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Degradation in vitro and in sacco of red clover leaves and stems
Animal Feed Science and Technology, 17 (1987) 295-304
295
Elsevier Science Publishers B.V., Amsterdam-- Printed in The Netherlands
D e g r a d a t i o n in Vitro and in Sacco of Red Clover L e a v e s and Stems ERIK NORDKVIST, HADDEN GRAHAMand PER ]kMAN Department of Animal Nutrition and Management, Swedish University of Agricultural Sciences, S-750 07 Uppsala (Sweden)
(Received 6 October 1986; accepted for publication 5 February 1987)
ABSTRACT
Nordkvist, E., Graham, H. and/~man, P., 1987. Degradation in vitro and in sacco of red clover leaves and stems. Anita. Feed Sci. Technol., 17: 295-304. Methods of estimating degradabilityof red clover leaves and stems in vitro and in sacco were compared. An initial experiment revealed that the in vitro system underestimated dry matter dsgradability of mixtures of leaves and stems which had a leaf content higher than about 50%. Degradation in sacco of both leaves and stems was faster and more extensivethan that in vitro, while the differencebetweendry matter disappearance in sacco and in vitro was greater for leaves than for stems. In addition, the pattern of disappearance of individualcomponentsdifferedmarkedlybetween the two methods when leaves,but not stems, were degraded.
INTRODUCTION
Accurate estimation of the nutritive value of feed is of great importance to animal production. Apart from large-scale feeding trials, which are expensive and time-consuming, two approaches of biological significance are commonly used for r u m i n a n t feeds; the in sacco or nylon bag technique (Lindberg, 1983 ) and various in vitro systems (Tilley and Terry, 1963; Lindgren, 1979). Both methods have been shown to be highly reliable and to correlate closely with digestion in vivo ( W a n a p a t et al., 1986). It has been shown t h a t even though both the speed and the extent of degradation differ, the patterns of degradation of straw with these two methods are in good agreement ( G r a h a m and Aman, 1984). In studies u n d e r t a k e n in this laboratory on the composition and degradability in vitro of different botanical fractions of legumes, there have been indications t h a t it may not be possible to obtain a correct estimate of the degradability of the whole p l a n t by adding data from the anatomical parts. This led us to perform a more extensive study of the patterns of degradation
0377-8401/87/$03.50
© 1987 Elsevier Science Publishers B.V.
296 of red clover leaves and stems in vitro and in sacco and the results are presented in this paper. MATERIALSAND METHODS
Plant material Red clover ( Trifolium pratense L., cv. Hermes II) was sown in May 1983 in an experimental field at the Swedish University of Agricultural Sciences, Uppsala. A sample of 26.5 kg fresh weight was harvested on 19 June, 1984, before the plants had flowered. From this, a 7.7-kg subsample was quickly separated into leaves, petioles and stems and was frozen. These fractions were freeze-dried, and the leaves and stems used for further studies. All material was ground in a Wiley mill to pass a 1-mm screen prior to degradability studies and chemical analysis. All analyses were carried out in duplicate. Two samples of 0.5 m 2 were also harvested for estimating yield.
Degradability studies In sacco A non-lactating cow was used which was fitted with a rumen cannula and fed on 3 kg hay and 1.5 kg concentrate per day in 2 equal meals at 08.00 and 16.00 h. Samples of leaves and stems of red clover (1.0 g freeze-dried weight), in bags of precision-woven nylon cloth (Ziirich Bolting Cloth Mfg. Co. Ltd., CH-8803 Ruschlikon) with a pore size of 2 0 / l m and internal dimensions 100 X 50 mm, were placed in the ventral portion of the rumen just prior to morning feeding (Lindberg, 1983 ). The bags were incubated in the rumen for 4, 8, 16, 24, 36, 48 or 72 h for two consecutive periods, with at least 3 replicates for each incubation time in each period. At the end of each incubation, the bags were washed in cold running tap water for 15 min, squeezed to remove excess water and freeze-dried. Residues from the respective incubation times were pooled before chemical analysis. In vitro Samples (0.5 g freeze-dried weight) of leaves and stems of red clover were incubated in vitro with buffered rumen fluid from two sheep fed on a hay/concentrate diet according to Lindgren (!979). However, the rumen liquor content was increased to 10% (v:v) of the buffered incubate to minimize the initial lag in digestion. Samples were incubated for 8, 16, 24, 36, 48, 72 or 96 h for two consecutive periods with at least 3 replicates for each incubation time in each period. After incubation, the residues were washed with 3 X 50 ml hot distilled water and 2 X 25 ml acetone, and air-dried. The residues from the respective incubation times were pooled prior to chemical analysis.
297 The zero-time dry matter disappearance values (see Fig. 2 ) were determined by extracting with the buffer used for digestion in vitro (excluding calcium chloride and ferrous sulphate) for 4 h at 38 ° C.
