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In Vitro Digestion Rate of Forage Cell Wall Components
In Vitro Digestion Rate of Forage Cell Wall Components L. W. SMITH, H. K. GOERING, D. R. WALDO, and C. H. GORDON
Animal Science Research Division, USDA Beltsville, Maryland 2070.5 Abstract
Objectives of this p a p e r were to demonstrate with forages of widely differing lignin:cellulose ratios that a) rates of cell wall digestion are first order, b) rates of cell wall digestion are not as well related to lignification of fiber as to cell solubles content of dry matter, and e) a similarity of lignin:cellulose ratios in the theoretically indigestible residue strongly supports the extent-of-digestion-limiting role of lignin.
Linearity of the semilog plots of remaining digestible fiber on time and their correlations (r = .98 approximately) indicated first order digestion kinetics for each of the six forages even though composition and observed rates of fiber digestion were markedly different. Immature rye cell walls digested fastest (27.03 ± . 8 1 % / hour, r 2 -- .999) and mature timothy cell walls digested slowest (5.65 -~+ . 3 1 % / hour, r 2 : .985). Similarity of lignin to cellulose ratios in theoretically indigestible residues from rye (.77), alfalfa (.79), orchardgrass (.79), and timothy (.63) suggest a dlgestion-extent-limiting role of ]ignin. Rates of cell wall digestion were positively (r -- .77, P < .05) related to cell solubles content of the forage. Cell solubles do not directly contribute to faster rates of cell wall digestion.
Experimental Procedure
Forage samples of total dry matter, approximating a half gram, were incubated (9) with rumen fluid for 6, ]2, 18, 24, 30, 48, and 72 hours.. To determine the influence of soluble dry matter on rate as well as extent of cell wall digestion, approximately half gram samples of isolated cell walls from the same forages were also incubated similarly. Ground filter paper, which is > 9 0 % digestible in the in vitro system, was a control. Original compositional analyses were performed exactly as outlined by Goering and Van Soest (9). Hemicellulose was determined as the differences between neutral-detergent fiber and acid-detergent fiber, lignin as the weight loss due to permanganate treatment of acid detergent fiber residue, and cellulose as the weight loss upon ashing the permanganate treated acid-detergent fiber residue. Cell walls, acid-detergent fiber and permanganate lignin analyses were sequentially performed on the same initial samples as well as on the fermentation residues, whether d r y matter or isolated cell walls were incubated, to calculate digestion rates of individual fiber constituents with no corrections for lignin recovery. In contrast, tabulated compositions of the 72-hour indigestible residue were from hemicellulose and cellulose in the 72-hour sample plus the lignin in the zero hour sample by sequential analyses. Digestion of fiber fractions approached an asymptote in 72 hours and was, thus, considered complete. Rates of fiber digestion were regressions of the percentages potentially digestible remaining on hours of fermentation.
Introduction
¥ o l u n t a r y intake of forage has been recognized as more important than nutrient digcsti~ bility in affecting animal performance, but both are highly interrelated to total digestible intake. Waldo, Smith, and Cox (17) recently proposed a tbeoretical separation of fiber into two fractions, that potentially digestible and that potentially indigestible. Several papers support such a concept (10, 11, ]3, ]8). BMdwin (2) has proposed wbat appe:/rs to be a similar concept. The rates of passage of the undigested portion of potentially digestible and indigestible residues as well as the rate of digestion of the potentially digestible fraction arc factors involved with voluntary intake. I n recent times in vitro digestibility has beer~ used widely to estimate in vivo digestion. Deinum, Yah Es and Yah Soest (4) have shown that in vitro cell wall digestibility was the most accurate of several laboratory methods for predicting in rive digestibility. Gill, Conrad and t i i b b s (8) showed that rate of digestible cellulose disappearance from in vitro digestion of alfalfa-grass silage was first order and related to digestible dry matter intake.
Results and Discussion
The compositions of hays are in Table 1. On a dry matter basis cell solubles ranged from 67 to 31; hemicellulose, 30 to 12; cellulose, 32
:Received for pub]icatlon Ju]y 28, 1970. 71
72
s ~ I T ~ ET A~.
TABLE 1. Chemical composition of hays.
Hay
Cell solubles
Hemieellu- Cellulose lose Lignin
10(
X "\.'~x. \', \
DZ~)--
--(% Rye, 23 cm tall, Nov. Alfalfa, prebud, Aug. Alfalfa, ½ bloom, Aug• Alfalfa Orchardgrass, 2nd cutting Timothy, mature
Invltro Ratesof Fiber Digestion(k)
67
16
15
2
61
12
20
7
55 52
12 12
24 27
9 9
40
25
29
6
31
30
32
7
Timothy (nm~re|
~ .~ \\" ~
b =.0243 Sb=.0015
\
"\.
