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Investigation of dry matter degradation and acidotic effect of some feedstuffs by means of in sacco and in vitro incubations
ANIMAL FEED XIENCE AND TECHNOLOGY
ELSEVlER
Animal Feed Science and Technology 5 1 ( 1995) 297-3 15
Investigation of dry matter degradation and acidotic effect of some feedstuffs by means of in sacco and in vitro incubations’ A.M. de Smet*, J.L. de Boever, D.L. de Brabander, J.M. Vanacker, Ch. V. BoucquC National Institutefor Animal Nutrition, Agricultural Research Centre-Ghent, Scheldeweg 68, 9090 Melle-Gontrode, Belgium Received 25 August 1993; accepted 17 May 1994
Abstract The dry matter degradation in the rumen and pH decrease of nine primary feedstuffs, three compound feeds and three combinations of each two primary feedstuffs, were evaluated by means of in sacco and in vitro incubations. The ingredients included barley, wheat, maniac, maize, sorghum, sugar beet pulp, soya-bean meal, soya-bean hulls and maize gluten feed. The compound feeds differed in content and composition of the starch and sugars fraction, as well as in tibre content. The three combinations each consisted of a rapidly and slowly degradable feedstuff. The feedstuffs were incubated for different times to examine optimal incubation conditions for both estimating methods. For the in sacco technique, best predictions were obtained after 3 h of incubation. In this way, ingredients were ranked in order of decreasing dry matter degradation: maniac, wheat, barley, maize gluten feed, beet pulp, soya-bean meal, maize, sorghum, soya-bean hulls. With the in vitro method, better results were observed when rumen fluid was taken after rather than before feeding. After 5 h of incubation, ingredients were ranked in order of declining pH decrease: maniac, wheat, beet pulp, maize gluten feed, barley, maize, soyabean meal, sorghum, soya-bean hulls. Except for beet pulp, in vitro ranking agreed fairly well with in sacco results. The validity of these techniques was examined by limited results of in vivo experiments, where the effect of the nature of the compound feed on rumen fermentation was investigated. In vivo, a clear difference in risk for rumen disturbances could be observed between the three compound feeds. In sacco and in vitro, however, the compound feed with the * Corresponding author. ’Communication no. 866 of the National Institute for Animal Nutrition. 0377-8401/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SsDIO377-8401(94)00680-8
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highcontent of slowly degradable starch, could hardly be distinguished from the crude fibre rich compound feed. As only a limited amount of in vivo material was available,the estimating value of these methods require confirmation. Keyworuk Acidotic effect; Dry matter degradation
1.Introduction
To meet their production potential, cows have to consume the required amounts of energy, proteins, minerals and vitamins. With regard to ration optimisation, the supply of sufficient physical structure should also be considered. As production levels have increased, concentrate need has increased substantially. However, the intake of large amounts of concentrates can disturb rumen fermentation. In circumstances of suboptimal roughage supply, the type of concentrate (chemical composition and degradation rate) is a determining factor in the occurrence of rumen disturbances. Large amounts of concentrates with a high content of easily fermentable substances, generally, rapidly depress pH of rumen fluid and alter volatile fatty acids (VFA) pattern in the rumen. Fibre rich concentrates, in contrast, are characterised by a slower degradation and a reduced postprandial pH drop (Malestein et al., 198 1; Varga and Hoover, 1983; Van Beukelen et al., 1983; Sutton, 1986; Robinson et al., 1986). The acidotic effect of feedstuffs is an important characteristic and must be taken into account when rations are composed. Measuring the acidotic effect in vivo, however, is an expensive and time-consuming method. To evaluate the influence of feedstuffs on rumen fermentation of hundreds of primary feedstuffs or compound feeds, laboratory methods are needed to predict this effect. As starch or tibre rich feedstuffs encompass a heterogeneous group of components, the risk for rumen disturbance cannot solely be predicted from their chemical composition (Nocek, 1988; Giger et al., 1988; Graham and Aman, 199 1). Beside chemical composition, feedstuffs are also characterised by the rate and extent of digestion. These characteristics are often evaluated by means of in vitro and in sacco incubations (Varga and Hoover, 1983; Sauvant et al., 1985; Cullen et al., 1986; Nocek, 1988; van der Koelen et al., 1992). Comparison of the various results, however, is complicated by numerous factors, e.g. differences in experimental feedstuffs, incubation techniques (sample size, particle size, bag porosity, washing procedure, etc.), animals, and feeding pattern of the animals. The objective of the present study was to examine optimal conditions for in sacco and in vitro incubations as tools to predict the acidotic effect in the rumen of primary feedstuffs and compound feeds.
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2. Materials and methods 2. I. Feeds&& The rumen dry matter (DM) degradability of nine primary feedstuffs, three compound feeds and three combinations each of two primary feedstuffs, was determined by means of in sacco incubations. The pH decrease of rumen fluid caused by these feedstuffs was evaluated by means of in vitro experiments. The ingredients were: sugar beet pulp, soya-bean hulls, maize, sorghum, barley, wheat, maniac, maize gluten feed and soya-bean meal. The compound feeds were composed to differ in content and composition of the carbohydrate fraction. A first compound feed, with a low content of starch and sugars, was mainly composed of beet pulp and soya-bean hulls (BpSh). The other two compound feeds had high starch and sugars contents, but differed in degradation rate: MaiSo was based on maize and sorghum as slowly degradable ingredients; BaWhMan, based on barley, wheat and maniac, was rapidly degradable. All compound feeds contained maize gluten feed and soya-bean meal as protein sources and were pelleted. The composition of the compound feeds is presented in Table 1. Each of the three combinations of feedstuffs consisted of both a rapidly and a slowly degradable component, in a SO/SO ratio (fresh matter basis): maniac/ beet pulp, barley/soya-bean hulls, wheat/maize. These combinations were not pelleted. Table 1 Composition
of the compound feeds (g kg-‘) BpSh
Barley Wheat Maniac Maize Sorghum Beet pulp Soya-bean hulls Soya-bean meal Maize gluten feed Beet molasses Tallow Trace elements MgO Dicalcium phosphate Limestone NaCl Vit. A+D,+E Lignosulphonate
MaiSo
BaWhMan 200 170 140
230 230 351 120 205 140 70 30 18 4 24 5 4 3 2
185 207 70 _ 18 4 20 9 4 3 2
207 120 70 I5 18 4 22 7 4 3 2
BpSh, compound feed based on beet pulp and soya-bean hulls; MaiSo, compound feed based on maize and sorghum; BaWhMan, compound feed based on barley, wheat and maniac.
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Table 2 Chemical composition of the experimental feedstuffs Dry matter (g kg-r)
Barley Wheat Maniac Maize Sorghum Beet pulp Soya-bean hulls Soya-bean meal Maize gluten feed BpSh MaiSo BaWhMan
853 885 888 847 859 894 870 87 1 889 876 868 876
Chemical composition
(g kg-’ DM)
Crude protein
Crude Crude Ash NDF tibre fat
ADF
Lignin Cellu- Starch Sugars lose
121 136 28 107 115 107 135 517 227 198 194 199
64 19 50 18 15 194 392 62 74 137 41 45
97 29 107 25 49 294 505 103 118 181 61 70
20 9 27 8 19 48 36 14 18 26 14 15
18 17 4 42 40 4 22 16 39 34 15 28
28 19 60 14 13 65 48 68 70 84 76 82
166 97 111 97 78 360 603 123 281 258 107 126
75 24 49 21 34 214 471 89 88 156 48 51
521 675 716 710 737 4 67 55 218 85 423 377
22 28 17 12 7 103 14 104 33 83 64 72
BpSh, compound feed based on beet pulp and soya-bean hulls; MaiSo, compound feed based on maize and sorghum; BaWhMan, compound feed based on barley, wheat and maniac.