Chemical analysis Dry matter was determined by drying at 105 ° C for 16 h and all results were calculated on a dry matter basis. Ash was determined by a standard A.O.A.C. (1980) method and minerals were determined by atomic absorption at the National Swedish Laboratory for Agricultural Chemistry, Uppsala. Crude protein was analyzed by standard Kjeldahl routine and permanganate lignin according to Robertson and Van Soest (1981). Samples were extracted with 80% aqueous ethanol and chloroform by Soxhlet extraction, and the insoluble residues were air-dried. Neutral constituents of non-starch polysaccharides were analyzed as alditol acetates by gas chromatography (GLC) on an OV-225 fused silica capillary column (Theander and Aman, 1979), while uronic acid constituents were determined by decarboxylation (Theander and ~.man, 1979). Acetyl substituents were released with 0.25 M sodium methoxide (Bacon and Gordon, 1980) and analyzed as benzyl acetates by GLC on a CP Sil 5 glass capillary column. Bound phenolic acids were analyzed by reversedphase HPLC (Nordkvist et al., 1984) on a CP Spher C18 column after hydrolysis overnight at room temperature with 1.0 M sodium hydroxide, neutralization and centrifugation. RESULTS AND DISCUSSION The total dry matter yield was estimated at about 5200 kg h a - 1 of which 42% was stems, 40% leaves and 18% petioles. Compared with lucerne at approximately the same harvest date, the yield of red clover was lower, but the ratio of leaves to stems was greater (Nordkvist and Aman, 1986). Degradability in vitro and in sacco of mixtures of various proportions of red clover leaves to stems is shown in Fig. 1. In agreement with previous results (~,man, 1985; Nordkvist and Aman, 1986), the extent of degradability was directly related to the fraction of stems. Degradability in sacco was approximately linear, with a degradability of over 93% for pure leaves, decreasing to about 82% for pure stems. Degradability in vitro, however, was close to linear only at fractions of stems over about 50%. With higher proportions of leaves, the curve levelled out and degradability was underestimated. The composition of the red clover leaves and stems is presented in Table I. Both contained similar amounts of extractives (sequentially extracted with 80% ethanol and chloroform) and of ash. The main component of the latter was calcium in the leaves and potassium in the stems. Crude protein content was almost three times higher in leaves than in stems. Both leaves and stems
298
92 9O
88 c
P 86 fo
84 ~ 82 E
m 80 78 2'0
/~0
6'0
(~0
I00 Stems (%)
Fig. 1. Degradability in vitro (96 h) (- - - ) and in sacco (48h) ( ) of mixtures of different proportions of red clover leaves and stems. Bars show standard error of mean; n = 3.
contained about 1% of bound acetyl groups and small amounts of cinnamic acids. The content of permanganate lignin was four times higher and that of cell wall polysaccharides nearly twice as high in the stems as in the leaves. The composition of the material was similar to that observed previously (~.man and Nordkvist, 1983; Aman, 1985) although the levels of components will differ between years, cultivars and husbandry conditions. The main constituents of the polysaccharides were glucose and uronic acid residues in both anatomical fractions, with a notably higher content of xylose residues in the stems than in the leaves. Figure 2 shows dry matter disappearance of red clover leaves and stems in vitro and in sacco at various incubation times. For both, the disappearance of dry matter was faster and more extensive in sacco and reached a plateau soon after about 20 h. The degradation in vitro of dry matter was much slower, and no difference between leaves and stems could be detected before 60 h of incubation. For the leaves, fermentation did not exceed solubilization (plotted at time zero) until after first sampling time, while for the stems a significant degradation occurred both in sacco and in vitro during the same period. This is a remarkable effect, since the contents of extractives were similar for both fractions. The composition of residues of leaves and stems at the normal sampling times for both methods ( i.e. after incubation in vitro for 96 h and in sacco for
299 TABLE I Chemical composition (g/100 g dry matter) of red clover leaves and stems Component
Leaves
Stems
Extractives 1 Crude protein Ash Calcium Magnesium Phosphorus Potassium Sodium Bound acetyl groups Bound p-coumaric acid Bound ferulic acid Permanganate lignin Polysaccharides Rhamnose residues Arabinose residues Xylose residues Mannose residues Galactose residues Glucose residues Uronic acid residues
27.8 27.4 8.5 2.75 0.38 0.26 1.52 0.019 0.8 0.018 0.010 1.4 26.5 0.5 2.0 1.4 0.6 1.4 11.5 9.1
26.3 11.0 7.0 0.85 0.29 0.17 2.69 0.013 1.0 0.014 0.010 6.3 45.9 0.4 3.7 5.7 1.1 2.7 22.6 9.7
~Sequentiallyextracted with 8 0 % ethanol and chloroform.
90 /i
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70
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I
I
I
20
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60
80
I
100 Time (h)
Fig. 2. Dry matter disappearance of red clover leaves incubated in sacco (- - • - -) and in vitro (- - C) - -) and stems incubated in sacco ( - - • - - ) and in vitro (-- [] - - ) for different periods of time. Bars show standard error of mean; n-- 6.