"\
"",,. ,~
'\
i161 zi
\
X
\ to 15; and lignin, 9 to 2% for the six hays. Lignin: acid-detergent fiber ratios X 190 were from 27 to 9 and are in Table 2. To demonstrate that these hays had no unusual characteristics, 48-hour digestibilities of cell walls in vitro ( I V C W D ) on total dry matter samples were compared to predicted cell wall digestibilities (9). The maximum deviation of the two estimates of digestibility was five digestibility units for one hay. This result was expected since digestibility of rye was out of the range of data for the prediction equation. F o u r of the six hays were predicted within three digestibility units which indicates that forages were normal and that digestibility of these hays was predicted quite well from chemical compositions. The forages varied widely in initial composition of cell walls (Table 3). However, after 72 hours of digestion, compositions of the indigestible residues were much more similar• The lignin:acid-detergent fiber and lignin: cellulose ratios of the initial hays had a 3.3and 3.6-fold range whereas the lignin:aciddetergent fiber and lignin;cellulose ratios of
\.
X X .'
,~
~"
24
+'o
•
\
\
"\
t "\ 48
36
Time {hours)
FIG. 1. Percent digestible timothy cell walls remaining versus time. the residues had only a 1.2 and 1.3 fold range indicating a similar extent-of-digestion-limiting role of lignin in these hays. Lignin recovery was standardized to 100 for calculations involving lignin in the residues because of the low and variable lignin for the rye. The two mature grasses, orchardgrass and timothy, were distinctly different from the other forages as indicated by the higher content o f hemieellulose. These two forages also gained about foltr additional digestion units, whereas the other more rapidly digested forages gained less than one unit between 48 and 72 hours. Linearity of the semilog plots of remaining digestible fiber on time and the correlation (r ~ .98) of the two values indicated first order digestion kinetics for each of the six for-
TABLm 2. Comparison of predicted cell wall digestibilities with 48-hour in vitro cell wall digestibilities of total dry matter samples. Hay
L/ADF × 100
Predicted CWD
48-hr a IVCWD
72-hr ~ I V C W indigestibility
Rye, 23 em tall, Nov. Alfalfa, prebud, Aug. Alfalfa, ~ bloom, Aug. Alfalfa Orchardgrass, 2nd cutting Timothy, mature
9 24 26 27 17 16
86 48 44 42 62 65
91.1 48.7 42.9 38.0 65•7 61.7
8.4 51.3 55.9 61.0 31.5 34.1
a Mean and sm of duplicate determinations•
JOUR)rAL OF DAI~Y SCI~NO~ VOL. 54, ~0. 1
± .1 ± .4 ± .04 -+- .7 ± .7 ± .3
± .2 - - .4 H- .7 ± .1 -+- .2 H- .3
FORAGE
CELL WALLS
73
T ~ L ~ 3. Initial versus residual composition of the cell walls in hay. Initial
Residual (72 hr) a
Itay
Hemicellulose
Celinlose
Lignin
Rye, 23 em tall, Nov. Alfalfa, prebud, Aug. Alfalfa, ~ bloom, Aug. Alfalfa Orchardgrass, 2nd cutting Timothy, mature
51 32 28 25 42 43
45 51 53 55 49 47
4 17 19 19 9 10
Hemicellulose
Cellulose
Lignin
I
R
22 24 21 21 32 35
44 42 44 45 38 40
34 34 35 34 30 25
10 32 36 35 20 20
77 80 80 76 79 63
L / C × 100
(% cw)
a Assuming full recovery of initial lignin. ages even though the composition and rates of fiber digestion were markedly different. Representative data for the timothy hay are in F i g u r e 1. The regression coefficient (b) X 2.303 X 100 gives the rate of digestion (k) in p e r cent digestion per hour. Theoretical lines for 10 and 20% digestion per hour are shown for the range in this paper. Mean rates of celt wall digestion for each of the six forages are in Table 4. L i g n i n : a c i d detergent fiber, lignin: cellulose, and cell solubles are also presented for each of the hays. The small grain rye (27.03%/hour) had the fastest cell wall digestion and timothy (5.65%/ hour) the slowest. The rye was sampled in November 1969 from plots at Dairy Cattle Research Branch, Beltsville, Maryland, when the plants were about 23 cm tall. The rye, therefore, was immature whereas the timothy was mature. Little additional information is available for the timothy since it w~s from a commercial source. The available maturity data f o r the other hays are in Table 1. Difficulties were encountered in obtaining reproducible rate constants for the third alfalfa listed and the orchardgrass. I n two trials,
these two hays had higher and nearly similar rates. Therefore, additional fermentations were done on different days to ascertain the cause of variation. This problem has not yet been resolved nor were similarly high rates observed again. Nevertheless, the means of all estimates (n) are in the table. The rates of cell wall digestion ( % / h o u r ) shown were from incubation of whole hay d r y matter. I n Table 4 rates of cell wall digestion are not closely associated with corresponding lignin: acid-detergent fiber ratios nor lignin:cellulose ratios. However, it may be more realistic, nutritionally, to use the lignin:cellulose ratio with a single component in both the numerator and denominator if both are equally accurate determinations. Rates of cell wall digestion were negatively correlated with lignin:acid-detergent fiber (r ----- --.60), lignin:cellulose (r = --.57), and positively correlated with cell solubles content of the dry matter (r = + . 7 7 ) . The last two correlations are in :Figure 2. Lignin: aciddetergent fiber and lignin:eellulose are both nonsignificant correlations. However, the correlation with cell solubles content was significant (P < .05).
