to compare these three groups of methods for predicting digestibility - chemical, biological and enzymatic - solely according to their residual standard deviation. Whereas for the first two groups (chemical and biological ) the comparison uses the same dosage methods (van Soest, 1967 and Tilley and Terry, 1963), there are infinite variations for enzymatic methods, variations which give differing but generally small residual standard deviations (1.7-2.7) ( de Boever et al., 1984). REFERENCES Abe, A., Horii, S. and Kameoka, K.I., 1974. Development and application of cellulase hydrolysis for predicting digestibility of roughage. 1 - Digestion of cell wall carbohydrates by cellulase. Jpn. J. Zootechn. Sci., 43: 141-145. AFNOR, 1981. Cellulose brute, M~thode CCE 4~me Directive Norme NF V 03-040 in Aliments des animaux. M~thodes d'analyse. Ed AFNOR. Aufr~re, J., 1982. Etude de la pr~vision de la digestibilit~ des fourrages par une m~thode enzymatique. Ann. Zootech., 31: 111-130.
217
Aufrbre,J. and Michalet-Doreau, B., 1983. In vivo digestibilityand predictionof digestibilityof some by-products.Feeding Value of By-Products and Their Use by Beef Cattle,E E C seminar, Gontrode, September 27-29. Bergen, L., Klopfenstein,T. and Britton, R., 1979. Effect of sodium hydroxide on efficiencyof rumen digestion.J. Anita. Sci.,49: 1317-1323. Castagna, A., Sauvant, D., Dorleans,M. and Giger,S.,1984.Etude de lad~gradabilit~enzymatique des aliments concentrds et sous-produits.Ann. Zootech.,33: 265-290. Cerning, J. and Guilbot,A., 1974. Carbohydrate composition of wheat. In: G.E. Inglett (Editor), Wheat: Production and Utilisation,Avi. Publis.Comp., West Port. Conn., Chapter 9. Clark,J. and Beard, J.,1977. Predictionof the digestibilityof ruminant feedsfrom theirsolubility in enzyme solutions.Anita. Feed. Sci.Technol.,2: 153-159. Collins, M., Ferreira, A.M., Rohneder, D.A. and Jorgensen, N.A., 1980. Laboratory analyses for predicting digestibility and intake of corn silage. Agron. J., 82: 889-892. De Boever, J.L., Cottyn, B.G., Vanacker, J.M. and Buysse, F.X., 1984. Test of different enzymatic methods on compound feeds. CEC Workshop on Methodology of Feedingstuffs for Ruminants. Demarquilly, C. and Jarrige, R., 1981. Panorama des mdthodes de pr~vision de la digestibilit~ et de la valeur ~nergdtique des fourrages. In: Pr~vision de la valeur nutritive des aliments des ruminants, INRA, Versailles, pp. 41-59. Demarquilly, C., Andrieu, J. and Sauvant, D., 1978. Composition et valeur nutritive des aliments. In: Alimentation des Ruminants, INRA 1978, Versailles, Chapter 16: 469-518. Dowman, M.G. and Collins, F., 1982. The use of enzymes to predict the digestibility of animal feeds. J. Sci. Food Agric., 33: 689-696. Fonnesbeck, P.V., 1976. Estimating nutritive value from chemical analysis. In: P.V. Fonnesbeck, L.E. Harris and L.C. Kearl (Editors), Feed Composition, Animal Nutrient Requirements and Computerization of Diets, Utah Agr. Exp., Utah State Univ., Jogan, Utah, pp. 219-231. Giger, S., Sauvant, D., Dorleans, M. and Morand-Fehr, P., 1979. D~termination semi automatique des constituants membranaires des aliments concentrds par la m~thode de van Soest. 30th Annual Meeting of the European Association for Animal Production, Hungate, England, 23-26 July 1979. Goering, H.K. and van Soest, P.J., 1970. Forage fiber analyses. Agricultural Handbook 379, UPSDA. Goto, I. and Minson, D.J., 1977. Prediction of the dry matter digestibility of tropical grasses using a pepsin-cellulase assay. Anita. Feed Sci. Technol., 2: 247-253. Hungate, R.E., 1966. The Rumen and its Microbes. Academic Press, New York. Israelsen, M., Rexen, B. and Vestergaard Thomsen, K., 1978. Cellulase insoluble fibre as a measure of unavailable organic matter in cattle compounds containing alkali-treated straw. Anita. Feed Sci. Technol., 3: 227-234. Istasse, L., Van Eenaeme, C., Lambot, 0., Gielen, M. and Bienfait, J.M., 1981. Etude de quelques facteurs de variation de la digestibilit~ in vitro. Applications ~ un foin trait~ ou non ~ la soude. Ann. Zootech., 30: 183-196. Jones, D.H. and Hayward, M.V., 1975. The effect of pepsin pre-treatment of herbage on the prediction of dry matter