The chemical composition of all samples, except the three combinations, was determined according to standard methods (Table 2). Dry matter was determined by drying a sample in a ventilated oven at 60-70°C followed by ovendrying at 103-105 “C during 3 h. Crude protein was determined with an automatic Kjelfoss. The extraction of crude fat was carried out in a Soxlet apparatus with petroleum ether for 6 h. Ash content was determined after calcination in a furnace at 600°C. Crude tibre was analysed by Fibertec. Neutral detergent tibre (NDF) was determined according to Van Soest and Wine ( 1967). For acid detergent tibre (ADF) and lignin, the methods of Van Soest ( 1963) were applied. NDF and ADF were expressed on ash-free basis. Celhtlose was calculated as the difference between ADF and l&in. For all feedstuffs, except beet pulp and the compound feed with a large amount of beet pulp (BpSh), starch was polarimetrically determined by the method of Ewers (Commission of the European Communities, 1972). For beetpulp and BpSh, starch was enzymatically determined (Van Gelder et al., 1992). Sugars were determined according to the method of Luff-Schoorl (Anonymous, 197 1) . 2.2. In sacco incubation Two lactating Holstein cows, fitted with a rumen cannula, were used to determine the in sacco DM degradation of the feedstuffs. The cows were fed ad libitum a diet consisting of 55% maize silage and 45% compound feed (on DM basis). The diets were offered in two equal meals at 08:30 and 20:30 h. The nylon bags, measuring 8 cm x 8 cm and having a pore size of 50 pm, contained 2 g of fresh material, which was ground to pass a 2 mm sieve. Prior to
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ruminal incubation, all bags were soaked in a buffer solution according to Tilley and Terry ( 1963 ) for 3 min. Sets of bags were incubated into the rumen for 3,6, 12, 24 and 48 h. After each of the respective incubation periods, the bags were washed for 10 min and rinsed for 10 min in a washing machine, dried at 105 ‘C for 3 h and weighed. To determine the immediately disappearing fraction, bags were only soaked in the buffer solution, rinsed and washed. All samples were incubated in two cows and on two different days, so for each feedstuff, four degradation values were obtained. 2.3. In vitro incubation Fresh samples ( 1 g; ground to pass a 2 mm sieve) were incubated in plastic tubes with 50 ml of a rumen fluid/buffer solution (SO/50 v/v). The buffer solution was composed according to the method of Tilley and Terry ( 1963). The rumen fluid originated from the two previously mentioned fistulated cows and was mixed in advance. The solution was thoroughly saturated with C02. After adding the sample and the rumen fluid/buffer solution to the tube, the space above the liquid in each tube was flushed out with CO1 and the tube was then sealed with a rubber cork filled with a gas release valve. Tubes were then incubated at 39’C in the dark, being shaken hourly. To evaluate the influence of the time of rumen sampling, rumen fluid was taken once just before morning feeding and once 3 h after feeding. The pH of the diluted fluid was measured before adding the samples and 1,2, 3, 4 and 5 h after incubation with one of the 15 feedstuffs. To correct for the influence of the feed particles present, the pH course of the rumen fluid/buffer mixture as such was also measured. After 1,3 and 5 h of incubation, toluene was added to the test-tube to stop the fermentation. The solution was frozen for a later determination of the volatile fatty acids. All determinations were carried out in duplicate.
3. Results and discussion 3.1. In sacco incubation
The variation in DM degradability, owing to differences in incubation period within the same cow, was larger than the variation between cows. As the duration of incubation increased, variation coefficients diminished. For 3, 6, 12, 24 and 48 h of incubation, the variation coefficient between periods ( VP) amounted to respectively 7.396, 8.2%, 4.5%, 2.6% and 0.636, the between-cows variation (V,) amounted to respectively 3.8%, 2.5%, 4.2%, 1.5% and 0.7%. A decrease in VPand V, with prolonged incubation time was also reported by Rodriguez ( 1968) and Van der Koelen et al. ( 1992). The rather small V, and VP values after 48 h of incubation indicate that at this incubation time, disappearance reflects plant
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characteristics and is hardly influenced by other factors (Van der Koelen et al., 1992). The degradation of the nine primary feedstuffs (as a mean of four measurements) is represented in Fig. 1. The percentage of DM, which disappeared out of the nylon bag during the short period of immersion in the buffer solution and the washing procedure, considerably differed between feedstuffs, varying from 12% for soya-bean hulls to 68% for maniac. The early disappearance is due to solubilisation and losses of very small feed particles through the bag pores (Orskov et al., 1980). In our study, the immediately disappearing fraction is considered as completely degraded. This is not totally correct as a small part of the particles, which escaped through the bag pores, pass to the omasum before being degraded. As this small fraction leaves the rumen immediately, it does not contribute to the acidification of the rumen content. However, the inaccuracy caused by not correcting for this fraction is very small and may be ignored. In most studies, the feed particles escaping through 1w
80
00
E 6 B
I
% %
40
20
0
3 0
i 10
20
M
40
50 time(h)
Fig. 1. In sacmdry matter degradation of nine ingredients.
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303
the bag pores are assumed to be totally digestible (Orskov et al., 1980; Lindberg and Varvikko, 1982; Sauvant et al., 1985; Van der Koelen et al., 1992). During the first 9-l 2 h, all ingredients are degraded at a higher rate than during
t
--t -*
Maso
Bpsh
--
SaWhMan
Fig. 2. In sacco dry matter degradation of three compound feeds. Table 3 Measured and calculated DM degradation of the three compound feeds after 3 h in sacco incubation
BpSh MaiSo BaWhMan
Measured degradation” @1
Calculated degradation (%l
49.4f 5.7 48.4f4.3 66.6 f 3.3
48.6 43.3 63.7
BpSh, compound feed based on beet pulp and soya-bean hulls; MaiSo, compound feed based on maize and sorghum; BaWhMan, compound feed based on barley, wheat and maniac.
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A.M. de Smet et al. /Animal Feed Science and Technology 51(1995) 297-315 100 T
I
50 time(h)
60
z fs so E %
40
B
0
10
20
30
40
50
time(h)
Fig. 3. In sacco dry matter degradation of three combinations of feedstuffs.
further incubation times. However, the degradation pattern varied considerably according to the feedstuff. The primary feedstuffs could roughly be divided into two categories: the first being rich in cytoplasmatic content and therefore being rapidly degradable, the second possessing a high content of slowly degradable cell wall material. Within these groups, however, there is also a range in degradation rate. Barley, wheat and maniac are more easily degradable than maize and sorghum, although they have all a high content of starch and sugars (see Table 2). As starch comprises 50-75% of these feedstuffs, the DM degradability is mainly determined by this fraction. The lower degradability of maize and sorghum is espe-
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305
cially due to the difference in granular structure of the starch. The starch granules of maize and sorghum are almost completely embedded in a protein matrix and this starch-protein interaction reduces the susceptibility of the starch to enzyme hydrolysis. Although the starch granules of maize and sorghum are very similar in size, shape and composition, sorghum is less degradable than maize. The major difference relates to the higher proportion of peripheral endosperm in sorghum, which is extremely dense, hard and resistant to digestion (Rooney and Pflugfelder, 1986). According to several authors (Johnson, 1976; Tamminga et al., 1978; Michalet-Doreau and Sauvant, 1989; Weiss et al., 1989) primary feedstuffs, rich in cell walls, have a rather low degradation rate. In our study, this was the case for soya-bean hulls, whereas beet pulp showed an even higher degradability than maize and sorghum. This can be explained by the high pectin content of the crude libre fraction. Although pectin belongs to the structural carbohydrates, it is a highly degradable component (Marounek et al., 1985 ) . The two protein rich feedstuffs, maize gluten feed and soya-bean meal, showed an intermediate degradation rate. Considering the compound feeds, the degradation corresponded to the characteristics of the incorporated ingredients. BaWhMan was degraded very easily, while MaiSo and BpSh showed a rather similar, but lower degradation rate (Fig. 2). The degradation values, calculated with the results of the single ingredients, were somewhat lower, especially for the starch rich compound feeds (Table 3 ) . This is probably the result of pelleting the compound feeds, since this effect is not taken into account for the calculations. The positive effect of processing feedstuffs on the degradability of starch is mentioned by several authors (Hale, 1973; Rooney and Pflugfelder, 1986; Malestein et al., 1988; Cone, 1991). The incubations with the feedstuff combinations indicated the additivity of the individual degradation values of the ingredients when predicting the degradability of a compound feed. As illustrated in Fig. 3, the measured degradation values were fairly well in accordance with the mathematical means of the degradations of the components in the mixture. 3.2. In vitro incubation As rumen environment evolves with time after feeding, the moment of rumen sampling can affect the results of in vitro measurements. In our study, the pH of the rumen fluid/buffer solution before incubation amounted to 6.59 and 6.23 when fluid was taken respectively before and 3 h after feeding. Because of the stronger microbial activity, lower pH values were obtained when fluid was taken after feeding. While the rumen fluid, taken before feeding, showed a constant pH during incubation without sample, the pH of the fluid taken after feeding decreased, due to fermentation of feed particles present in the rumen fluid. Hence, corrections were carried out on the in vitro results with fluid taken after feeding. The variation in pH decrease between periods ( IX’,) was more apparent for incubations with fluid taken after than before feeding. Probably, fluctuations in
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297-315
rumen fluid composition will be larger when fluid is taken after feeding, which results in a higher variation in pH decrease. For 1,2, 3,4 and 5 h of incubation with fluid taken before feeding, VC, amounted to, respectively, 0.3%, 0.6%, 0.696, 0.7% and 3.5%. When fluid was taken after feeding, VC, values were, respectively, 0.6%, 0.6%, 1.096, 1.2% and 1.0%. Fig. 4 represents the pH decrease of the different primary feedstuffs, when incubations were carried out with fluid taken after feeding. Maniac, wheat, beet pulp, maize gluten feed and barley showed the strongest pH decrease. For soyabean meal and maize intermediate values were measured, while the smallest changes were observed with sorghum and soya-bean hulls. The very fast pH decrease with beet pulp and maize gluten feed was rather surprising. A similar pH pattern was observed for MaiSo and BpSh. BaWhMan showed a faster and deeper pH decrease (Fig. 5 ) . pH decreases of the combined feeds differed from what could be expected from the pH decreases of the composing feedstuffs (Fig. 6). Comparable experiments of Malestein et al. ( 1982) showed that,
a.4
a
T
J-
5.8 --
5.8
--
5.4
--
5.2
--
5
--
4.8
--
lp
Fig. 4. In vitro pH decrease of nine ingredients.