TABLE 4. Comparisons of in vitro rates of cell wall digestion with lignin:cellulose ratios and cell solubles content from total dry matter samples.
Hay
n
CW dig. a %/hr
Rye, 23 em tall, Nov. Alfalfa, prebud, Aug. Alfalfa, 1/~ bloom, Aug. Alfalfa Orchardgrass, 2nd cutting Timothy, mature
1 2 2 6 13 1
27.03 11.38 10.31 8.75 7.81 5.65
~ .81 ± 2.23 -~ .62 ± .65 ± .56 ± .31
L/ADF × 100
L/C × 100
Cell solubles % DM
9 24 26 27 17 16
10 32 36 35 20 20
67 61 55 52 40 31
a Mean and average SD [B] where applicable. JOURNAL OF DAIRY SCIENCE ¥ 0 L . 54, NO. I
74
SMITH
ET AL.
Invitro Rate o f Cell W a l l s Digestion vs. Cell Solubles Contents and Lillnin tCal|ulosa Ratios x=Lignin tCalluloJe y=22.5 4 2 x x Sbt.31 ""
I= 30
o
• ~ Call Soluble8 Content • y-'-10.6~.44x
r=-.57
Sb m.|8 r,~.77
| 20 I
X
10
|
X
•
X
@l
0
10
•
•
Jc
•
20
30
40
50
I 70
60
Percent
Fro. 2. Relationships of the rates of cell walls digestion to lignin:cellulose ratios and cell solubles contents. When the rye sample was deleted and correlations recalculated (Fig. 3), lignin:aciddetergent fiber (r : 4-.76, P ~ .05), ]ignin: cellulose (r ---- +.78, P ~ .05), and cell solubles (r ------5.98, P ( . 0 1 ) all have positive and significant correlations. This might be interpreted as indicating that lignin has a positive influence on rate of digestion in spite of the well established negative relationship of ligni-
fication on extent of digestion. A more plausible explanation, though, in view of other published data (3, 5, 7, 12, 16) as well as data in this paper, is that alfalfa cell walls digest more rapidly than grass cell walls at maturities for which these forages are harvested for hay. Nevertheless, forages with the higher cell solubles contents digested most rapidly in vitro. Cell solubles may be contributing directly to
Invitro Rate of Cell Walls Digestion vs. Cell Solubles Contents and
Lignin:
Cellulose R a t i o s - S u m m e r Forages 40
30 .0 -
x - Lignin:Cellulose Y= 5.35~ .12x Sb= .10 r= +.78
e-Cell Solubles Content Y=-.20÷.18x Sb= .02 r= ~.98
e o'J ut
m
10
g
!0
FIG. 3. Relationships of lignin:cellulose ratios and cell solubles contents to the rates of cell walls digestion of summer forages. JOURNAL OF DAIRY SCIEI~'CE V 0 L
54 w NO. 1
FORAGE
¢q
r-~
+l 41 +l 41 O9
.
.
.
.
+~ 41 +1 41
+1 +1 +1 +l
+l 41 +1 41 ~
ke~
~
u'3
41 +1 ~1 41
41 41 41 +1
+l +1 +1 +l
r~
¢I 41 41 41
o
r~
r~
CELL
75
WALLS
faster rates of cell wall digestion. To test this hypothesis, both whole hay dry matter and isolated cell walls were fermented simultaneously. Rates of cell wall, acid-detergent fiber, beretcellulose, and cellulose digestion were determined on whole dry matter and on isolated cell walls for an alfalfa, rye, orehardgrass, and timothy. These results are in Table 5. Generally little, if any difference, was found in rate constants between fermentation of whole dry matter or isolated cell walls. Also, little influence was found on the rate of fermentation o f cell walls, acid-detergent fiber, hemicellulose or cellulose within a particular hay. The larger standard deviation of the rye rate constants and the greater variation between digestion rates of the fiber components was probably the result of selecting suboptimal fermentation intervals for this rapidly digesting forage. These data strongly preclude the possibility of cell solubles per se contributing to more rapid digestion of cell walls in vitro. However, theories may be generated to attempt an explanation of the relationship of faster rates of cell wall digestion for hays as related to greater solubles content. The first may be simply physical as suggested by Donefer (6). Forages of high-cell solubles contents may have less well developed structural components, thus allowing ruminal bacteria greater access to the cell wall for attack. Both surface availability for attack and fragility of the cell wall could be crucial considerations. Another equally plausible and not entirely unrelated explanation may be that degree of crystallinity (1, 14) of structural carbohydrates slay be different for legumes as opposed to grasses as well as different relative to maturity. Donefer (6) found a high positive correlation of cell solubles with nutritive value index, and Yah Soest (15) showed a high negative relationship between cell wall content and voluntary intake. Their results (6, 15) show a direct relationship of cell solubles to increased voluntary intake. Thus, higher solubles content and the associated faster rate of digestion should be additive in diminishing rumen fill. Tomlin (14) was unable to establish a relationship of crystallinity with extent of forage digestion. However, his inability to show this relationship may have resulted from X-ray measurements on improperly prepared samples. Further investigation is required to determine the mechanisms involved which allow more rapid rates of fiber digestion in forages of high cell solubles content. Estimates of indigestible fiber and rate of digestion of potentially digestible fiber in forage should have practical application in predicting J0~RNAL
O F D A I R Y S c I ~ l v ~ ] ~ VOW. 5 4 , NO. 1
76
S M I T H ET AL.