300 TABLE II Disappearance of dry matter (g/100 g) and composition (g/100 g DM) of red clover leaves and stems incubated in vitro (96 h ) or in sacco ( 48 h) Component
Dry matter disappearance1 Crude protein Ash Calcium Magnesium Phosphorus Potassium Sodium Bound acetyl groups Bound p-coumaric acid Bound ferulic acid Permanganate lignin Polysaccharides Rhamnose residues Arabinose residues Xylose residues Mannose residues Galactose residues Glucoseresidues Uronic acid residues
Leaves
Stems
In vitro
In sacco
In vitro
In sacco
78.7 + 1.62 17.4 26.1 7.43 0.75 4.80 0.42 0.53 1.08 0.024 0.015 5.6 22.6 0.58 0.65 1.97 0.31 0.81 14.35 3.93
90.4 ± 0.21 14.5 9.8 3.63 0.16 1.37 0.035 0.20 1.88 0.027 0.029 6.7 41.7 1.03 0.79 3.46 0.66 1.22 28.78 5.78
75.1 ± 0.47 4.7 4.9 1.25 0.12 0.64 0.096 0.14 3.8 0.016 0.009 28.6 48.6 0.46 0.91 11.71 0.93 1.08 26.75 6.78
81.4 ± 0.33 9.0 2.8 0.69 0.11 0.15 0.043 0.19 3.6 0.012 0.010 26.0 49.9 0.35 0.95 13.23 0.95 1.13 26.32 6.95
~Mean± standard error of mean; n-- 6. 48 h) are shown in Table II. In t he leaves, the c o n t e n t of crude prot ei n was higher in the residues in vitro t h a n in those in sacco, while t he opposite was found for the stems. T h e most striking difference in composition of residues was the high c o n t e n t of ash p r e s e n t in leaf residues in vitro. T h e stems also had a higher c o n t e n t of ash after t r e a t m e n t in vitro t h a n in sacco. Of the elements analyzed, calcium a nd phos phor us d o m i n a t e d and about half of t he ash c o n t e n t was account e d for by the five elements analysed. C o n ten ts of b o u n d acetyl groups and cinnamic acid derivatives were generally enriched during all digestions. In a gr eem ent with other investigations ( S m i t h et al., 1972; B u x t o n and H or ns t e i n, 1986), p e r m a n g a n a t e lignin and cell wall polysaccharides were main constituents in the indigestible residues of red clover. Of th e polysaccharides, glucose, uronic acid and xylose were the major residues. T h e main differences in polysaccharide composition from the original material (Table I) were lower cont ent s of uronic acid residues and higher c o n t e n t s of glucose and, in particular, xylose residues. T hi s resembles the p a t t e r n of degradation of lucerne in vitro ( N o r d k v i s t and Aman, 1986 ).
301
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Fig. 3. Disappearance of totalpolysaccharides (• + ), uronic acid (~k- • ), glucose ( × • ) and xylose ( LX [] ) residues in red clover leaves incubated in sacco ( ) and in vitro (- - -) relative to dry matter disappearance (DMD).
The disappearance of some components of cell walls from red clover leaves in relation to dry matter disappearance is presented in Fig. 3, and corresponding data from stems are shown in Fig. 4. When these figures are compared it is clear that in the stems there are only minor differences between incubations in sacco and in vitro in the disappearance of total polysaccharides, glucose, uronic acid and xylose residues. In the leaves, however, degradation in sacco caused total polysaccharides, glucose, uronic acid and xylose residues to disappear at a significantly slower rate relative to dry matter than did treatment in vitro.