digestibility from solubility in fungal cellulase solubility. J. Sci. Food Agric., 26: 711-718. Kellner, R.J. and Kirgessner, M., 1976. Zur Methodick der in vitro Verdaulichkeitobestimmung von Griin- und Rauhfutter mir Cellulase. Landwirtsh. Forsch, 29: 204-210. Kumeno, F., Dehority, B.A. and Johnson, R.R., 1967. Development of an in vitro fermentation technique for estimating the nutritive value of high energy mixed rations for ruminants. J. Anita. Sci., 26: 867-871. Marten, G.C., Goodrich, R.D., Schmid, A.R., Maiske, J.C., Jordan, R.M. and Linn, J.C., 1975. Evaluation of laboratory methods for determining quality of corn and sorghum silages. 2. Chemical methods for predicting in vivo digestibility. Agron. J. (U.S.A.), 67: 247-251. Mathiesen, H.P. and Moiler, J., 1983. Enzymatisk in vitro oploselighed. Beretning, 108: 1-17.
218 McQueen, R.E. and Nicholson, J.W.G., 1979. Fiber analysis.Modification of the neutral detergent fiber procedure for cereals and vegetables by using amylase. J. Ass. OfficialAnn. Chem., 62: 676-680. Morrison, I.M., 1976. N e w laboratory methods for predicting the nutritivevalue of forage crops. World Rev. Anita. Prod., 12: 75-82. Nik-Khah, A. and Beard, J., 1977. A comparison of methods for determining the digestibilityof ration for sheep. Aust. J. Exp. Agric. Anita. Husb., 17: 746-750. Ololade, B.G., Movat, D.N. and Winch, J.E., 1970. Effect of processing methods on the in vitro digestibilityof sodium hydroxide treated roughages. Can. J. Anita. Sci.,50: 657-662. Osbourn, D.F. and Siddons, R.C., 1980. Enzymatic methods to predict the value of the energy and protein in feedingstuffs.Ann. Zootech., 29: no. Hors Sdrie,325-336. Rexen, F.P. and Vestergaard Thomsen, K., 1976. The effectof digestibilityof a new technique for alkalitreatment of straw. Anita. Feed Sci. Technol., 1: 73-83. Riquet, A.M., 1979. Etude des constituants membranaires de divers produits v6g6taux destinds ralimentation animale. Significationet limitesdes principalesm6thodes de dosage. Th~se Doctorat 3~me cycle,Universitd de Paris XI, 85 pp. Salo, M.L., 1957. Lignin studies.I. Investigation concerning lignin determinations. Maataloust. Aikakansk, 29: 186-193. Sauvant, D., 1980. Laboratory methods for predictingthe energy and protein value of concentrates for ruminants. In: A.J.H. Van Es and J.M. Van Der Meer (Editors),Methods of Analysis for Predicting the Energy and Protein Value of Feeds for Farm Animals, Predoc, Wageningen, pp. 93-106. Sauvant, D., 1981. Pr~vision de la valeur 6nergdtique des aliments concentrds et composds pour les ruminants. In: Pr~vision de la Valeur Nutritive des Aliments des Ruminants, INRA, Versailles,pp. 237-258. Schaller, D., 1978. Fiber content and structure in foods. Am. J. Clin. Nutr., 31: $99-$102. Schmid, A.R., Goodrich, R.D., Marten, G.C., Meiske, J.C., Jordan, R.M. and Halgerson, J.L., 1975. Evaluation of laboratory methods for determining quality of corn and sorghum silages. I.Biological methods for predicting in vivo digestibility.Agron. J.,67: 243-251. Tilley, J.M.A. and Terry, R.A., 1963. A two stage technique for the in vitro digestion of forage crops. J. Br. Grassl. Soc., 18: 104-111. Van Der Meer, J.M., 1982. European "in vitro"ring test.Manuel, C E C Workshop on Methodology of Feedingstuffs for Ruminants. Van Der Meet, J.M., 1983. European "in vitro" ring test.Statisticalreport, C E C Workshop on Methodology of Feedingstuffs for Ruminants. Van Soest, P.J., 1963. Use of detergent in the analysis of fibrous feed. I - A rapid method for the determination of fiber and lignin. J. Ass. Off. Agric. Chem., 46: 829-835. Van Soest, P.J., 1967. Development of a comprehensive system of feed analysis and application to forages. J. Anim. Sci., 26: 119-128. Van Soest, P.J. and Wine, R.H., 1967. Use of detergents in the analysis of fibrous feeds. IV. Determination of plant cell-wall constituents. J. Assoc. Off. Agric. Chem., 50: 50-55. Wainman, F.W., Dewey, P.J.S. and Boyne, A.N., 1981. Feedingstuffs Evaluation Unit Third. Report 1981, Rowett Research Institute. Wilkins, R.J., 1967. The application of the in vitro digestion technique to some aride-zone fodders. J. Br. Grassl. Soc., 22: 65-69.