n u t r i t i v e value. E v e n i f p a s s a g e is c o n s t a n t , the sum o f t h e p o t e n t i a l l y indigestible fiber plus t h e r e m a i n i n g digestible sometime a f t e r f e e d i n g s h o u l d r e s u l t in a u s e f u l estimate of r u m e n fill which should be h i g h l y r e l a t e d to v o l u n t a r y c o n s u m p t i o n . Such estimates b a s e d on data in Figure I indicate that approximately 5 0 % t i m o t h y , 3 3 % a l f a l f a , a n d 5 % r y e digestible cell wall would be r e m a i n i n g a f t e r 12 hours.
Acknowledgments The authors acknowledge the laboratory assistance of J o h n O'Connor, William Kramer, and Elaine Bierman in conducting this study.
References (1) Baker, T. I., G. ¥ . Quicke, O. G. Bentley, R. R. Johnson, and A. L. Moxon. 1959. The influence of certain physical properties of purified celluloses and forage cellulose on this digestibility by rumen microorganisms in vitro. J. Animal Sci., 18: 655. (2) Baldwin, R. L., H. L. Lucas, and R. Coburn. 1969. Energetic relationships in the formation and utilization of fermentation endproducts. Third Int. Syrup. Physiol. of Digestion and Metabolism in the Ruminant. Cambridge, England, Aug. 18-23. (3) Baumgardt, B. R., M. W. Taylor, and J. L. Cason. 1962. Evaluation of forages in the laboratory. II. Simplified artificial rumen procedures for obtaining repeatable estimates of forage nutritive value. J. Dairy Sci., 45: 62. (4) Deinum, B., A. J. A. ¥ a n Es, and P. J. Yah Soest. 1968. Climate, nitrogen and grass. II. The influence of light intensity, temperature and nitrogen on in vivo digestibility of grass and the prediction of these effects from some chemical procedures. Netherlands J. Agr. Sci., 16: 217. (5) Demarquilly, C. 1965. Factors affecting the voluntary intake of green forage by sheep. Proc. I X Int. Grassland Congr., p. 877. (6) Donefer, E. 1969. Forage solubility measurements in relation to nutritive value. National Conf. on Forage Quality Evaluation and Utilization, Sept. 2-5, Lincoln, Nebraska.
JOURI~AL OF DAIRY ScIl~l~CE VOL. 54, NO. 1
(7) Donefer, E., E. W. Crampton, a n d L. E. Lloyd. 1960. Prediction of the nutritive value index of a forage from in vitro rumen fermentation data. J. Animal Sci., 19: 545. (8) Gill, S. S., H. R. Conrad, a n d J. W. Hibbs. 1969. Relative rate of in vitro cellulose disappearance as a possible estimator of digestible dry matter intake. J. Dairy Sci., 52 : 1687. (9) Goering, H. K., and P. J. V a n Soest. 1970. Forage fiber analyses (apparatus, reagents, procedures and some application). Agr. handbook No. 379. ARS, USDA. (10) Johnson, R. R., H. W. Scott, A. L. Moxan, and O. G. Bentley. 1959. The digestible cellulose remaining in the feces of sheep fed different roughages as determined by in vitro rumen fermentation. J. Animal Sci., 18: 520. (11) McAnally, R. H. 1942. Digestion of straw by the ruminant. Bioehem. J., 36: 392. (12) Reid, R. L., and G. A. Jung. 1965. Influence of fertilized treatment on the intake, digestibility, and palatability of tall rescue hay. J. Animal Sci., 24: 615. (13) Smith, L. W., H. K. Goering, and C. H. Gordon. 1969. Influence of chemical treatments upon digestibility of r u m i n a n t feces. Proc. Conf. Animal Waste Management, Cornell Univ., Ithaca, N.Y., pp. 88. (14) Tomlin, D. C. 1960. Crystallinity of cellulose and digestibility of fecdstuff cellulose in the bovine tureen. Ph.D. Thesis, Univ. of Florida, University Microfilms 60-5148. (15) Van Soest, P. J. 1965. Symposium on factors influencing the voluntary intake of herbage by ruminants: Voluntary intake in relation to chemical composition and digestibility. J. Animal Sci., 24: 834. (16) Van Soest, P. J. 1968. Chemical estimates of the nutritional value of feeds. Proc. Cornell Nutr. Conf., p. 38. (17) Waldo, D. R., L. W. Smith, and E. L. Cox. 1969. Developments on a theore*ical model of fiber digestion in relation to lignification and unresolved problems. Presented at the Tenth Conference on Rumen Function, Chicago, Illinois, DCRB 69-10. (18) Wilkens, R. J. 1969. The potential digestibility of cellulose in forage and feces. J . Agr. Sci., 73: 57.