A.M. deSmet et al. /Animal FeedScience and Technology 51(1995) 297-315
48
,~~~_~~+~~~
0
307
_--,-_-_
1
2
3
4
5 time (II)
Fig. 5. In vitro pH decrease of three compound feeds.
when feedstuffs were combined and incubated in rumen fluid, the final pH usually was lower, than when the feedstuffs were incubated separately. A possible explanation is that, when the substrate has a more complex composition, more species of microorganisms can each ferment part of it and as a consequence more VFA will be formed, resulting in a lower pH (Malestein et al., 1982). Moreover, a synergism between different species can occur, when fermentation products of one species are used as substrate by another one. In our study however, no change in VFA composition was observed, when incubations were carried out with fluid taken after feeding. This is probably due to the high amount of VFA already present in the rumen fluid, as a result of the fermentation of the ration fed. Compared with this fraction, the amount of VFA coming from the feed samples was too small to be detected. With fluid taken before feeding, changes in VFA composition caused by the degradation of the feed samples were actually noted. The largest changes occurred with maniac, barley and wheat, and, rather surprisingly, also
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5.6
IO
54
4 0
1
2
3
4
5 time(h)
Fig. 6. In vitro pH decrease of three combinations of feedstuffs.
with soya-bean meal, Within the three compound feeds, changes in VFA composition were similar, but a difference in VFA level was observed (Table 4). As expected from the chemical composition of the compound feeds, the lowest acetic acid and the highest propionic acid level is observed with the compound feeds rich in starch and sugars. Although maize and sorghum did not induce large VFA changes when incubated separately, Ma230 showed considerable changes in VFA
A.M. deSmet et al. /Animal FeedScience and Technology 51(1995) 297-31.5 Table 4 Evolution of pH, VFA pattern and total acids concentration feeds in rumen fluid taken before feeding
Blanc0
BpSh
MaiSo
BaWhMan
309
during incubation of the three compound
Duration of incubation (h)
pH
AC. ac. (mol. Oh)
Pr. ac. (mol. W)
But. ac. (mol. %)
Total acids (meq per 100 ml)
1 3 5 1 3 5 1 3 5 1 3 5
6.61 6.62 6.61 6.35 6.12 5.76 6.43 6.21 5.93 6.35 6.07 5.74
68.5 68.1 68.0 68.0 65.3 63.5 66.7 64.2 60.8 65.2 61.4 61.0
14.8 14.7 14.4 17.7 20.9 21.3 18.0 21.0 23.1 19.6 24.2 23.1
12.1 12.3 12.6 10.9 11.1 12.7 11.8 11.9 13.3 11.5 11.5 12.6
4.4 4.3 3.3 5.9 7.0 9.0 6.1 5.7 8.4 6.2 8.2 9.0
BpSh, compound feed based on beet pulp and soya-bean hulls; MaiSo, compound feed based on maize and sorghum; BaWhMan, compound feed based on barley, wheat and maniac. AC. ac., acetic acid; Pr. ac., propionic acid; But. ac., butyric acid.
pattern. This can again be ascribed to the more complex composition of the substrate. 3.3. Predictive value of the in sacco and in vitro method To evaluate these estimating methods, in vivo experiments, concerning the influence of the composition of compound feeds on the physical structure requirements of dairy cattle, were taken as a reference. For an optimal rumen function, ruminants need a minimum amount of structural components in the ration. The structural value of a ration not only depends on the kind of roughage, but also on the kind of compound feed included in the ration. A suboptimal structure supply can induce problems with feed intake, a decrease in milk fat content or milk production, acidosis, etc. As part of our structure evaluation research, in vivo trials were carried out to determine the minimum amount of roughage needed for normal rumen function. Eight lactating Holstein cows received a diet consisting of maize silage and one of the three compound feeds (BpSh, MaiSo and BaWhMan) in a decreasing roughage/compound feed (R/C) ratio. After a reference period of 2 weeks on an R/C ratio of 60/40, R/C decreased weekly, first in steps of 10% and from the ratio 40/60 on in steps of 5%. The lowest roughage part which cows can tolerate before showing symptoms of lack of physical structure, was called the critical roughage part (R,,,). This index, which also depends on the compound feed, indirectly provides an indication of the fermentation rate and acidotic effect of the compound feeds. According to this index, the three compound feeds were ranked as follows when they were given in combination
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with maize silage: BpSh, MaiSo and BaWhMan with Rcrit of, respectively, 31%, 33% and 38%. In a second experiment when the three compound feeds were given with prewilted grass silage, the Rcti, values obtained were, respectively, 22%, 26% and 33%. For these experiments, however, very large standard deviations due to differences between animals ( 2 6 percentage units) have to be mentioned. While in these experiments a difference existed between the Rcrit of each of the three compound feeds, this pattern did not agree with the in sacco results, where hardly any difference between BpSh and MaiSo was observed (see Fig. 2). In comparable earlier in sacco experiments with the same three compound feeds, the compound feeds were ranked in the same order as in the in vivo experiments, when incubated for 3 h. BaWhMan was degraded most rapidly (64.8%)) followed by MaiSo (50.0%) and BpSh (47.9%). None of the other incubation times gave the same ranking order as the in vivo results. Hence, on the basis of these results, the disturbing effect of feedstuffs on rumen fermentation seemed to be best predicted by in sacco incubation for 3 h. Three hours seems an acceptable duration of incubation as the acidotic effect can mainly be expected in the first hours after feed intake, when degradation of feedstuffs is most intensive. Furthermore, differences between feedstuffs diminish with larger incubation times. Although, based on the earlier results, feedstuffs were best evaluated after 3 h of in sacco incubation, in vivo and in sacco results did not agree completely. In vivo behaviour of MaiSo and BpSh was different, while in sacco degradation patterns differed little. The earlier in sacco results, however, agree fairly well with the degradation percentages obtained in the experiments described here. In these experiments, the difference in DM degradation between MaiSo (48.4%) and BpSh (49.4%) was almost negligible. Because of the large similarity between earlier and last degradation patterns and because a rather short incubation time allows a better evaluation of the differences between feedstuffs, 3 h is assumed as being the most suitable in sacco incubation time. According to this method, feedstuffs can be ranked in order of decreasing degradability. In our experiments, primary feedstuffs were classified as follows (DM degradation percentages are given in parentheses) : maniac ( 75.3 ) , wheat (73.9), barley (55.5), maize gluten feed (52.0), beet pulp (39.6), soyabeanmeal (34.0),maize (29.1),sorghum (20.5),soya-beanhulls (19.7). When the in vitro results of the compound feeds are compared with the in vivo data, a discrepancy similar to that of the in sacco incubations was observed. The in vitro method is a well reproducible, fast and cheap incubation technique. To evaluate these methods, correlations were calculated between the DM degradation after 3 h of in sacco incubation and in vitro pH decreases. If a good correlation exists between the two techniques, the in vitro method can replace the in sacco incubations as the estimating method. Better correlations were obtained when in vitro incubations were carried out with fluid taken after rather than before feeding. This can be ascribed to the greater microbial activity of the fluid, when sampled on a moment of active fermentation. The highest correlation coefficient (r= 0.9 1) was noted for 5 h of in vitro incubation with fluid taken after feeding (Table 5, Fig. 7 ) . Therefore, these in vitro incubation conditions seem to
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Table 5 Evolution of pH, VFA pattern and total acids concentration feedstuffs in rumen fluid taken before feeding
Barley
pH
AC. ac. (mol. %)
Pr. ac. (mol. %)
But. ac. (mol. %)
Total acids (meq per 100 ml)
1
6.37 6.21 5.92 6.37 6.24 5.87 6.32 5.91 5.36 6.41 6.28 6.13 6.48 6.35 6.22 6.18 5.90 5.64 6.39 6.29 6.20 6.43 6.30 6.15 6.18 6.07 5.83
64.0 61.2 58.8 63.9 61.2 59.0 65.4 61.7 59.8 65.2 63.7 63.1 66.6 64.4 64.2 69.6 67.8 64.2 68.6 67.1 66.1 61.9 65.5 59.0 63.8 60.0 62.0
18.7 20.9 22.7 18.3 20.9 23.9 18.4 22.6 24.6 16.5 16.9 17.5 15.7 15.4 16.3 15.7 18.6 20.4 15.9 18.1 17.7 17.4 19.3 23.2 18.6 23.1 21.5
13.2 14.2 15.1 13.6 13.9 13.6 12.9 13.3 13.5 14.3 15.6 15.8 13.4 16.2 15.7 11.8 11.2 12.9 11.7 11.4 12.6 11.1 11.6 13.9 13.6 13.2 13.4
5.1 6.6 9.4 5.0 7.3 7.7 5.0 7.5 10.1 5.3 5.7 5.5 4.5 5.5 5.3 5.6 7.5 8.7 5.3 6.4 7.1 5.8 6.6 9.0 5.4 7.0 9.4
1 3 5
Maniac
1 3 5
Maize
1 3 5
Sorghum
1
Beet pulp
3 5 1 3 5
Soya-bean hulls
Soya-bean meal
Maize @uteri feed
of the nine primary
Duration of incubation (h)
3 5 Wheat
during incubation
311
1 3 5 1 3 5 1 3 5
AC. ac., acetic acid; Pr. ac., propionic acid; But. ac., butyric acid.