203
Comparison of Methods for Predicting Digestibility of Feeds JOCELYNE
AUFRERE
and B R I G I T T E M I C H A L E T - D O R E A U
InstitutNational de la Recherche Agronomique (INRA ), Laboratoire des Aliments, Centre de Recherche Zootechnique et Vdtdrinaire (CRZV), Theix-63122 Ceyrat (France) (Received 29 May 1986; accepted forpublication13 November 1987)
ABSTRACT Aufr~re,J. and Michalet-Doreau, B., 1988. Comparison of methods for predictingdigestibilityof feeds.Anita. Feed Sci. Technol.,20: 203-218. Five techniques were investigated to find a laboratory method for the prediction of the in vivo digestibility of feeds and mixed feeds: two chemical techniques (Weende crude fibre and van Soest methods), one biological technique (in vitro digestibility ) and two enzymatic techniques (digestion by pepsin-cellulase, normal or modified (HCI 0.1 N instead of 1 N) ). The enzymatic degradability (Method B, HC10.1 N) allowed a more accurate prediction of the in vivo organic matter digestibility (OMD) of 24 feeds than the separate data of crude fibre, neutral detergent fibre (NDF), acid detergent fibre (ADF) and acid detergent lignin (ADL) contents or in vitro digestibility. The selected equation obtained with the celhlase method could also be used for feed mixtures.
INTRODUCTION
Many laboratory methods for predicting the digestibility of energy and dry matter of forage exist. They can be classified into three classes: (1) chemical analyses only; (2) fermentation with rumen micro-organisms; (3) hydrolysis by enzymatic preparations. Variable correlations have been established between the indigestible fraction of forages and their crude fibre content Weende method (AFNOR, 1981) or lignocellulose (ADF) of van Soest (1963) content. In contrast with these methods, those involvingrumen micro-organisms or enzymes (such as in vitro or in sacco digestibility and cellulase attack followed or preceded by chemical and/or enzymatic attack) are based on an appreciation of the indigestible residue of forage. These methods for predicting forage digestibility have been the subject of several reviews (Fonnesbeck, 1976; Morrison, 1976; Osbourn and Siddons, 1980; Demarquilly and Jarrige, 1981 ). 0377-8401/88/$03.50
© 1988 Elsevier Science Publishers B.V.
204 These methods, all developed for use on forages, have been much less studied with energy feeds (except Fonnesbeck, 1976). The official method of calculating the energy value of energy feeds or compound feeds in France was proposed by Institut National de la Recherche Agronomique (INRA). It is based on some of the chemical constituents of the feed studied: crude protein, crude fibre and fat (Demarquilly et al., 1978). However, this chemical method has limitations, especially with feeds rich in unusually digestible cell wall constituents such as sugar beet and citrus pulps and soya hulls. For this type of feed, measurement of the lignin content (determined by van Soest's method (van Soest, 1963)) improves the prediction ( Sauvant, 1981 ). The work presented here compares various chemical, biological and enzymatic methods of predicting the digestibility of feeds or feed mixtures. MATERIALSAND METHODS The 24 feeds studied include (Table I): 2 starch-rich cereals; 3 leguminous seeds and protein-rich seed cakes; 13 industrial by-products (citrus pulp, soya hulls) high in easily-digestible cellulose; 6 forages and straw high in poorlydigestible cellulose. The in vivo digestibility (apparent digestibility) of these 24 feeds has been determined earlier. Samples representing 13 diets made up of hay or straw and single concentrates in variable proportions (concentrate feeds ranged from 15 to 60% ) were studied in order to verify the cumulative effect of the enzymatic method. In addition 24 mixture feeds evaluated at the Rowett Research Institute were studied in order to verify the enzymatic method chosen.