302 gO
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Fig. 4. Disappearance of total polysaccharides ( • + ), uronic acid (~A-• ), glucose ( × • ) and xylose ( [] A ) residuesin red cloverstems incubated in sacco ( ) and in vitro (- - -) relative to dry matter disappearance (DMD). The p a t t e r n of disappearance of non-starch polysaccharide residues from the stems, with arabinose, galactose and uronic acids the most degradable and xylose the least, was identical for both incubation methods. This pattern has been observed for legumes in earlier studies (Gaillard, 1962; Aman, 1985; Nordkvist and ,~man, 1986). In leaves, arabinose and uronic acids were again the most degraded residues both in vitro and in sacco. However, the pattern of disappearance of the other polysaccharide residues from the leaves differed between the two methods, with xylose the least degraded residue in vitro and glucose in sacco. Recalculation of leaf dry matter disappearance on an organic matter basis would reduce the differences between the two methods by an aver-
303 age of 3 percentage units, but would not eliminate the divergence between the two methods, either in extent or in pattern of degradation. Wilman and Altimimi (1984) and Buxton and Hornstein (1986) observed a lower degradability in vitro of red clover leaves than in white clover and lucerne leaves, which was not explained by chemical composition. The latter authors also found that dry matter disappearance in vitro was closely correlated with neutral detergent fibre and permanganate lignin contents of legume stems and whole plants, but not of leaves. Visual assessment of incubations in the present study suggested that the difference in wetting properties between leaves and stems may play a major role. These differences may result from waxes on the leaf surface, and could have caused the prolonged lag time and initial slow degradation of the leaves. The accumulation in vitro of, for example, degradation end products, minerals and antimicrobial substances could influence the number and type of microorganisms in the incubation, and thus affect the extent and course of degradation. However, an accumulation of volatile fatty acids was unlikely to be involved, as the pH of leaf incubations in vitro did not fall below 6.7 and was not significantly different from that of stem incubations. In conclusion, the extent and pattern of degradation of red clover stems in vitro could be predicted from in sacco data. However, disappearance of leaf dry matter in vitro was slower and less than would have been predicted from figures obtained in sacco. Furthermore, the pattern of degradation of feed components differed between these two methods. Care should therefore be exercised when interpreting values obtained in vitro from feeds containing high proportions of legume leaves. ACKNOWLEDGEMENTS Professor Bengt Torsell is acknowledged for supplying the plant material, and May-Britt GrSn Rydberg for technical assistance. This work was financed through a grant from the Swedish Council for Forestry and Agricultural Research.
REFERENCES Aman, P., 1985. Chemicalcompositionand in vitro degradabilityof majorchemicalconstituents in botanicalfractionsof red cloverharvestedat differentstagesof maturity.J. Sci.FoodAgric., 36: 775-780. Aman, P. and Nordkvist, E., 1983. Chemicalcomposition and in vitro degradabilityof major chemicalconstituentsof redcloverharvestedat differentstagesofmaturity.J. Sci.FoodAgric., 34: 1185-1189. A.O.A.C., 1980. OfficialMethodsof Analysis. 13th edn. Associationof OfficialAnalyticalChemists, Washington,DC.
304
Bacon, J.S.D. and Gordon, A.H., 1980. Effects of various deacetylation procedures on the nylon bag digestibilityof barley straw and of grass cellwalls recovered from sheep faeces.J. Agric. Sci.,94: 361-367. Buxton, D.R. and Hornstein, J.S., 1986. Cell-wall concentration and components in stratified canopies of alfalfa,birdsfoot trefoiland red clover.Crop. Sci.,26: 180-184. Gaillard, B.D.E., 1962. The relationship between the cell-wallconstituents of roughages and the digestibilityof organic matter. J. Agric. Sci.,59: 369-373. Graham, H. and Aman, P., 1984. A comparison between degradation in vitro and in sacco of constituents of untreated and ammonia-treated barley straw. Anita. Feed Sci. Technol., 10: 199-211. Lindberg, J.E., 1983. Factors affectingpredictions of rumen degradabilityusing the nylon bag (in sacco) technique and a comparison between in vivo and in sacco degradability measurements. Swedish University of AgriculturalSciences,Uppsala, Department of Animal Nutrition,Report No. 59. pp. 1-36. Lindgren, E., 1979. The nutritionalvalue of roughages determined in vivo and by laboratory methods. Swedish University of Agricultural Sciences, Uppsala, Department of Animal Nutrition, Report No. 45, I~P.1-61. Nordkvist, E. and Aman, P., 1986. Changes during growth in anatomical and chemical composition and in vitro degradability of lucerne. J. Sci. Food Agric.,37: 1-7. Nordkvist, E., Salomonsson, A.-C. and Aman, P., 1984. Distribution of insolublebound phenolic acids in barley grain.J. Sci. Food Agric.,35: 657-661. Robertson, J.R. and Van Soest, P.J., 1981. The detergent system of analysis and its application to human foods. In: W.P.T. James and O. Theander (Editors), The Analysis of Dietary Fiber in Food. Marcel Dekker, N e w York, pp. 123-158. Smith, L.W., Goering, H.K. and Gordon, C.H., 1972. Relationships of forage composition with rates of cellwall digestion and indigestibilityof cellwalls.J. Dairy Sci.,55: 1140-1147. Theander, O. and Aman, P., 1979. Studies on dietary fibres,i. Analysis and chemical characterization of water-soluble and water-insoluble dietary fibres.Swedish J. Agric. Res., 9: 97-106. Tilley,J.M.A. and Terry, R.A., 1963. A two-stage technique for in vitro digestion of forage crops. J. Br. Grassl. Soc., 18: 104-111. Wanapat, M., Sundstol, F. and Hall, I.M.R., 1986. A comparison of alkalitreatment methods used to improve the nutritivevalue of straw. II.In sacco and in vitro degradation relativeto in vivo digestibility.Anita. Feed Sci. Technol., 14: 215-220. Wilman, D. and Altimimi, N.A.K., 1984. The in-vitrodigestibilityand chemical composition of plant parts in white clover,red clover and lucerne during primary growth. J. Sci. Food Agric., 35: 133-138.