be most appropriate to predict feedstuff degradation. However, when the VFA, formed during fermentation were considered, more information was obtained from incubations with fluid taken before feeding (see section 3.2. ). If a close simulation of in vivo degradation is aimed at, rumen fluid has to be sampled after feeding. So, the time of rumen sampling depends on the kind of information desired. For the in vitro incubation with fluid taken after feeding, the primary feedstuffs were ranked in order of declining pH decrease as follows (relative pH decrease is given in parentheses): maniac (14.9), wheat (12.5), beet pulp (11.4), maize gluten feed (10.6), barley (10.4), maize (8.0), soya-bean.meal (6.6), sorghum (4.5 ), soya-bean hulls (4.2 ). A weakness of the validation procedure is the restricted reference material. Thus, to give a final conclusion on the predictive value of the in sacco and in vitro methods would be hasty. One can ask if any method can predict accurately the
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Fig. 7. Correlation between in vitro pH (5 h incubation) tion) of the nine primary feedstuffs.
and in sacco DM degradation
Table 6 Correlation coefficients between degradation after 3 h in sacco incubation during 1,2,3,4 and 5 h incubation in fluid taken before and after feeding Duration of incubation (h)
Fluid taken before feeding
Fluid taken after feeding
1 2 3 4 5
0.37 0.47 0.53 0.70 0.77
0.49 0.62 0.65 0.87 0.91
(3 h incuba-
and in vitro pH decrease
acidotic effect of feedstuffs on rumen fermentation, as this is a very complex system which is not only influenced by the feedstuff itself, but also by the animal and environmental circumstances. Some disagreement between in vivo and in sacco results is also reported by Michalet-Doreau and Sauvant ( 1989). In in vivo experiments with three compound feeds based on beet pulp, maize or barley, the percentage of organic matter actually degraded in the rumen was the same for beet pulp and barley and exceeded that for maize, while in sacco, the degradation rate of beet pulp and maize were rather low and that of barley was higher. Therefore, in sacco behaviour of beet pulp did not accord with in vivo degradation. In our experiments, beet pulp neither showed a similar degradation pattern during in sacco and in vitro incubations. This feedstuff caused a very fast in vitro pH decrease, while in sacco an intermediate DM degradation was observed. This discrepancy can be related to observations of De Visser et al. ( 199 1). They reported a very small soluble fraction for dried beet pulp, which was ascribed to the swelling of the ground dry material during the washing procedure. As a consequence, particle size increased and the number of very small particles which could be rinsed out of the nylon
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bags was reduced. In this way, the in sacco procedure does not seem to predict DM degradation of beet pulp accurately, while for other ingredients reliable information is obtained. 4. Conclusion In sacco DM degradation and in vitro pH decrease were investigated, in order to rank feedstuffs following their disturbing influence on rumen fermentation. In our study, these methods showed some differences with in vivo experiments where the influence of different compound feeds on rumen function is investigated. A possible reason for this discrepancy may be that some parameters, which also play a role in the rumen degradation processes, are not taken into account (e.g. particle size, chewing activity, rate of passage, absorption of VFA, rumen motility). It would be hasty to draw conclusions about the accuracy of these methods to predict the risk for rumen disturbances, because the reference material is limited. In vitro and in sacco incubations, however, can give interesting information about the degradation pattern of feedstuffs. It thus becomes clear that starch and fibre rich feedstuffs cannot be divided into two well-defined groups. Within each group, there is a large range in degradation rate, as illustrated by the difference between maniac, barley and wheat on the one hand, and maize and sorghum on the other hand, or between beet pulp and soya-bean meal. Fibre rich feedstuffs can even show a higher degradation rate than feedstuffs with a high content of starch and sugars. Although some information is obtained from these estimating methods, no judgement can be made about the acidotic effect of feedstuffs on rumen fermentation. For better evaluation of these estimating techniques, more in vivo observations should be carried out. In further investigations, this will form an area of special attention. Acknowledgements
The authors wish to thank all the laboratory personnel of the institute for their valuable technical assistance. The research was financially supported by the Institute for the Encouragement of Scientific Research in Industry and Agriculture, Brussels. References Anonymous, 1971. Determination of sugar. Community methods of analysis for the official control of feedingstuffs. Off. J. Eur. Commun. L: 13. Commission of the European Communities, 1972. Determination of starch. Oftic. J. No. L123: 6, Brussels.
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Cone, J.W., 1991, Degradation of starch in feed concentrates by enzymes, rumen fluid and rumen enzymes. J. Sci. Food Agric., 54: 23-34. Cullen, A.J., Harmon, D.L. and Nagaraja, T.G., 1986. In vitro fermentation of sugars, grains and byproduct feeds in relation to initiation of ruminal lactate production. J. Dairy Sci., 69: 26 16-262 1. De Visser, H., Huisert, H. and Ketelaar, R.S., 199 1. Dried beet pulp, pressed beet pulp and maize silage as substitutes for concentrates in dairy cow rations. 2. Feed intake, fermentation pattern and ruminal degradation characteristics. Neth. J. Agric. Sci., 39: 21-30. Giger, S., Sauvant, D., Durand, M. and Hervieu, J., 1988. Influence de la nature de l’aliment concentre sur quelques paramttres de la digestion dans le rumen. Reprod. Nutr. Develop., 28: 117118. Graham, H. and Aman, P., 199 1. Nutritional aspects of dietary tibres. Anim. Feed Sci. Technol., 32: 143-158. Hale, W.H., 1973. Influence of processing on the utilization of grains (starch) by ruminants. J. Anim. Sci., 37: 1075-1083. Johnson, R.R., 1976. Influence of carbohydrate solubility on non-protein nitrogen utilization in the ruminant. J. Anim. Sci., 43: 184-191. Lindberg, J.E. and Varvikko, T., 1982. The effect of bag pore size on the ruminal degradation of dry matter, nitrogenous compounds and cell walls in nylon bags. Swed. J. Agric. Res., 12: 163- 17 1. Malestein, A., van ‘t Klooster, A.Th., Counotte, G.H.M. and Prins, R.A., 1981. Concentrate feeding and ruminal fermentation. 1. Influence of the frequency of feeding concentrates on rumen acid composition, feed intake and milk production. Neth. J. Agric. Sci., 29: 239-248. Malestein, A., van ‘t Klooster, A.Th., Counotte, G.H.M. and Prins, R.A., 1982. Concentrate feeding and ruminal fermentation. 2. Influence of concentrate ingredients on pH and L-lactate concentration in incubations in vitro with rumen fluid. Neth. J. Agric. Sci., 30: 259-273. Malestein, A., van ‘t Klooster, A.Th. and Cone, J.W., 1988. Degradability of various types of starch by incubation with rumen fluid or with bacterial a-amylase. J. Anim. Physiol. Anim. Nutr., 59: 225-232. Marounek, M., Bartos, S. and Brezina, P., 1985. Factors influencing the production of volatile fatty acids from hemicellulose, pectin and starch by mixed culture of rumen microorganisms. Z. Tierphysiol., Tieremahr. Futtermittelkd., 53: 50-58. Michalet-Doreau, B. and Sauvant, D., 1989. Infuence de la nature du concentre, &males ou pulpe de betterave sur la digestion chez les ruminants. INRA Prod. Anim., 2: 235-244. Nocek, J.E., 1988. In situ and other methods to estimate ruminal protein and energy digestibility: a review. J. Dairy Sci., 7 1: 205 l-2069. Orskov, E.R., Hovell, F.D.D. and Mould, F., 1980. The use of the nylon bag technique for the evaluation of feedstuffs. Trop. Anim. Prod., 5: 198-2 13. Robinson, P.H., Tamminga, S. and van Vuuren, A.M., 1986. Influence of declining level of feed intake and varying the proportion of starch in the concentrate on rumen fermentation in dairy cows. Livest. Prod. Sci., 15: 173-l 89. Rodriguez, H., 1968. The in vivo bag technique indigestibility studies. Rev. Cubana Cien Agric., 2: 77-81. Rooney, L.W. and Pflugfelder, R.L., 1986. Factors affecting starch digestibility with special emphasis on sorghum and corn. J. Anim. Sci., 63: 1607-1623. Sauvant, D., Bertrand, D. and Giger, S., 1985. Variations and prevision of the in sacco dry matter digestion of concentrates and by-products. Anim. Feed Sci. Technol., 13: 7-23. Sutton, J.D., 1986. Rumen fermentation and gastro-intestinal absorption: carbohydrates. Proc. Seminar EEC, 1986/06/25-27, Foulum (Denmark), pp. 21-38. Tamminga, S., van Vuuren, A.M. and van der Koelen, C.J., 1978. De betekenis van de pensfermentatie bij herkauwers. Landbouwkundig Tijdschrift, 90: 197-203. Tilley, J.M.A. and Terry, R.A., 1963. A two-stage technique for the in vitro digestion of forage crops. J. Br. Grassl. Sot., 18: 104-l 11. Van Beukelen, P., Wensing, Th. and Breukink, H.J., 1983. Postprandial changes in ruminal fluid and some blood metabolites during induction and recovery of milk fat depression in high yielding dairy cows. Z. Tierphysiol., Tieren%. Futtermittelkd., 49: 137-l 52.