Determining in vivo digestibility Each in vivo digestibility measurement was realized on 6 mature castrated wethers, 3 or 4 years old. Digestibility measures were done with total collection of faeces for 6 days after a preliminary period of 15 days. Feeds involved in this study were associated with hay and represented 60% of the diet dry matter, a level established to avoid disturbing rumen functions. Some diets were supplemented with urea to obtain a nitrogen content (about 2% ) necessary to meet the requirements of rumen micro-organisms. Diets were fed twice a day (23 g digestible organic matter kg-1 po.75) at near maintenance level. This method reduced or minimized associative digestibility between feed and hay, so feed digestibility was calculated using the difference method. In vivo digestibility coefficients of the organic matter varied from 46.3 for straw to 91.0 for soya bean meal for the separate feedstuffs and from 53.1 to 78.9 for the complete rations. The accuracy of prediction of these digestibility measurements was on average + 2.5 (variation coefficient 3% ).
205
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Methods for estimating feed digestibility Chemical methods The 24 feeds were analysed for ash, total protein (N. Kjeldahl X 6.25), lipid (ether extract) and crude fibre (Weende). In addition, cell-wall constituents: neutral detergent fibre (NDF), acid detergent fibre (ADF) and acid detergent lignin (ADL), were determined according to van Soest (1963) and van Soest and Wine {1967), adapted by Giger et al. (1979).
In vitro digestibility In vitro digestibility was determined according to the method of Tilley and Terry (1963). The feeds with protein contents below 10% were completed with urea, glucose and yeast (Wilkins, 1967).
Enzymatic degradability The technique used was that generally employed in the laboratory for forages (Aufr~re, 1982), modified due to the presence of starch in certain feeds (acid hydrolysis of starch at 80 ° C ). This technique involves three stages: (1) pre-treatment with pepsin (pepsin Merck no. 7190; 1:10 000) in hydrochloric acid (0.2% pepsin in 1 N HC1 (method A) or 0.1 N HC1 (method B)) in a water bath at 40 ° C for 24 h; (2) starch hydrolysis in a water bath in the same mixture for exactly 30 min at 80 ° C; (3) attack by cellulase (cellulase Onozuka R 10 extracted from Trichoderma viride, Yakult Honsha Co. Ltd, Japan) after filtration and rinsing, for 24 h in a water bath at 40 ° C. RESULTS
Predicting digestibility from chemical composition The chemical compositions of the feeds studied are given in Table I. Average total protein content (TNC) is 16.3%, but certain feeds have contents up to 52%. Crude fibre content also varies considerably, from 2 to 51%. As for forage, the organic matter digestibility ( OMD, in % ) was estimated from the crude fibre content (CBO expressed in g kg - 1 0 M ) but with poor accuracy (eqn. (1)). OMD = 84.5 - 0.54 CBO R = 0.575, RSD = 12.5, N= 24
(1)
Introduction in the above regression equation of other chemical constituents in addition to crude fibre (total crude protein, ash or starch) does not much improve accuracy in predicting digestibility. Differences between measurements and estimated values are large, on av-
207 in vtvo organic matter d i g e s t i b i l i t y (Oil))
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90
80
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~ 40
:
10
',
,
20
30
40
50
!
I
60
70
I
I
80 90 Calculated 014D
Fig. 1.The relationbetween organic matter digestibilitymeasured in vivo ( O M D ) and that estimated from the crude fibre content (CBO) and/or the cell-wallcontent (NDFO). • = C B O ; /x = N D F O . For key to abbreviationssee Table I.
erage 11.4 points, 16% in relative value. For a given crude fibre content, organic matter digestibility varies considerably according to the basic material. Wheat straw and citrus pulp both have crude fibre contents of about 40% but their digestibilities are 46.3 and 75.4, respectively. Similarly, rapeseed hulls and sugar beet pulp have, for the same crude fibre content (approximately 19% ), very different digestibilities (58.9 and 85.3, respectively). Further, this predicting equation largely underestimates the digestibility of feeds rich in highly digestible cell walls, particularly pea and soya hulls, sugar beet and citrus pulps (Fig. 1 ). If these 4 feeds are excluded, the average difference between measurements and estimates is reduced, from 11.4 to 7.7; the variation coefficient drops from 16 to 13%, which is still rather high. Since Weende's crude fibre does not properly represent cell wall in a feed, other analytical methods were tried. The results of cell-wall constituents according to van Soest's method, shown in Table I, provide an improved appreciation of the importance of cell walls and their composition. The cell-wall fraction is better represented by NDF (expressed in g k g - 1 0 M ) than crude fibre content, giving an improved prediction of digestibility (RSD=9.2).