295
Elsevier Science Publishers B.V., Amsterdam-- Printed in The Netherlands
D e g r a d a t i o n in Vitro and in Sacco of Red Clover L e a v e s and Stems ERIK NORDKVIST, HADDEN GRAHAMand PER ]kMAN Department of Animal Nutrition and Management, Swedish University of Agricultural Sciences, S-750 07 Uppsala (Sweden)
(Received 6 October 1986; accepted for publication 5 February 1987)
ABSTRACT
Nordkvist, E., Graham, H. and/~man, P., 1987. Degradation in vitro and in sacco of red clover leaves and stems. Anita. Feed Sci. Technol., 17: 295-304. Methods of estimating degradabilityof red clover leaves and stems in vitro and in sacco were compared. An initial experiment revealed that the in vitro system underestimated dry matter dsgradability of mixtures of leaves and stems which had a leaf content higher than about 50%. Degradation in sacco of both leaves and stems was faster and more extensivethan that in vitro, while the differencebetweendry matter disappearance in sacco and in vitro was greater for leaves than for stems. In addition, the pattern of disappearance of individualcomponentsdifferedmarkedlybetween the two methods when leaves,but not stems, were degraded.
INTRODUCTION
Accurate estimation of the nutritive value of feed is of great importance to animal production. Apart from large-scale feeding trials, which are expensive and time-consuming, two approaches of biological significance are commonly used for r u m i n a n t feeds; the in sacco or nylon bag technique (Lindberg, 1983 ) and various in vitro systems (Tilley and Terry, 1963; Lindgren, 1979). Both methods have been shown to be highly reliable and to correlate closely with digestion in vivo ( W a n a p a t et al., 1986). It has been shown t h a t even though both the speed and the extent of degradation differ, the patterns of degradation of straw with these two methods are in good agreement ( G r a h a m and Aman, 1984). In studies u n d e r t a k e n in this laboratory on the composition and degradability in vitro of different botanical fractions of legumes, there have been indications t h a t it may not be possible to obtain a correct estimate of the degradability of the whole p l a n t by adding data from the anatomical parts. This led us to perform a more extensive study of the patterns of degradation
0377-8401/87/$03.50
© 1987 Elsevier Science Publishers B.V.
296 of red clover leaves and stems in vitro and in sacco and the results are presented in this paper. MATERIALSAND METHODS
Plant material Red clover ( Trifolium pratense L., cv. Hermes II) was sown in May 1983 in an experimental field at the Swedish University of Agricultural Sciences, Uppsala. A sample of 26.5 kg fresh weight was harvested on 19 June, 1984, before the plants had flowered. From this, a 7.7-kg subsample was quickly separated into leaves, petioles and stems and was frozen. These fractions were freeze-dried, and the leaves and stems used for further studies. All material was ground in a Wiley mill to pass a 1-mm screen prior to degradability studies and chemical analysis. All analyses were carried out in duplicate. Two samples of 0.5 m 2 were also harvested for estimating yield.
Degradability studies In sacco A non-lactating cow was used which was fitted with a rumen cannula and fed on 3 kg hay and 1.5 kg concentrate per day in 2 equal meals at 08.00 and 16.00 h. Samples of leaves and stems of red clover (1.0 g freeze-dried weight), in bags of precision-woven nylon cloth (Ziirich Bolting Cloth Mfg. Co. Ltd., CH-8803 Ruschlikon) with a pore size of 2 0 / l m and internal dimensions 100 X 50 mm, were placed in the ventral portion of the rumen just prior to morning feeding (Lindberg, 1983 ). The bags were incubated in the rumen for 4, 8, 16, 24, 36, 48 or 72 h for two consecutive periods, with at least 3 replicates for each incubation time in each period. At the end of each incubation, the bags were washed in cold running tap water for 15 min, squeezed to remove excess water and freeze-dried. Residues from the respective incubation times were pooled before chemical analysis. In vitro Samples (0.5 g freeze-dried weight) of leaves and stems of red clover were incubated in vitro with buffered rumen fluid from two sheep fed on a hay/concentrate diet according to Lindgren (!979). However, the rumen liquor content was increased to 10% (v:v) of the buffered incubate to minimize the initial lag in digestion. Samples were incubated for 8, 16, 24, 36, 48, 72 or 96 h for two consecutive periods with at least 3 replicates for each incubation time in each period. After incubation, the residues were washed with 3 X 50 ml hot distilled water and 2 X 25 ml acetone, and air-dried. The residues from the respective incubation times were pooled prior to chemical analysis.
297 The zero-time dry matter disappearance values (see Fig. 2 ) were determined by extracting with the buffer used for digestion in vitro (excluding calcium chloride and ferrous sulphate) for 4 h at 38 ° C.