Animal Science Research Division, USDA Beltsville, Maryland 2070.5 Abstract
Objectives of this p a p e r were to demonstrate with forages of widely differing lignin:cellulose ratios that a) rates of cell wall digestion are first order, b) rates of cell wall digestion are not as well related to lignification of fiber as to cell solubles content of dry matter, and e) a similarity of lignin:cellulose ratios in the theoretically indigestible residue strongly supports the extent-of-digestion-limiting role of lignin.
Linearity of the semilog plots of remaining digestible fiber on time and their correlations (r = .98 approximately) indicated first order digestion kinetics for each of the six forages even though composition and observed rates of fiber digestion were markedly different. Immature rye cell walls digested fastest (27.03 ± . 8 1 % / hour, r 2 -- .999) and mature timothy cell walls digested slowest (5.65 -~+ . 3 1 % / hour, r 2 : .985). Similarity of lignin to cellulose ratios in theoretically indigestible residues from rye (.77), alfalfa (.79), orchardgrass (.79), and timothy (.63) suggest a dlgestion-extent-limiting role of ]ignin. Rates of cell wall digestion were positively (r -- .77, P < .05) related to cell solubles content of the forage. Cell solubles do not directly contribute to faster rates of cell wall digestion.
Experimental Procedure
Forage samples of total dry matter, approximating a half gram, were incubated (9) with rumen fluid for 6, ]2, 18, 24, 30, 48, and 72 hours.. To determine the influence of soluble dry matter on rate as well as extent of cell wall digestion, approximately half gram samples of isolated cell walls from the same forages were also incubated similarly. Ground filter paper, which is > 9 0 % digestible in the in vitro system, was a control. Original compositional analyses were performed exactly as outlined by Goering and Van Soest (9). Hemicellulose was determined as the differences between neutral-detergent fiber and acid-detergent fiber, lignin as the weight loss due to permanganate treatment of acid detergent fiber residue, and cellulose as the weight loss upon ashing the permanganate treated acid-detergent fiber residue. Cell walls, acid-detergent fiber and permanganate lignin analyses were sequentially performed on the same initial samples as well as on the fermentation residues, whether d r y matter or isolated cell walls were incubated, to calculate digestion rates of individual fiber constituents with no corrections for lignin recovery. In contrast, tabulated compositions of the 72-hour indigestible residue were from hemicellulose and cellulose in the 72-hour sample plus the lignin in the zero hour sample by sequential analyses. Digestion of fiber fractions approached an asymptote in 72 hours and was, thus, considered complete. Rates of fiber digestion were regressions of the percentages potentially digestible remaining on hours of fermentation.
Introduction
¥ o l u n t a r y intake of forage has been recognized as more important than nutrient digcsti~ bility in affecting animal performance, but both are highly interrelated to total digestible intake. Waldo, Smith, and Cox (17) recently proposed a tbeoretical separation of fiber into two fractions, that potentially digestible and that potentially indigestible. Several papers support such a concept (10, 11, ]3, ]8). BMdwin (2) has proposed wbat appe:/rs to be a similar concept. The rates of passage of the undigested portion of potentially digestible and indigestible residues as well as the rate of digestion of the potentially digestible fraction arc factors involved with voluntary intake. I n recent times in vitro digestibility has beer~ used widely to estimate in vivo digestion. Deinum, Yah Es and Yah Soest (4) have shown that in vitro cell wall digestibility was the most accurate of several laboratory methods for predicting in rive digestibility. Gill, Conrad and t i i b b s (8) showed that rate of digestible cellulose disappearance from in vitro digestion of alfalfa-grass silage was first order and related to digestible dry matter intake.
Results and Discussion
The compositions of hays are in Table 1. On a dry matter basis cell solubles ranged from 67 to 31; hemicellulose, 30 to 12; cellulose, 32
:Received for pub]icatlon Ju]y 28, 1970. 71
72
s ~ I T ~ ET A~.
TABLE 1. Chemical composition of hays.