208 TABLE II Equations predicting organic matter digestibility from chemical composition (expressed in % of OM) Constant
Crudefibre (CBO)
84.50 94.13 81.03 80.68 93:88 96.01 85.37 95.50
-0.54*
Cell walls (NDFO)
Lignocellulose (ADFO)
Lignin (ADLO)
-0.49* -0.29* - 1.70" -0.51" - 0.40*
0.03 - 1.13" -1.49" -1.16"
-0.18 +0.08
-0.44*
R
RSD
0.58 0.79 0.44 0.66 0.80 0.89 0.71 0.90
12.5 9.2 13.7 11.5 9.4 6.9 11.0 6.9
*P < 0.05. U s e of the N D F c o n t e n t instead of crude fibre c o n t e n t in digestibility-predicting e q u a t i o n s results in a reduction in the difference b e t w e e n calculated values and m e a s u r e m e n t s , generally 6.2 c o m p a r e d w i t h 11.4. Precision of prediction from N D F values ( R S D = 13.7) is slightly better t h a n that o b t a i n e d in vivo organic matter digestibility
BEET /
90
SOYB.M • CORN BEET.pLUP Q •
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70
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IRAWQcORN'BT
n = 24
6b
do
RSD = 5.4
r : 0.936
in vitro digestibility (TILLEY et TERRY)
Fig. 2. The relationbetween in vivo organic matterdigestibilityand in vitro digestibility of Tilley and Terry. For keytoabbreviations see Table I.
209
from ADF contents (RSD = 13.7) or ADL contents ( RSD = 11.5) but this difference is not significant (Table II ). Two-by-two combinations of parameters give a further slight increase in predicting accuracy, the residual standard deviation being reduced from 9.2 to 6.9 when both lignin and NDF contents are taken into account, but again this improvement is not significant and the standard deviation remains high. Using the summative model of Goering and van Soest (1970), the predicting equation results in a difference between calculated values and measurements (7.0) which is slightly higher than that between predicting values from NDF content and measurements (6.2). However it must be taken into account that the summative model was developed upon forages and is applied here on concentrate feeds.
Predicting digestibility from in vitro digestibility ( Tilley and Terry, 1963) For the 24 feeds studied, the relation between in vivo organic matter digestibility ( OMD ) and in vitro dry matter digestibility (IVMD, in % ) is given by eqn. (2) OMD = 13.0 + 0.807 IVMD R = 0.936, RSD -- 5.4, N-- 24
(2)
The mean difference between measured and calculated values is 4.2, a considerable improvement in comparison with that obtained by chemical methods, although straw treated with sodium hydroxide deviates greatly from the regression line, at a distance of 13.7 (Fig. 2). Many studies (Ololade et al., 1970; Rexen and Vestergaard Thomsen, 1976; Bergen et al., 1979; Istasse et al., 1981 ) have shown that digestibility of cereal straw or maize cobs treated with sodium hydroxide is largely overestimated by this method. A revised equation linking in vitro digestibility with in vivo digestibility, after excluding the straw data, was calculated (eqn, ( 3 ) ) O M D = 14.6+0.789 IVMD R = 0.955, RSD = 4.4, N = 23
(3)
Exclusion of the straw data improved precision. The residual standard deviation was reduced from 5.4 for 24 samples to 4.4 for the 23 remaining.
Estimating digestibility from enzymatic degradability The enzymatic degradability method used for forage (Aufr~re, 1982) was applied to the 24 feeds studied. With forage, cellulase attack was preceded by hydrolysis with pepsin in an acid medium (1 N HC1, Method A). However, this method had to be slightly modified to analyse energy feeds of high starch contents. It was necessary to hydrolyse the starch before enzymatic attack.
210 in vivo organic matter digestibihty (OMD)
BEET SOYB.M O ~ PEA,H "
COR N LURP" %~m~GLF
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80
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I
go
Pepsin-cellulase d i g e s t i b i l i t y (DCO) HCl 0 . I N
Fig. 3. The relation between in vivo organic matter digestibility and pepsin-cellulase digestibility with 1 N hydrochloric acid and 0.1 N hydrochloric acid. For key to abbreviations see Table I.