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Van der Koelen, C.J., Goedhart, P.W., van Vuuren, A.M. and Savoini, G., 1992. Sources of variation of the in situ nylon bag technique. Anim. Feed Sci. Technol., 38: 35-42. Van Gelder, A.H., Te Brinke, E.M., Cone, J.W., Van Lonkhuysen, H.J., Jelten, J.M. and Lichtendonk, W.J. (Editors), 1992. Protocol voor de analyse van niet-zetmeel koolhydraten (nsp). Enzymatische bepaling van zetmeel amyloglucosidase/hexokinase methode met behulp van een autoanalyser (of spectrofotometer). Produktschap voor veevoeder, ‘s-Gravenhage, The Netherlands, pp. 32-35. Van Soest, P.J., 1963. Use of detergents in the analysis of fibrous feeds. II. A rapid method for the determination of fiber and lignin. J. Assoc. Off. Anal. Chem., 45: 829. 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. Anal. Chem., 50:50. Varga, G.A. and Hoover, W.H., 1983. Rate and extent of neutral detergent fibre degradation of feedstuffs in situ. J. Dairy Sci., 66: 2109-2 115. Weiss, W.P., Fisher, G.R. and Erickson, G.M., 1989. Effect of source of neutral detergent fiber and starch on nutrient utilization by dairy cows. J. Dairy Sci., 72:2308-2315.
ELSEVlER
Animal Feed Science and Technology 5 1 ( 1995) 297-3 15
Investigation of dry matter degradation and acidotic effect of some feedstuffs by means of in sacco and in vitro incubations’ A.M. de Smet*, J.L. de Boever, D.L. de Brabander, J.M. Vanacker, Ch. V. BoucquC National Institutefor Animal Nutrition, Agricultural Research Centre-Ghent, Scheldeweg 68, 9090 Melle-Gontrode, Belgium Received 25 August 1993; accepted 17 May 1994
Abstract The dry matter degradation in the rumen and pH decrease of nine primary feedstuffs, three compound feeds and three combinations of each two primary feedstuffs, were evaluated by means of in sacco and in vitro incubations. The ingredients included barley, wheat, maniac, maize, sorghum, sugar beet pulp, soya-bean meal, soya-bean hulls and maize gluten feed. The compound feeds differed in content and composition of the starch and sugars fraction, as well as in tibre content. The three combinations each consisted of a rapidly and slowly degradable feedstuff. The feedstuffs were incubated for different times to examine optimal incubation conditions for both estimating methods. For the in sacco technique, best predictions were obtained after 3 h of incubation. In this way, ingredients were ranked in order of decreasing dry matter degradation: maniac, wheat, barley, maize gluten feed, beet pulp, soya-bean meal, maize, sorghum, soya-bean hulls. With the in vitro method, better results were observed when rumen fluid was taken after rather than before feeding. After 5 h of incubation, ingredients were ranked in order of declining pH decrease: maniac, wheat, beet pulp, maize gluten feed, barley, maize, soyabean meal, sorghum, soya-bean hulls. Except for beet pulp, in vitro ranking agreed fairly well with in sacco results. The validity of these techniques was examined by limited results of in vivo experiments, where the effect of the nature of the compound feed on rumen fermentation was investigated. In vivo, a clear difference in risk for rumen disturbances could be observed between the three compound feeds. In sacco and in vitro, however, the compound feed with the * Corresponding author. ’Communication no. 866 of the National Institute for Animal Nutrition. 0377-8401/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SsDIO377-8401(94)00680-8
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highcontent of slowly degradable starch, could hardly be distinguished from the crude fibre rich compound feed. As only a limited amount of in vivo material was available,the estimating value of these methods require confirmation. Keyworuk Acidotic effect; Dry matter degradation
1.Introduction
To meet their production potential, cows have to consume the required amounts of energy, proteins, minerals and vitamins. With regard to ration optimisation, the supply of sufficient physical structure should also be considered. As production levels have increased, concentrate need has increased substantially. However, the intake of large amounts of concentrates can disturb rumen fermentation. In circumstances of suboptimal roughage supply, the type of concentrate (chemical composition and degradation rate) is a determining factor in the occurrence of rumen disturbances. Large amounts of concentrates with a high content of easily fermentable substances, generally, rapidly depress pH of rumen fluid and alter volatile fatty acids (VFA) pattern in the rumen. Fibre rich concentrates, in contrast, are characterised by a slower degradation and a reduced postprandial pH drop (Malestein et al., 198 1; Varga and Hoover, 1983; Van Beukelen et al., 1983; Sutton, 1986; Robinson et al., 1986). The acidotic effect of feedstuffs is an important characteristic and must be taken into account when rations are composed. Measuring the acidotic effect in vivo, however, is an expensive and time-consuming method. To evaluate the influence of feedstuffs on rumen fermentation of hundreds of primary feedstuffs or compound feeds, laboratory methods are needed to predict this effect. As starch or tibre rich feedstuffs encompass a heterogeneous group of components, the risk for rumen disturbance cannot solely be predicted from their chemical composition (Nocek, 1988; Giger et al., 1988; Graham and Aman, 199 1). Beside chemical composition, feedstuffs are also characterised by the rate and extent of digestion. These characteristics are often evaluated by means of in vitro and in sacco incubations (Varga and Hoover, 1983; Sauvant et al., 1985; Cullen et al., 1986; Nocek, 1988; van der Koelen et al., 1992). Comparison of the various results, however, is complicated by numerous factors, e.g. differences in experimental feedstuffs, incubation techniques (sample size, particle size, bag porosity, washing procedure, etc.), animals, and feeding pattern of the animals. The objective of the present study was to examine optimal conditions for in sacco and in vitro incubations as tools to predict the acidotic effect in the rumen of primary feedstuffs and compound feeds.
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2. Materials and methods 2. I. Feeds&& The rumen dry matter (DM) degradability of nine primary feedstuffs, three compound feeds and three combinations each of two primary feedstuffs, was determined by means of in sacco incubations. The pH decrease of rumen fluid caused by these feedstuffs was evaluated by means of in vitro experiments. The ingredients were: sugar beet pulp, soya-bean hulls, maize, sorghum, barley, wheat, maniac, maize gluten feed and soya-bean meal. The compound feeds were composed to differ in content and composition of the carbohydrate fraction. A first compound feed, with a low content of starch and sugars, was mainly composed of beet pulp and soya-bean hulls (BpSh). The other two compound feeds had high starch and sugars contents, but differed in degradation rate: MaiSo was based on maize and sorghum as slowly degradable ingredients; BaWhMan, based on barley, wheat and maniac, was rapidly degradable. All compound feeds contained maize gluten feed and soya-bean meal as protein sources and were pelleted. The composition of the compound feeds is presented in Table 1. Each of the three combinations of feedstuffs consisted of both a rapidly and a slowly degradable component, in a SO/SO ratio (fresh matter basis): maniac/ beet pulp, barley/soya-bean hulls, wheat/maize. These combinations were not pelleted. Table 1 Composition
of the compound feeds (g kg-‘) BpSh
Barley Wheat Maniac Maize Sorghum Beet pulp Soya-bean hulls Soya-bean meal Maize gluten feed Beet molasses Tallow Trace elements MgO Dicalcium phosphate Limestone NaCl Vit. A+D,+E Lignosulphonate
MaiSo
BaWhMan 200 170 140
230 230 351 120 205 140 70 30 18 4 24 5 4 3 2
185 207 70 _ 18 4 20 9 4 3 2
207 120 70 I5 18 4 22 7 4 3 2
BpSh, compound feed based on beet pulp and soya-bean hulls; MaiSo, compound feed based on maize and sorghum; BaWhMan, compound feed based on barley, wheat and maniac.