Hay
Cell solubles
Hemieellu- Cellulose lose Lignin
10(
X "\.'~x. \', \
DZ~)--
--(% Rye, 23 cm tall, Nov. Alfalfa, prebud, Aug. Alfalfa, ½ bloom, Aug• Alfalfa Orchardgrass, 2nd cutting Timothy, mature
Invltro Ratesof Fiber Digestion(k)
67
16
15
2
61
12
20
7
55 52
12 12
24 27
9 9
40
25
29
6
31
30
32
7
Timothy (nm~re|
~ .~ \\" ~
b =.0243 Sb=.0015
\
"\.
"\
"",,. ,~
'\
i161 zi
\
X
\ to 15; and lignin, 9 to 2% for the six hays. Lignin: acid-detergent fiber ratios X 190 were from 27 to 9 and are in Table 2. To demonstrate that these hays had no unusual characteristics, 48-hour digestibilities of cell walls in vitro ( I V C W D ) on total dry matter samples were compared to predicted cell wall digestibilities (9). The maximum deviation of the two estimates of digestibility was five digestibility units for one hay. This result was expected since digestibility of rye was out of the range of data for the prediction equation. F o u r of the six hays were predicted within three digestibility units which indicates that forages were normal and that digestibility of these hays was predicted quite well from chemical compositions. The forages varied widely in initial composition of cell walls (Table 3). However, after 72 hours of digestion, compositions of the indigestible residues were much more similar• The lignin:acid-detergent fiber and lignin: cellulose ratios of the initial hays had a 3.3and 3.6-fold range whereas the lignin:aciddetergent fiber and lignin;cellulose ratios of
\.
X X .'
,~
~"
24
+'o
•
\
\
"\
t "\ 48
36
Time {hours)
FIG. 1. Percent digestible timothy cell walls remaining versus time. the residues had only a 1.2 and 1.3 fold range indicating a similar extent-of-digestion-limiting role of lignin in these hays. Lignin recovery was standardized to 100 for calculations involving lignin in the residues because of the low and variable lignin for the rye. The two mature grasses, orchardgrass and timothy, were distinctly different from the other forages as indicated by the higher content o f hemieellulose. These two forages also gained about foltr additional digestion units, whereas the other more rapidly digested forages gained less than one unit between 48 and 72 hours. Linearity of the semilog plots of remaining digestible fiber on time and the correlation (r ~ .98) of the two values indicated first order digestion kinetics for each of the six for-
TABLm 2. Comparison of predicted cell wall digestibilities with 48-hour in vitro cell wall digestibilities of total dry matter samples. Hay
L/ADF × 100
Predicted CWD
48-hr a IVCWD
72-hr ~ I V C W indigestibility
Rye, 23 em tall, Nov. Alfalfa, prebud, Aug. Alfalfa, ~ bloom, Aug. Alfalfa Orchardgrass, 2nd cutting Timothy, mature
9 24 26 27 17 16
86 48 44 42 62 65
91.1 48.7 42.9 38.0 65•7 61.7
8.4 51.3 55.9 61.0 31.5 34.1
a Mean and sm of duplicate determinations•
JOUR)rAL OF DAI~Y SCI~NO~ VOL. 54, ~0. 1
± .1 ± .4 ± .04 -+- .7 ± .7 ± .3
± .2 - - .4 H- .7 ± .1 -+- .2 H- .3
FORAGE
CELL WALLS
73
T ~ L ~ 3. Initial versus residual composition of the cell walls in hay. Initial
Residual (72 hr) a
Itay
Hemicellulose
Celinlose
Lignin
Rye, 23 em tall, Nov. Alfalfa, prebud, Aug. Alfalfa, ~ bloom, Aug. Alfalfa Orchardgrass, 2nd cutting Timothy, mature
51 32 28 25 42 43
45 51 53 55 49 47
4 17 19 19 9 10
Hemicellulose
Cellulose
Lignin
I
R
22 24 21 21 32 35
44 42 44 45 38 40
34 34 35 34 30 25
10 32 36 35 20 20
77 80 80 76 79 63
L / C × 100
(% cw)
a Assuming full recovery of initial lignin. ages even though the composition and rates of fiber digestion were markedly different. Representative data for the timothy hay are in F i g u r e 1. The regression coefficient (b) X 2.303 X 100 gives the rate of digestion (k) in p e r cent digestion per hour. Theoretical lines for 10 and 20% digestion per hour are shown for the range in this paper. Mean rates of celt wall digestion for each of the six forages are in Table 4. L i g n i n : a c i d detergent fiber, lignin: cellulose, and cell solubles are also presented for each of the hays. The small grain rye (27.03%/hour) had the fastest cell wall digestion and timothy (5.65%/ hour) the slowest. The rye was sampled in November 1969 from plots at Dairy Cattle Research Branch, Beltsville, Maryland, when the plants were about 23 cm tall. The rye, therefore, was immature whereas the timothy was mature. Little additional information is available for the timothy since it w~s from a commercial source. The available maturity data f o r the other hays are in Table 1. Difficulties were encountered in obtaining reproducible rate constants for the third alfalfa listed and the orchardgrass. I n two trials,
these two hays had higher and nearly similar rates. Therefore, additional fermentations were done on different days to ascertain the cause of variation. This problem has not yet been resolved nor were similarly high rates observed again. Nevertheless, the means of all estimates (n) are in the table. The rates of cell wall digestion ( % / h o u r ) shown were from incubation of whole hay d r y matter. I n Table 4 rates of cell wall digestion are not closely associated with corresponding lignin: acid-detergent fiber ratios nor lignin:cellulose ratios. However, it may be more realistic, nutritionally, to use the lignin:cellulose ratio with a single component in both the numerator and denominator if both are equally accurate determinations. Rates of cell wall digestion were negatively correlated with lignin:acid-detergent fiber (r ----- --.60), lignin:cellulose (r = --.57), and positively correlated with cell solubles content of the dry matter (r = + . 7 7 ) . The last two correlations are in :Figure 2. Lignin: aciddetergent fiber and lignin:eellulose are both nonsignificant correlations. However, the correlation with cell solubles content was significant (P < .05).