211 TABLE III Enzymaticdegradabilityof diet (DCO) Rations (constituentsas % DM)
DCO DCO additive= global DCOi×li%
Straw (84) +soyabean meal (15) NaOH straw (84) +soya bean meal (15) Straw (84)+maize (5)+soya bean (11) NaOH straw (84) +maize (5) +soya bean meal Barley (59.9) +hay (40.1) Barley (29.7) +hay (70.3) Maize (62)+hay (38) Lupin (60.6) +hay (39.4) Beet pulp (30) +hay (70) Beet pulp (39.9) + hay (70) Rapeseedmeal (60.3)+hay (39.7) Soyabean meal (30) +hay (70) Soyabean meal (60) +hay (40)
39.1 51.5 42.6 49.9 75.4 64.0 75.5 74.2 65.2 74.9 69.8 65.1 79.6
39.5 53.1 40.2 53.9 74.2 64.5 76.5 76.0 64.5 76.1 73.4 65.3 78.6
The equation associating organic matter digestibility (OMD) and enzymatic degradability as a proportion of the organic matter (DCO, in % ) is shown in eqn. (4), Method A OMD = 12.3 + 0.781 DCO
(4)
R = 0.905, RSD = 6.5, N = 24 Study of the deviations from the regression suggest that enzymatic degradability underestimates the digestibility of feeds rich in easily digestible cellulose such as pea, horse bean and soya hulls and citrus pulp (Fig. 3). In contrast, Method A overestimates digestibility when the basic product has a high hemicellulose content (hemicellulose calculated by the difference N D F - ADF) as is the case for maize cobs and whole plant residues and bran. For these feeds, the difference between the values measured and calculated is high (11.2, 9.7 and 11.8, respectively). However, their high hemicellulose contents do not appear to be the direct cause of their deviation from the model: straw, which also contains a considerable proportion of hemicellulose does not deviate from the regression line. The factor involved appears rather to be the pentosans which constitute most of the hemicellulose fraction of maize cobs and wheat bran (Cerning and Guilbot, 1974; Riquet, 1979). The explanation for this may be that pentosans, considered as poorly digestible by ruminants (Hungate, 1966), are hydrolysed by hydrochloric acid (Salo, 1957; van Soest, 1967; Schaller, 1978; McQueen and Nicholson, 1979). The hydrochloric acid attack (1 N HC1) which precedes cellulase action could be the cause of overestimation of digestibility for pentosan-rich feeds.
212 in vivo organic matter d i g e s t i b i l i t y (OMD)
90
Y= X
85
80
~O
75
i
70
!
75
|
80
~
85
$
90
-b
OMDestimated by pepsin-cellulase digestibility
Fig. 4. T h e relation between in vivo organic m a t t e r digestibility a n d organic m a t t e r digestibility estimated by pepsin-cellulase m e t h o d for 24 concentrate mixtures of the Rowett Institute.
Following this hypothesis, the action of acid hydrolysis was reduced before the enzyme attack with a weaker concentration of hydrochloric acid (0.1 N, Method B). Applied to the 24 feeds, degradability with 0.1 N acid is on average 5.9 lower TABLE IV Comparison of m e t h o d of predicting organic m a t t e r digestibility Variables
NDF +ADL
Residual s t a n d a r d deviation
7.4
Correlation between deviations measured OMD - calculated OMD with NDF+ADL DIV DCO
1.000 ---
DIV 5.4
- 0.098 1.000 --
DCO 3.2
0.233 0.252 1.000
213 than that obtained with I N acid. However, the reduction varies with the feed considered, 26.6 and 11.9, respectively, for maize cobs and plant residues and 15.2 for bran, compared with 4.2 for the other feeds. The equation relating organic matter digestibility (OMD) and enzymatic degradability, measured with the weaker hydrochloric analysis (0.1 N HC1) and compared, as before, with the organic matter content (DCO, in % ) is eqn. (5), Method B OMD -- 22.6 + 0.699 DCO R = 0.978, RSD -- 3.2, N= 24
(5)
As might be expected, the difference between measured and calculated values is reduced, 2.5 compared with 5.0 for Method A (Fig. 3b), particularly as a result of reduced deviations for maize cobs, and plant residues and bran ( 3.8 against a previous 10.9). To determine the cumulative effect of this enzymatic method on mixed feeds, measurements of degradability by cellulase (with pre-treatment in 0.1 N acid medium) were made on representative samples of 13 rations of forage (hay or straw) and various concentrates. First, we calculated the difference between degradability by cellulase either measured on the rations or calculated from the degradabilities of each of the basic feeds (Table III). The measurements and calculations are not significantly different, deviations varying from - 4.0 to + 2.4 according to the feeds considered; enzymatic degradability is thus an additive measurement. Secondly, for 24 mixed feeds of the Rowett Institute we calculated the variations between digestibilities measured and those estimated from eqn. (5). They ranged from - 2 . 5 to + 3.5 (Fig. 4). Enzymatic degradability can thus be used also to predict digestibility of concentrate mixtures. The average accuracy of the estimation judged by the mean difference between measured and calculated values is better than that obtained for feeds, respectively 1.!0 compared with 2.5 found for the 24 feeds.