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Table 2 Chemical composition of the experimental feedstuffs Dry matter (g kg-r)
Barley Wheat Maniac Maize Sorghum Beet pulp Soya-bean hulls Soya-bean meal Maize gluten feed BpSh MaiSo BaWhMan
853 885 888 847 859 894 870 87 1 889 876 868 876
Chemical composition
(g kg-’ DM)
Crude protein
Crude Crude Ash NDF tibre fat
ADF
Lignin Cellu- Starch Sugars lose
121 136 28 107 115 107 135 517 227 198 194 199
64 19 50 18 15 194 392 62 74 137 41 45
97 29 107 25 49 294 505 103 118 181 61 70
20 9 27 8 19 48 36 14 18 26 14 15
18 17 4 42 40 4 22 16 39 34 15 28
28 19 60 14 13 65 48 68 70 84 76 82
166 97 111 97 78 360 603 123 281 258 107 126
75 24 49 21 34 214 471 89 88 156 48 51
521 675 716 710 737 4 67 55 218 85 423 377
22 28 17 12 7 103 14 104 33 83 64 72
BpSh, compound feed based on beet pulp and soya-bean hulls; MaiSo, compound feed based on maize and sorghum; BaWhMan, compound feed based on barley, wheat and maniac.
The chemical composition of all samples, except the three combinations, was determined according to standard methods (Table 2). Dry matter was determined by drying a sample in a ventilated oven at 60-70°C followed by ovendrying at 103-105 “C during 3 h. Crude protein was determined with an automatic Kjelfoss. The extraction of crude fat was carried out in a Soxlet apparatus with petroleum ether for 6 h. Ash content was determined after calcination in a furnace at 600°C. Crude tibre was analysed by Fibertec. Neutral detergent tibre (NDF) was determined according to Van Soest and Wine ( 1967). For acid detergent tibre (ADF) and lignin, the methods of Van Soest ( 1963) were applied. NDF and ADF were expressed on ash-free basis. Celhtlose was calculated as the difference between ADF and l&in. For all feedstuffs, except beet pulp and the compound feed with a large amount of beet pulp (BpSh), starch was polarimetrically determined by the method of Ewers (Commission of the European Communities, 1972). For beetpulp and BpSh, starch was enzymatically determined (Van Gelder et al., 1992). Sugars were determined according to the method of Luff-Schoorl (Anonymous, 197 1) . 2.2. In sacco incubation Two lactating Holstein cows, fitted with a rumen cannula, were used to determine the in sacco DM degradation of the feedstuffs. The cows were fed ad libitum a diet consisting of 55% maize silage and 45% compound feed (on DM basis). The diets were offered in two equal meals at 08:30 and 20:30 h. The nylon bags, measuring 8 cm x 8 cm and having a pore size of 50 pm, contained 2 g of fresh material, which was ground to pass a 2 mm sieve. Prior to
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ruminal incubation, all bags were soaked in a buffer solution according to Tilley and Terry ( 1963 ) for 3 min. Sets of bags were incubated into the rumen for 3,6, 12, 24 and 48 h. After each of the respective incubation periods, the bags were washed for 10 min and rinsed for 10 min in a washing machine, dried at 105 ‘C for 3 h and weighed. To determine the immediately disappearing fraction, bags were only soaked in the buffer solution, rinsed and washed. All samples were incubated in two cows and on two different days, so for each feedstuff, four degradation values were obtained. 2.3. In vitro incubation Fresh samples ( 1 g; ground to pass a 2 mm sieve) were incubated in plastic tubes with 50 ml of a rumen fluid/buffer solution (SO/50 v/v). The buffer solution was composed according to the method of Tilley and Terry ( 1963). The rumen fluid originated from the two previously mentioned fistulated cows and was mixed in advance. The solution was thoroughly saturated with C02. After adding the sample and the rumen fluid/buffer solution to the tube, the space above the liquid in each tube was flushed out with CO1 and the tube was then sealed with a rubber cork filled with a gas release valve. Tubes were then incubated at 39’C in the dark, being shaken hourly. To evaluate the influence of the time of rumen sampling, rumen fluid was taken once just before morning feeding and once 3 h after feeding. The pH of the diluted fluid was measured before adding the samples and 1,2, 3, 4 and 5 h after incubation with one of the 15 feedstuffs. To correct for the influence of the feed particles present, the pH course of the rumen fluid/buffer mixture as such was also measured. After 1,3 and 5 h of incubation, toluene was added to the test-tube to stop the fermentation. The solution was frozen for a later determination of the volatile fatty acids. All determinations were carried out in duplicate.
3. Results and discussion 3.1. In sacco incubation
The variation in DM degradability, owing to differences in incubation period within the same cow, was larger than the variation between cows. As the duration of incubation increased, variation coefficients diminished. For 3, 6, 12, 24 and 48 h of incubation, the variation coefficient between periods ( VP) amounted to respectively 7.396, 8.2%, 4.5%, 2.6% and 0.636, the between-cows variation (V,) amounted to respectively 3.8%, 2.5%, 4.2%, 1.5% and 0.7%. A decrease in VPand V, with prolonged incubation time was also reported by Rodriguez ( 1968) and Van der Koelen et al. ( 1992). The rather small V, and VP values after 48 h of incubation indicate that at this incubation time, disappearance reflects plant
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characteristics and is hardly influenced by other factors (Van der Koelen et al., 1992). The degradation of the nine primary feedstuffs (as a mean of four measurements) is represented in Fig. 1. The percentage of DM, which disappeared out of the nylon bag during the short period of immersion in the buffer solution and the washing procedure, considerably differed between feedstuffs, varying from 12% for soya-bean hulls to 68% for maniac. The early disappearance is due to solubilisation and losses of very small feed particles through the bag pores (Orskov et al., 1980). In our study, the immediately disappearing fraction is considered as completely degraded. This is not totally correct as a small part of the particles, which escaped through the bag pores, pass to the omasum before being degraded. As this small fraction leaves the rumen immediately, it does not contribute to the acidification of the rumen content. However, the inaccuracy caused by not correcting for this fraction is very small and may be ignored. In most studies, the feed particles escaping through 1w
80
00
E 6 B
I
% %
40
20
0
3 0
i 10
20
M
40
50 time(h)
Fig. 1. In sacmdry matter degradation of nine ingredients.
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303
the bag pores are assumed to be totally digestible (Orskov et al., 1980; Lindberg and Varvikko, 1982; Sauvant et al., 1985; Van der Koelen et al., 1992). During the first 9-l 2 h, all ingredients are degraded at a higher rate than during
t
--t -*
Maso
Bpsh
--
SaWhMan
Fig. 2. In sacco dry matter degradation of three compound feeds. Table 3 Measured and calculated DM degradation of the three compound feeds after 3 h in sacco incubation
BpSh MaiSo BaWhMan
Measured degradation” @1
Calculated degradation (%l
49.4f 5.7 48.4f4.3 66.6 f 3.3
48.6 43.3 63.7
BpSh, compound feed based on beet pulp and soya-bean hulls; MaiSo, compound feed based on maize and sorghum; BaWhMan, compound feed based on barley, wheat and maniac.