Chemical analysis Dry matter was determined by drying at 105 ° C for 16 h and all results were calculated on a dry matter basis. Ash was determined by a standard A.O.A.C. (1980) method and minerals were determined by atomic absorption at the National Swedish Laboratory for Agricultural Chemistry, Uppsala. Crude protein was analyzed by standard Kjeldahl routine and permanganate lignin according to Robertson and Van Soest (1981). Samples were extracted with 80% aqueous ethanol and chloroform by Soxhlet extraction, and the insoluble residues were air-dried. Neutral constituents of non-starch polysaccharides were analyzed as alditol acetates by gas chromatography (GLC) on an OV-225 fused silica capillary column (Theander and Aman, 1979), while uronic acid constituents were determined by decarboxylation (Theander and ~.man, 1979). Acetyl substituents were released with 0.25 M sodium methoxide (Bacon and Gordon, 1980) and analyzed as benzyl acetates by GLC on a CP Sil 5 glass capillary column. Bound phenolic acids were analyzed by reversedphase HPLC (Nordkvist et al., 1984) on a CP Spher C18 column after hydrolysis overnight at room temperature with 1.0 M sodium hydroxide, neutralization and centrifugation. RESULTS AND DISCUSSION The total dry matter yield was estimated at about 5200 kg h a - 1 of which 42% was stems, 40% leaves and 18% petioles. Compared with lucerne at approximately the same harvest date, the yield of red clover was lower, but the ratio of leaves to stems was greater (Nordkvist and Aman, 1986). Degradability in vitro and in sacco of mixtures of various proportions of red clover leaves to stems is shown in Fig. 1. In agreement with previous results (~,man, 1985; Nordkvist and Aman, 1986), the extent of degradability was directly related to the fraction of stems. Degradability in sacco was approximately linear, with a degradability of over 93% for pure leaves, decreasing to about 82% for pure stems. Degradability in vitro, however, was close to linear only at fractions of stems over about 50%. With higher proportions of leaves, the curve levelled out and degradability was underestimated. The composition of the red clover leaves and stems is presented in Table I. Both contained similar amounts of extractives (sequentially extracted with 80% ethanol and chloroform) and of ash. The main component of the latter was calcium in the leaves and potassium in the stems. Crude protein content was almost three times higher in leaves than in stems. Both leaves and stems
298
92 9O
88 c
P 86 fo
84 ~ 82 E
m 80 78 2'0
/~0
6'0
(~0
I00 Stems (%)
Fig. 1. Degradability in vitro (96 h) (- - - ) and in sacco (48h) ( ) of mixtures of different proportions of red clover leaves and stems. Bars show standard error of mean; n = 3.
contained about 1% of bound acetyl groups and small amounts of cinnamic acids. The content of permanganate lignin was four times higher and that of cell wall polysaccharides nearly twice as high in the stems as in the leaves. The composition of the material was similar to that observed previously (~.man and Nordkvist, 1983; Aman, 1985) although the levels of components will differ between years, cultivars and husbandry conditions. The main constituents of the polysaccharides were glucose and uronic acid residues in both anatomical fractions, with a notably higher content of xylose residues in the stems than in the leaves. Figure 2 shows dry matter disappearance of red clover leaves and stems in vitro and in sacco at various incubation times. For both, the disappearance of dry matter was faster and more extensive in sacco and reached a plateau soon after about 20 h. The degradation in vitro of dry matter was much slower, and no difference between leaves and stems could be detected before 60 h of incubation. For the leaves, fermentation did not exceed solubilization (plotted at time zero) until after first sampling time, while for the stems a significant degradation occurred both in sacco and in vitro during the same period. This is a remarkable effect, since the contents of extractives were similar for both fractions. The composition of residues of leaves and stems at the normal sampling times for both methods ( i.e. after incubation in vitro for 96 h and in sacco for
299 TABLE I Chemical composition (g/100 g dry matter) of red clover leaves and stems Component
Leaves
Stems
Extractives 1 Crude protein Ash Calcium Magnesium Phosphorus Potassium Sodium Bound acetyl groups Bound p-coumaric acid Bound ferulic acid Permanganate lignin Polysaccharides Rhamnose residues Arabinose residues Xylose residues Mannose residues Galactose residues Glucose residues Uronic acid residues
27.8 27.4 8.5 2.75 0.38 0.26 1.52 0.019 0.8 0.018 0.010 1.4 26.5 0.5 2.0 1.4 0.6 1.4 11.5 9.1
26.3 11.0 7.0 0.85 0.29 0.17 2.69 0.013 1.0 0.014 0.010 6.3 45.9 0.4 3.7 5.7 1.1 2.7 22.6 9.7
~Sequentiallyextracted with 8 0 % ethanol and chloroform.
90 /i
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C
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~ 60
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60
80
I
100 Time (h)
Fig. 2. Dry matter disappearance of red clover leaves incubated in sacco (- - • - -) and in vitro (- - C) - -) and stems incubated in sacco ( - - • - - ) and in vitro (-- [] - - ) for different periods of time. Bars show standard error of mean; n-- 6.