Comparison of different methods of predicting the OMD The enzymatic method used (pepsin-cellulase) predicts organic matter digestibility of feeds with greater accuracy than the chemical methods, when cellwall constituents are considered separately or for the combination NDF and ADL (Table IV). In addition, the differences between the observed and calculated values were compared for each of the three models used (principal component analysis). The differences calculated from NDF and ADL contents vary in opposite direction to in vitro digestibility especially for feeds rich in highly digestible cell walls. The digestibility of these feeds is underestimated with the cell-wall contents and overestimated by in vitro digestibility. Never-
214 theless, overall there is no significant correlation between the deviations calculated by any one of the three methods used. DISCUSSION In France, to predict energy feed digestibility and thus energy value, equations involving the chemical composition of these feeds were proposed by Demarquilly et al. (1978) from a series of data concerning the composition and digestibility of 61 common basic feeds given in the 1978 INRA Tables. However, the precision of prediction obtained with crude fibre content remains very poor (RSD -- 12.5, N = 24). As shown by Sauvant (1981), deviations from predictions may be very large for certain feeds. This is particularly true for feeds with easily digestible cellulose (pea and soya hulls, sugar beet and citrus pulp ), for which digestibility is considerably underestimated. The regression equation involving cell-wall contents determined by the method of van Soest improves prediction accuracy (RSD = 9.2 with NDF content), especially for feeds with a large amount of easily digestible cellulose. Sauvant (1981), Wainman et al. (1981) and Castagna et al. (1984) found that the residual standard deviation from the OMD estimate of different feeds from NDF contents is further improved if the ADL is introduced into the model. Nevertheless, the precision obtained remains lower (RSD -- 6.9) than that of Sauvant (1981) ( RSD = 3.2, N = 45) and Castagna et al. (1984) (RSD = 3.4, N - 18). Use of the summative model of Goering and van Soest (1970) does not improve prediction accuracy; however such comparison is not totally possible, since the summative model is applied upon data from which the equations were not developed. Biological or enzymatic methods have also been proposed for predicting concentrate digestibility. The in vitro digestibility of Tilley and Terry (1963) has been used: (1) to compare basic materials by Dowman and Collins (1982) for barley ( RSD = 2.1, N = 28 ) and oats (RSD = 3.8, N = 16 ) and by Schmid et al. (1975) and Collins et al. (1980) for cereal silages; (2) to compare mixed rations by Kumeno et al. (1967), Nik-Khah and Beard (1977), Giger et al. (1979) and Mathiesen and Moller (1983). For the 24 feeds used in the present study, in vitro digestibility gives a better prediction of the feed in vivo digestibility than the combination NDF and ADL ( RSD -- 4.4 and 6.9, respectively). In contrast, Marten et al. (1975) obtained, for cereals and sorghum silage, equally good digestibility estimates with ADF residues and in vitro digestibility. The use of methods measuring feed degradability under enzyme action has recently been developed for concentrates (Abe et al., 1974; Clark and Beard, 1977; Ilsraelsen et al., 1978; Dowman and Collins, 1982; Castagna et al., 1984). They vary considerably,but all are based on two steps: (1) a pre- or post-treatment by chemicals (HCI, detergent) and/or enzymes (amylase, pepsin, pron-
215
TABLE V Comparison of differentmethods of enzymatic degradation (de Boever et al.,1984) References
Measurement method
Type of cellulaseused
Kellner and Kirchgessner (1976)
2 N HCI Cellulase Pepsin
30 rain,I000C 24 h, 40°C 48 h, 40°C
D o w m a n and Collins (1982) Standard method ADAS, U.K. (Method chain CEE)
NDS Cellulase With amyloglucosidase A (pre-treatment)
I h, 100°C 24 h, 40°C
Castagna et al. (1984)
{Method chain CEE)
H20 Cocktail enzyme I Pepsin Cocktail enzyme II
I0 min, 700C 24 h, 40°C 4 h, 40°C 24 h, 40 °C
Jones and Hayward {1975) GotoandMinson (1977) Aufrbre (1982) (Method chain CEE)
AmyloglucosidaseB 16h, 55°C Pepsin 24h, 40°C Cellulase 24 h, 40°C Reducing volume
Sauvant (1980)
Pepsin H20 Cellulase
16 h, 60°C
24h, 40°C 48 h, 40°C
Aufrbre and Doreau (1983)
Pepsin Pepsin Cellulase
24 h, 40°C 45 rain, 80°C 24h, 40°C
Prediction of OMD from celluiase digestibility r2
RSD
Roth
0.64
2.2
BDH Roth Onozuka Roth Onozuka
0.46 0.59 0.60 0.64 0.66
2.7 2.4 2.3 2.2 2.2
Novoenzymes
0.57
2.4
Novoenzymes
0.57
2.6
BDH Onozuka BDH + Sigma BDH Onozuka
0.75 0.79 0.73 0.71 0.74
1.8 1.7 1.9 2.O 1.9
BDH Onozuka BDH Onozuka
0.75 0.77 0.72 0.77
1.8 1.8 1.9 1.8
Onozuka
0.79
1.7
R S D = residualstandard deviation.