TABLE 4. Comparisons of in vitro rates of cell wall digestion with lignin:cellulose ratios and cell solubles content from total dry matter samples.
Hay
n
CW dig. a %/hr
Rye, 23 em tall, Nov. Alfalfa, prebud, Aug. Alfalfa, 1/~ bloom, Aug. Alfalfa Orchardgrass, 2nd cutting Timothy, mature
1 2 2 6 13 1
27.03 11.38 10.31 8.75 7.81 5.65
~ .81 ± 2.23 -~ .62 ± .65 ± .56 ± .31
L/ADF × 100
L/C × 100
Cell solubles % DM
9 24 26 27 17 16
10 32 36 35 20 20
67 61 55 52 40 31
a Mean and average SD [B] where applicable. JOURNAL OF DAIRY SCIENCE ¥ 0 L . 54, NO. I
74
SMITH
ET AL.
Invitro Rate o f Cell W a l l s Digestion vs. Cell Solubles Contents and Lillnin tCal|ulosa Ratios x=Lignin tCalluloJe y=22.5 4 2 x x Sbt.31 ""
I= 30
o
• ~ Call Soluble8 Content • y-'-10.6~.44x
r=-.57
Sb m.|8 r,~.77
| 20 I
X
10
|
X
•
X
@l
0
10
•
•
Jc
•
20
30
40
50
I 70
60
Percent
Fro. 2. Relationships of the rates of cell walls digestion to lignin:cellulose ratios and cell solubles contents. When the rye sample was deleted and correlations recalculated (Fig. 3), lignin:aciddetergent fiber (r : 4-.76, P ~ .05), ]ignin: cellulose (r ---- +.78, P ~ .05), and cell solubles (r ------5.98, P ( . 0 1 ) all have positive and significant correlations. This might be interpreted as indicating that lignin has a positive influence on rate of digestion in spite of the well established negative relationship of ligni-
fication on extent of digestion. A more plausible explanation, though, in view of other published data (3, 5, 7, 12, 16) as well as data in this paper, is that alfalfa cell walls digest more rapidly than grass cell walls at maturities for which these forages are harvested for hay. Nevertheless, forages with the higher cell solubles contents digested most rapidly in vitro. Cell solubles may be contributing directly to
Invitro Rate of Cell Walls Digestion vs. Cell Solubles Contents and
Lignin:
Cellulose R a t i o s - S u m m e r Forages 40
30 .0 -
x - Lignin:Cellulose Y= 5.35~ .12x Sb= .10 r= +.78
e-Cell Solubles Content Y=-.20÷.18x Sb= .02 r= ~.98
e o'J ut
m
10
g
!0
FIG. 3. Relationships of lignin:cellulose ratios and cell solubles contents to the rates of cell walls digestion of summer forages. JOURNAL OF DAIRY SCIEI~'CE V 0 L
54 w NO. 1
FORAGE
¢q
r-~
+l 41 +l 41 O9
.
.
.
.