300 TABLE II Disappearance of dry matter (g/100 g) and composition (g/100 g DM) of red clover leaves and stems incubated in vitro (96 h ) or in sacco ( 48 h) Component
Dry matter disappearance1 Crude protein Ash Calcium Magnesium Phosphorus Potassium Sodium Bound acetyl groups Bound p-coumaric acid Bound ferulic acid Permanganate lignin Polysaccharides Rhamnose residues Arabinose residues Xylose residues Mannose residues Galactose residues Glucoseresidues Uronic acid residues
Leaves
Stems
In vitro
In sacco
In vitro
In sacco
78.7 + 1.62 17.4 26.1 7.43 0.75 4.80 0.42 0.53 1.08 0.024 0.015 5.6 22.6 0.58 0.65 1.97 0.31 0.81 14.35 3.93
90.4 ± 0.21 14.5 9.8 3.63 0.16 1.37 0.035 0.20 1.88 0.027 0.029 6.7 41.7 1.03 0.79 3.46 0.66 1.22 28.78 5.78
75.1 ± 0.47 4.7 4.9 1.25 0.12 0.64 0.096 0.14 3.8 0.016 0.009 28.6 48.6 0.46 0.91 11.71 0.93 1.08 26.75 6.78
81.4 ± 0.33 9.0 2.8 0.69 0.11 0.15 0.043 0.19 3.6 0.012 0.010 26.0 49.9 0.35 0.95 13.23 0.95 1.13 26.32 6.95
~Mean± standard error of mean; n-- 6. 48 h) are shown in Table II. In t he leaves, the c o n t e n t of crude prot ei n was higher in the residues in vitro t h a n in those in sacco, while t he opposite was found for the stems. T h e most striking difference in composition of residues was the high c o n t e n t of ash p r e s e n t in leaf residues in vitro. T h e stems also had a higher c o n t e n t of ash after t r e a t m e n t in vitro t h a n in sacco. Of the elements analyzed, calcium a nd phos phor us d o m i n a t e d and about half of t he ash c o n t e n t was account e d for by the five elements analysed. C o n ten ts of b o u n d acetyl groups and cinnamic acid derivatives were generally enriched during all digestions. In a gr eem ent with other investigations ( S m i t h et al., 1972; B u x t o n and H or ns t e i n, 1986), p e r m a n g a n a t e lignin and cell wall polysaccharides were main constituents in the indigestible residues of red clover. Of th e polysaccharides, glucose, uronic acid and xylose were the major residues. T h e main differences in polysaccharide composition from the original material (Table I) were lower cont ent s of uronic acid residues and higher c o n t e n t s of glucose and, in particular, xylose residues. T hi s resembles the p a t t e r n of degradation of lucerne in vitro ( N o r d k v i s t and Aman, 1986 ).
301
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Fig. 3. Disappearance of totalpolysaccharides (• + ), uronic acid (~k- • ), glucose ( × • ) and xylose ( LX [] ) residues in red clover leaves incubated in sacco ( ) and in vitro (- - -) relative to dry matter disappearance (DMD).
The disappearance of some components of cell walls from red clover leaves in relation to dry matter disappearance is presented in Fig. 3, and corresponding data from stems are shown in Fig. 4. When these figures are compared it is clear that in the stems there are only minor differences between incubations in sacco and in vitro in the disappearance of total polysaccharides, glucose, uronic acid and xylose residues. In the leaves, however, degradation in sacco caused total polysaccharides, glucose, uronic acid and xylose residues to disappear at a significantly slower rate relative to dry matter than did treatment in vitro.
302 gO
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Fig. 4. Disappearance of total polysaccharides ( • + ), uronic acid (~A-• ), glucose ( × • ) and xylose ( [] A ) residuesin red cloverstems incubated in sacco ( ) and in vitro (- - -) relative to dry matter disappearance (DMD). The p a t t e r n of disappearance of non-starch polysaccharide residues from the stems, with arabinose, galactose and uronic acids the most degradable and xylose the least, was identical for both incubation methods. This pattern has been observed for legumes in earlier studies (Gaillard, 1962; Aman, 1985; Nordkvist and ,~man, 1986). In leaves, arabinose and uronic acids were again the most degraded residues both in vitro and in sacco. However, the pattern of disappearance of the other polysaccharide residues from the leaves differed between the two methods, with xylose the least degraded residue in vitro and glucose in sacco. Recalculation of leaf dry matter disappearance on an organic matter basis would reduce the differences between the two methods by an aver-
303 age of 3 percentage units, but would not eliminate the divergence between the two methods, either in extent or in pattern of degradation. Wilman and Altimimi (1984) and Buxton and Hornstein (1986) observed a lower degradability in vitro of red clover leaves than in white clover and lucerne leaves, which was not explained by chemical composition. The latter authors also found that dry matter disappearance in vitro was closely correlated with neutral detergent fibre and permanganate lignin contents of legume stems and whole plants, but not of leaves. Visual assessment of incubations in the present study suggested that the difference in wetting properties between leaves and stems may play a major role. These differences may result from waxes on the leaf surface, and could have caused the prolonged lag time and initial slow degradation of the leaves. The accumulation in vitro of, for example, degradation end products, minerals and antimicrobial substances could influence the number and type of microorganisms in the incubation, and thus affect the extent and course of degradation. However, an accumulation of volatile fatty acids was unlikely to be involved, as the pH of leaf incubations in vitro did not fall below 6.7 and was not significantly different from that of stem incubations. In conclusion, the extent and pattern of degradation of red clover stems in vitro could be predicted from in sacco data. However, disappearance of leaf dry matter in vitro was slower and less than would have been predicted from figures obtained in sacco. Furthermore, the pattern of degradation of feed components differed between these two methods. Care should therefore be exercised when interpreting values obtained in vitro from feeds containing high proportions of legume leaves. ACKNOWLEDGEMENTS Professor Bengt Torsell is acknowledged for supplying the plant material, and May-Britt GrSn Rydberg for technical assistance. This work was financed through a grant from the Swedish Council for Forestry and Agricultural Research.