ase) and (2) attack by cellulase alone or mixed with other enzymes (amylase, hemicellulase). The enzymatic method presented here estimates digestibility of feeds with a better accuracy (RSD = 3.2, N = 24). When applied to concentrate mixtures, our method also gives acceptable results, the mean deviation between measured values and those estimated by eqn. (5) is smaller for the 13 rations (1.1) than for the 24 feeds studied (2.5). This improved accuracy for concentrate mixtures probably results from the fact that the estimation errors for each of the components usually compensate for each other. The repeatability of the method is satisfactory (1.2), and can be divided into within-series or within-
216
days (0.8) and between-series or between-days standard deviations (0.9). The simplicity of the method, all the operations being carried out in the same container, largely explains the good intra-series repeatability obtained. A series of analyses was organized within the European Economic Communities by Van Der Meet (1982, 1983) to evaluate the repeatability and reproducibility of an enzymatic degradation procedure: 34 laboratories were involved and 3 methods of enzyme degradation were tested. One of the latter, pepsin + cellulase, was very similar to the method proposed in this paper. The European study indicated that variation within a laboratory remains small, although a little higher than found previously, 1.8 compared with 1.2, but the variation between laboratories was large ( 5.3 ) whatever the method of enzyme degradation used. The use of control samples in each series is thus necessary to permit comparison between laboratories. In 1984 de Boever et al. compared 18 methods of enzymatic degradability on 31 concentrates with known digestibilities. The accuracy of estimation varied from 1.7 to 2.7 according to the method used: the best predictions were obtained with pepsin-cellulase methods (RSD = 1.7-2.0) (Table V ). The best method (RSD = 1.7 ) is an adaptation of the one proposed in this paper (pepsin hydrolysis in acid conditions and attack by cellulase) but differs by the duration of acid hydrolysis (45 min compared with 30 min), and the relative proportions of cellulase and substrate (30% compared with 10% expressed as a percentage). This study indicates that enzymatic degradability provides an estimation of the digestibility of energy feeds used in the manufacture of concentrates with greater precision than those obtained by other chemical or biological methods. Systematic comparisons of several methods for estimating feed digestibility have also shown this superiority of enzymatic degradability over in vitro digestibility (van der Meet, 1982; Mathiesen and Moller, 1983; de Boever et al., 1984). Nevertheless, it is difficult to compare these three groups of methods for predicting digestibility - chemical, biological and enzymatic - solely according to their residual standard deviation. Whereas for the first two groups (chemical and biological ) the comparison uses the same dosage methods (van Soest, 1967 and Tilley and Terry, 1963), there are infinite variations for enzymatic methods, variations which give differing but generally small residual standard deviations (1.7-2.7) ( de Boever et al., 1984). REFERENCES Abe, A., Horii, S. and Kameoka, K.I., 1974. Development and application of cellulase hydrolysis for predicting digestibility of roughage. 1 - Digestion of cell wall carbohydrates by cellulase. Jpn. J. Zootechn. Sci., 43: 141-145. AFNOR, 1981. Cellulose brute, M~thode CCE 4~me Directive Norme NF V 03-040 in Aliments des animaux. M~thodes d'analyse. Ed AFNOR. Aufr~re, J., 1982. Etude de la pr~vision de la digestibilit~ des fourrages par une m~thode enzymatique. Ann. Zootech., 31: 111-130.
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