304
A.M. de Smet et al. /Animal Feed Science and Technology 51(1995) 297-315 100 T
I
50 time(h)
60
z fs so E %
40
B
0
10
20
30
40
50
time(h)
Fig. 3. In sacco dry matter degradation of three combinations of feedstuffs.
further incubation times. However, the degradation pattern varied considerably according to the feedstuff. The primary feedstuffs could roughly be divided into two categories: the first being rich in cytoplasmatic content and therefore being rapidly degradable, the second possessing a high content of slowly degradable cell wall material. Within these groups, however, there is also a range in degradation rate. Barley, wheat and maniac are more easily degradable than maize and sorghum, although they have all a high content of starch and sugars (see Table 2). As starch comprises 50-75% of these feedstuffs, the DM degradability is mainly determined by this fraction. The lower degradability of maize and sorghum is espe-
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Technology 51(1995) 297-315
305
cially due to the difference in granular structure of the starch. The starch granules of maize and sorghum are almost completely embedded in a protein matrix and this starch-protein interaction reduces the susceptibility of the starch to enzyme hydrolysis. Although the starch granules of maize and sorghum are very similar in size, shape and composition, sorghum is less degradable than maize. The major difference relates to the higher proportion of peripheral endosperm in sorghum, which is extremely dense, hard and resistant to digestion (Rooney and Pflugfelder, 1986). According to several authors (Johnson, 1976; Tamminga et al., 1978; Michalet-Doreau and Sauvant, 1989; Weiss et al., 1989) primary feedstuffs, rich in cell walls, have a rather low degradation rate. In our study, this was the case for soya-bean hulls, whereas beet pulp showed an even higher degradability than maize and sorghum. This can be explained by the high pectin content of the crude libre fraction. Although pectin belongs to the structural carbohydrates, it is a highly degradable component (Marounek et al., 1985 ) . The two protein rich feedstuffs, maize gluten feed and soya-bean meal, showed an intermediate degradation rate. Considering the compound feeds, the degradation corresponded to the characteristics of the incorporated ingredients. BaWhMan was degraded very easily, while MaiSo and BpSh showed a rather similar, but lower degradation rate (Fig. 2). The degradation values, calculated with the results of the single ingredients, were somewhat lower, especially for the starch rich compound feeds (Table 3 ) . This is probably the result of pelleting the compound feeds, since this effect is not taken into account for the calculations. The positive effect of processing feedstuffs on the degradability of starch is mentioned by several authors (Hale, 1973; Rooney and Pflugfelder, 1986; Malestein et al., 1988; Cone, 1991). The incubations with the feedstuff combinations indicated the additivity of the individual degradation values of the ingredients when predicting the degradability of a compound feed. As illustrated in Fig. 3, the measured degradation values were fairly well in accordance with the mathematical means of the degradations of the components in the mixture. 3.2. In vitro incubation As rumen environment evolves with time after feeding, the moment of rumen sampling can affect the results of in vitro measurements. In our study, the pH of the rumen fluid/buffer solution before incubation amounted to 6.59 and 6.23 when fluid was taken respectively before and 3 h after feeding. Because of the stronger microbial activity, lower pH values were obtained when fluid was taken after feeding. While the rumen fluid, taken before feeding, showed a constant pH during incubation without sample, the pH of the fluid taken after feeding decreased, due to fermentation of feed particles present in the rumen fluid. Hence, corrections were carried out on the in vitro results with fluid taken after feeding. The variation in pH decrease between periods ( IX’,) was more apparent for incubations with fluid taken after than before feeding. Probably, fluctuations in
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A.M. de Smet et al. /Animal Feed Science and Technology Sl(l995)
297-315
rumen fluid composition will be larger when fluid is taken after feeding, which results in a higher variation in pH decrease. For 1,2, 3,4 and 5 h of incubation with fluid taken before feeding, VC, amounted to, respectively, 0.3%, 0.6%, 0.696, 0.7% and 3.5%. When fluid was taken after feeding, VC, values were, respectively, 0.6%, 0.6%, 1.096, 1.2% and 1.0%. Fig. 4 represents the pH decrease of the different primary feedstuffs, when incubations were carried out with fluid taken after feeding. Maniac, wheat, beet pulp, maize gluten feed and barley showed the strongest pH decrease. For soyabean meal and maize intermediate values were measured, while the smallest changes were observed with sorghum and soya-bean hulls. The very fast pH decrease with beet pulp and maize gluten feed was rather surprising. A similar pH pattern was observed for MaiSo and BpSh. BaWhMan showed a faster and deeper pH decrease (Fig. 5 ) . pH decreases of the combined feeds differed from what could be expected from the pH decreases of the composing feedstuffs (Fig. 6). Comparable experiments of Malestein et al. ( 1982) showed that,
a.4
a
T
J-
5.8 --
5.8
--
5.4
--
5.2
--
5
--
4.8
--
lp
Fig. 4. In vitro pH decrease of nine ingredients.
A.M. deSmet et al. /Animal FeedScience and Technology 51(1995) 297-315
48
,~~~_~~+~~~
0
307
_--,-_-_
1
2
3
4
5 time (II)
Fig. 5. In vitro pH decrease of three compound feeds.
when feedstuffs were combined and incubated in rumen fluid, the final pH usually was lower, than when the feedstuffs were incubated separately. A possible explanation is that, when the substrate has a more complex composition, more species of microorganisms can each ferment part of it and as a consequence more VFA will be formed, resulting in a lower pH (Malestein et al., 1982). Moreover, a synergism between different species can occur, when fermentation products of one species are used as substrate by another one. In our study however, no change in VFA composition was observed, when incubations were carried out with fluid taken after feeding. This is probably due to the high amount of VFA already present in the rumen fluid, as a result of the fermentation of the ration fed. Compared with this fraction, the amount of VFA coming from the feed samples was too small to be detected. With fluid taken before feeding, changes in VFA composition caused by the degradation of the feed samples were actually noted. The largest changes occurred with maniac, barley and wheat, and, rather surprisingly, also
308
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5.6
IO
54
4 0
1
2
3
4
5 time(h)
Fig. 6. In vitro pH decrease of three combinations of feedstuffs.
with soya-bean meal, Within the three compound feeds, changes in VFA composition were similar, but a difference in VFA level was observed (Table 4). As expected from the chemical composition of the compound feeds, the lowest acetic acid and the highest propionic acid level is observed with the compound feeds rich in starch and sugars. Although maize and sorghum did not induce large VFA changes when incubated separately, Ma230 showed considerable changes in VFA
A.M. deSmet et al. /Animal FeedScience and Technology 51(1995) 297-31.5 Table 4 Evolution of pH, VFA pattern and total acids concentration feeds in rumen fluid taken before feeding
Blanc0
BpSh
MaiSo
BaWhMan
309
during incubation of the three compound
Duration of incubation (h)
pH
AC. ac. (mol. Oh)
Pr. ac. (mol. W)
But. ac. (mol. %)
Total acids (meq per 100 ml)
1 3 5 1 3 5 1 3 5 1 3 5
6.61 6.62 6.61 6.35 6.12 5.76 6.43 6.21 5.93 6.35 6.07 5.74
68.5 68.1 68.0 68.0 65.3 63.5 66.7 64.2 60.8 65.2 61.4 61.0
14.8 14.7 14.4 17.7 20.9 21.3 18.0 21.0 23.1 19.6 24.2 23.1
12.1 12.3 12.6 10.9 11.1 12.7 11.8 11.9 13.3 11.5 11.5 12.6
4.4 4.3 3.3 5.9 7.0 9.0 6.1 5.7 8.4 6.2 8.2 9.0
BpSh, compound feed based on beet pulp and soya-bean hulls; MaiSo, compound feed based on maize and sorghum; BaWhMan, compound feed based on barley, wheat and maniac. AC. ac., acetic acid; Pr. ac., propionic acid; But. ac., butyric acid.