REFERENCES Aman, P., 1985. Chemicalcompositionand in vitro degradabilityof majorchemicalconstituents in botanicalfractionsof red cloverharvestedat differentstagesof maturity.J. Sci.FoodAgric., 36: 775-780. Aman, P. and Nordkvist, E., 1983. Chemicalcomposition and in vitro degradabilityof major chemicalconstituentsof redcloverharvestedat differentstagesofmaturity.J. Sci.FoodAgric., 34: 1185-1189. A.O.A.C., 1980. OfficialMethodsof Analysis. 13th edn. Associationof OfficialAnalyticalChemists, Washington,DC.
304
Bacon, J.S.D. and Gordon, A.H., 1980. Effects of various deacetylation procedures on the nylon bag digestibilityof barley straw and of grass cellwalls recovered from sheep faeces.J. Agric. Sci.,94: 361-367. Buxton, D.R. and Hornstein, J.S., 1986. Cell-wall concentration and components in stratified canopies of alfalfa,birdsfoot trefoiland red clover.Crop. Sci.,26: 180-184. Gaillard, B.D.E., 1962. The relationship between the cell-wallconstituents of roughages and the digestibilityof organic matter. J. Agric. Sci.,59: 369-373. Graham, H. and Aman, P., 1984. A comparison between degradation in vitro and in sacco of constituents of untreated and ammonia-treated barley straw. Anita. Feed Sci. Technol., 10: 199-211. Lindberg, J.E., 1983. Factors affectingpredictions of rumen degradabilityusing the nylon bag (in sacco) technique and a comparison between in vivo and in sacco degradability measurements. Swedish University of AgriculturalSciences,Uppsala, Department of Animal Nutrition,Report No. 59. pp. 1-36. Lindgren, E., 1979. The nutritionalvalue of roughages determined in vivo and by laboratory methods. Swedish University of Agricultural Sciences, Uppsala, Department of Animal Nutrition, Report No. 45, I~P.1-61. Nordkvist, E. and Aman, P., 1986. Changes during growth in anatomical and chemical composition and in vitro degradability of lucerne. J. Sci. Food Agric.,37: 1-7. Nordkvist, E., Salomonsson, A.-C. and Aman, P., 1984. Distribution of insolublebound phenolic acids in barley grain.J. Sci. Food Agric.,35: 657-661. Robertson, J.R. and Van Soest, P.J., 1981. The detergent system of analysis and its application to human foods. In: W.P.T. James and O. Theander (Editors), The Analysis of Dietary Fiber in Food. Marcel Dekker, N e w York, pp. 123-158. Smith, L.W., Goering, H.K. and Gordon, C.H., 1972. Relationships of forage composition with rates of cellwall digestion and indigestibilityof cellwalls.J. Dairy Sci.,55: 1140-1147. Theander, O. and Aman, P., 1979. Studies on dietary fibres,i. Analysis and chemical characterization of water-soluble and water-insoluble dietary fibres.Swedish J. Agric. Res., 9: 97-106. Tilley,J.M.A. and Terry, R.A., 1963. A two-stage technique for in vitro digestion of forage crops. J. Br. Grassl. Soc., 18: 104-111. Wanapat, M., Sundstol, F. and Hall, I.M.R., 1986. A comparison of alkalitreatment methods used to improve the nutritivevalue of straw. II.In sacco and in vitro degradation relativeto in vivo digestibility.Anita. Feed Sci. Technol., 14: 215-220. Wilman, D. and Altimimi, N.A.K., 1984. The in-vitrodigestibilityand chemical composition of plant parts in white clover,red clover and lucerne during primary growth. J. Sci. Food Agric., 35: 133-138.