pattern. This can again be ascribed to the more complex composition of the substrate. 3.3. Predictive value of the in sacco and in vitro method To evaluate these estimating methods, in vivo experiments, concerning the influence of the composition of compound feeds on the physical structure requirements of dairy cattle, were taken as a reference. For an optimal rumen function, ruminants need a minimum amount of structural components in the ration. The structural value of a ration not only depends on the kind of roughage, but also on the kind of compound feed included in the ration. A suboptimal structure supply can induce problems with feed intake, a decrease in milk fat content or milk production, acidosis, etc. As part of our structure evaluation research, in vivo trials were carried out to determine the minimum amount of roughage needed for normal rumen function. Eight lactating Holstein cows received a diet consisting of maize silage and one of the three compound feeds (BpSh, MaiSo and BaWhMan) in a decreasing roughage/compound feed (R/C) ratio. After a reference period of 2 weeks on an R/C ratio of 60/40, R/C decreased weekly, first in steps of 10% and from the ratio 40/60 on in steps of 5%. The lowest roughage part which cows can tolerate before showing symptoms of lack of physical structure, was called the critical roughage part (R,,,). This index, which also depends on the compound feed, indirectly provides an indication of the fermentation rate and acidotic effect of the compound feeds. According to this index, the three compound feeds were ranked as follows when they were given in combination
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with maize silage: BpSh, MaiSo and BaWhMan with Rcrit of, respectively, 31%, 33% and 38%. In a second experiment when the three compound feeds were given with prewilted grass silage, the Rcti, values obtained were, respectively, 22%, 26% and 33%. For these experiments, however, very large standard deviations due to differences between animals ( 2 6 percentage units) have to be mentioned. While in these experiments a difference existed between the Rcrit of each of the three compound feeds, this pattern did not agree with the in sacco results, where hardly any difference between BpSh and MaiSo was observed (see Fig. 2). In comparable earlier in sacco experiments with the same three compound feeds, the compound feeds were ranked in the same order as in the in vivo experiments, when incubated for 3 h. BaWhMan was degraded most rapidly (64.8%)) followed by MaiSo (50.0%) and BpSh (47.9%). None of the other incubation times gave the same ranking order as the in vivo results. Hence, on the basis of these results, the disturbing effect of feedstuffs on rumen fermentation seemed to be best predicted by in sacco incubation for 3 h. Three hours seems an acceptable duration of incubation as the acidotic effect can mainly be expected in the first hours after feed intake, when degradation of feedstuffs is most intensive. Furthermore, differences between feedstuffs diminish with larger incubation times. Although, based on the earlier results, feedstuffs were best evaluated after 3 h of in sacco incubation, in vivo and in sacco results did not agree completely. In vivo behaviour of MaiSo and BpSh was different, while in sacco degradation patterns differed little. The earlier in sacco results, however, agree fairly well with the degradation percentages obtained in the experiments described here. In these experiments, the difference in DM degradation between MaiSo (48.4%) and BpSh (49.4%) was almost negligible. Because of the large similarity between earlier and last degradation patterns and because a rather short incubation time allows a better evaluation of the differences between feedstuffs, 3 h is assumed as being the most suitable in sacco incubation time. According to this method, feedstuffs can be ranked in order of decreasing degradability. In our experiments, primary feedstuffs were classified as follows (DM degradation percentages are given in parentheses) : maniac ( 75.3 ) , wheat (73.9), barley (55.5), maize gluten feed (52.0), beet pulp (39.6), soyabeanmeal (34.0),maize (29.1),sorghum (20.5),soya-beanhulls (19.7). When the in vitro results of the compound feeds are compared with the in vivo data, a discrepancy similar to that of the in sacco incubations was observed. The in vitro method is a well reproducible, fast and cheap incubation technique. To evaluate these methods, correlations were calculated between the DM degradation after 3 h of in sacco incubation and in vitro pH decreases. If a good correlation exists between the two techniques, the in vitro method can replace the in sacco incubations as the estimating method. Better correlations were obtained when in vitro incubations were carried out with fluid taken after rather than before feeding. This can be ascribed to the greater microbial activity of the fluid, when sampled on a moment of active fermentation. The highest correlation coefficient (r= 0.9 1) was noted for 5 h of in vitro incubation with fluid taken after feeding (Table 5, Fig. 7 ) . Therefore, these in vitro incubation conditions seem to
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Table 5 Evolution of pH, VFA pattern and total acids concentration feedstuffs in rumen fluid taken before feeding
Barley
pH
AC. ac. (mol. %)
Pr. ac. (mol. %)
But. ac. (mol. %)
Total acids (meq per 100 ml)
1
6.37 6.21 5.92 6.37 6.24 5.87 6.32 5.91 5.36 6.41 6.28 6.13 6.48 6.35 6.22 6.18 5.90 5.64 6.39 6.29 6.20 6.43 6.30 6.15 6.18 6.07 5.83
64.0 61.2 58.8 63.9 61.2 59.0 65.4 61.7 59.8 65.2 63.7 63.1 66.6 64.4 64.2 69.6 67.8 64.2 68.6 67.1 66.1 61.9 65.5 59.0 63.8 60.0 62.0
18.7 20.9 22.7 18.3 20.9 23.9 18.4 22.6 24.6 16.5 16.9 17.5 15.7 15.4 16.3 15.7 18.6 20.4 15.9 18.1 17.7 17.4 19.3 23.2 18.6 23.1 21.5
13.2 14.2 15.1 13.6 13.9 13.6 12.9 13.3 13.5 14.3 15.6 15.8 13.4 16.2 15.7 11.8 11.2 12.9 11.7 11.4 12.6 11.1 11.6 13.9 13.6 13.2 13.4
5.1 6.6 9.4 5.0 7.3 7.7 5.0 7.5 10.1 5.3 5.7 5.5 4.5 5.5 5.3 5.6 7.5 8.7 5.3 6.4 7.1 5.8 6.6 9.0 5.4 7.0 9.4
1 3 5
Maniac
1 3 5
Maize
1 3 5
Sorghum
1
Beet pulp
3 5 1 3 5
Soya-bean hulls
Soya-bean meal
Maize @uteri feed
of the nine primary
Duration of incubation (h)
3 5 Wheat
during incubation
311
1 3 5 1 3 5 1 3 5
AC. ac., acetic acid; Pr. ac., propionic acid; But. ac., butyric acid.
be most appropriate to predict feedstuff degradation. However, when the VFA, formed during fermentation were considered, more information was obtained from incubations with fluid taken before feeding (see section 3.2. ). If a close simulation of in vivo degradation is aimed at, rumen fluid has to be sampled after feeding. So, the time of rumen sampling depends on the kind of information desired. For the in vitro incubation with fluid taken after feeding, the primary feedstuffs were ranked in order of declining pH decrease as follows (relative pH decrease is given in parentheses): maniac (14.9), wheat (12.5), beet pulp (11.4), maize gluten feed (10.6), barley (10.4), maize (8.0), soya-bean.meal (6.6), sorghum (4.5 ), soya-bean hulls (4.2 ). A weakness of the validation procedure is the restricted reference material. Thus, to give a final conclusion on the predictive value of the in sacco and in vitro methods would be hasty. One can ask if any method can predict accurately the
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Fig. 7. Correlation between in vitro pH (5 h incubation) tion) of the nine primary feedstuffs.
and in sacco DM degradation
Table 6 Correlation coefficients between degradation after 3 h in sacco incubation during 1,2,3,4 and 5 h incubation in fluid taken before and after feeding Duration of incubation (h)
Fluid taken before feeding
Fluid taken after feeding
1 2 3 4 5
0.37 0.47 0.53 0.70 0.77
0.49 0.62 0.65 0.87 0.91
(3 h incuba-
and in vitro pH decrease
acidotic effect of feedstuffs on rumen fermentation, as this is a very complex system which is not only influenced by the feedstuff itself, but also by the animal and environmental circumstances. Some disagreement between in vivo and in sacco results is also reported by Michalet-Doreau and Sauvant ( 1989). In in vivo experiments with three compound feeds based on beet pulp, maize or barley, the percentage of organic matter actually degraded in the rumen was the same for beet pulp and barley and exceeded that for maize, while in sacco, the degradation rate of beet pulp and maize were rather low and that of barley was higher. Therefore, in sacco behaviour of beet pulp did not accord with in vivo degradation. In our experiments, beet pulp neither showed a similar degradation pattern during in sacco and in vitro incubations. This feedstuff caused a very fast in vitro pH decrease, while in sacco an intermediate DM degradation was observed. This discrepancy can be related to observations of De Visser et al. ( 199 1). They reported a very small soluble fraction for dried beet pulp, which was ascribed to the swelling of the ground dry material during the washing procedure. As a consequence, particle size increased and the number of very small particles which could be rinsed out of the nylon
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bags was reduced. In this way, the in sacco procedure does not seem to predict DM degradation of beet pulp accurately, while for other ingredients reliable information is obtained. 4. Conclusion In sacco DM degradation and in vitro pH decrease were investigated, in order to rank feedstuffs following their disturbing influence on rumen fermentation. In our study, these methods showed some differences with in vivo experiments where the influence of different compound feeds on rumen function is investigated. A possible reason for this discrepancy may be that some parameters, which also play a role in the rumen degradation processes, are not taken into account (e.g. particle size, chewing activity, rate of passage, absorption of VFA, rumen motility). It would be hasty to draw conclusions about the accuracy of these methods to predict the risk for rumen disturbances, because the reference material is limited. In vitro and in sacco incubations, however, can give interesting information about the degradation pattern of feedstuffs. It thus becomes clear that starch and fibre rich feedstuffs cannot be divided into two well-defined groups. Within each group, there is a large range in degradation rate, as illustrated by the difference between maniac, barley and wheat on the one hand, and maize and sorghum on the other hand, or between beet pulp and soya-bean meal. Fibre rich feedstuffs can even show a higher degradation rate than feedstuffs with a high content of starch and sugars. Although some information is obtained from these estimating methods, no judgement can be made about the acidotic effect of feedstuffs on rumen fermentation. For better evaluation of these estimating techniques, more in vivo observations should be carried out. In further investigations, this will form an area of special attention. Acknowledgements
The authors wish to thank all the laboratory personnel of the institute for their valuable technical assistance. The research was financially supported by the Institute for the Encouragement of Scientific Research in Industry and Agriculture, Brussels. References Anonymous, 1971. Determination of sugar. Community methods of analysis for the official control of feedingstuffs. Off. J. Eur. Commun. L: 13. Commission of the European Communities, 1972. Determination of starch. Oftic. J. No. L123: 6, Brussels.
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