+~ 41 +1 41
+1 +1 +1 +l
+l 41 +1 41 ~
ke~
~
u'3
41 +1 ~1 41
41 41 41 +1
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o
r~
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75
WALLS
faster rates of cell wall digestion. To test this hypothesis, both whole hay dry matter and isolated cell walls were fermented simultaneously. Rates of cell wall, acid-detergent fiber, beretcellulose, and cellulose digestion were determined on whole dry matter and on isolated cell walls for an alfalfa, rye, orehardgrass, and timothy. These results are in Table 5. Generally little, if any difference, was found in rate constants between fermentation of whole dry matter or isolated cell walls. Also, little influence was found on the rate of fermentation o f cell walls, acid-detergent fiber, hemicellulose or cellulose within a particular hay. The larger standard deviation of the rye rate constants and the greater variation between digestion rates of the fiber components was probably the result of selecting suboptimal fermentation intervals for this rapidly digesting forage. These data strongly preclude the possibility of cell solubles per se contributing to more rapid digestion of cell walls in vitro. However, theories may be generated to attempt an explanation of the relationship of faster rates of cell wall digestion for hays as related to greater solubles content. The first may be simply physical as suggested by Donefer (6). Forages of high-cell solubles contents may have less well developed structural components, thus allowing ruminal bacteria greater access to the cell wall for attack. Both surface availability for attack and fragility of the cell wall could be crucial considerations. Another equally plausible and not entirely unrelated explanation may be that degree of crystallinity (1, 14) of structural carbohydrates slay be different for legumes as opposed to grasses as well as different relative to maturity. Donefer (6) found a high positive correlation of cell solubles with nutritive value index, and Yah Soest (15) showed a high negative relationship between cell wall content and voluntary intake. Their results (6, 15) show a direct relationship of cell solubles to increased voluntary intake. Thus, higher solubles content and the associated faster rate of digestion should be additive in diminishing rumen fill. Tomlin (14) was unable to establish a relationship of crystallinity with extent of forage digestion. However, his inability to show this relationship may have resulted from X-ray measurements on improperly prepared samples. Further investigation is required to determine the mechanisms involved which allow more rapid rates of fiber digestion in forages of high cell solubles content. Estimates of indigestible fiber and rate of digestion of potentially digestible fiber in forage should have practical application in predicting J0~RNAL
O F D A I R Y S c I ~ l v ~ ] ~ VOW. 5 4 , NO. 1
76
S M I T H ET AL.
n u t r i t i v e value. E v e n i f p a s s a g e is c o n s t a n t , the sum o f t h e p o t e n t i a l l y indigestible fiber plus t h e r e m a i n i n g digestible sometime a f t e r f e e d i n g s h o u l d r e s u l t in a u s e f u l estimate of r u m e n fill which should be h i g h l y r e l a t e d to v o l u n t a r y c o n s u m p t i o n . Such estimates b a s e d on data in Figure I indicate that approximately 5 0 % t i m o t h y , 3 3 % a l f a l f a , a n d 5 % r y e digestible cell wall would be r e m a i n i n g a f t e r 12 hours.
Acknowledgments The authors acknowledge the laboratory assistance of J o h n O'Connor, William Kramer, and Elaine Bierman in conducting this study.
References (1) Baker, T. I., G. ¥ . Quicke, O. G. Bentley, R. R. Johnson, and A. L. Moxon. 1959. The influence of certain physical properties of purified celluloses and forage cellulose on this digestibility by rumen microorganisms in vitro. J. Animal Sci., 18: 655. (2) Baldwin, R. L., H. L. Lucas, and R. Coburn. 1969. Energetic relationships in the formation and utilization of fermentation endproducts. Third Int. Syrup. Physiol. of Digestion and Metabolism in the Ruminant. Cambridge, England, Aug. 18-23. (3) Baumgardt, B. R., M. W. Taylor, and J. L. Cason. 1962. Evaluation of forages in the laboratory. II. Simplified artificial rumen procedures for obtaining repeatable estimates of forage nutritive value. J. Dairy Sci., 45: 62. (4) Deinum, B., A. J. A. ¥ a n Es, and P. J. Yah Soest. 1968. Climate, nitrogen and grass. II. The influence of light intensity, temperature and nitrogen on in vivo digestibility of grass and the prediction of these effects from some chemical procedures. Netherlands J. Agr. Sci., 16: 217. (5) Demarquilly, C. 1965. Factors affecting the voluntary intake of green forage by sheep. Proc. I X Int. Grassland Congr., p. 877. (6) Donefer, E. 1969. Forage solubility measurements in relation to nutritive value. National Conf. on Forage Quality Evaluation and Utilization, Sept. 2-5, Lincoln, Nebraska.
JOURI~AL OF DAIRY ScIl~l~CE VOL. 54, NO. 1
(7) Donefer, E., E. W. Crampton, a n d L. E. Lloyd. 1960. Prediction of the nutritive value index of a forage from in vitro rumen fermentation data. J. Animal Sci., 19: 545. (8) Gill, S. S., H. R. Conrad, a n d J. W. Hibbs. 1969. Relative rate of in vitro cellulose disappearance as a possible estimator of digestible dry matter intake. J. Dairy Sci., 52 : 1687. (9) Goering, H. K., and P. J. V a n Soest. 1970. Forage fiber analyses (apparatus, reagents, procedures and some application). Agr. handbook No. 379. ARS, USDA. (10) Johnson, R. R., H. W. Scott, A. L. Moxan, and O. G. Bentley. 1959. The digestible cellulose remaining in the feces of sheep fed different roughages as determined by in vitro rumen fermentation. J. Animal Sci., 18: 520. (11) McAnally, R. H. 1942. Digestion of straw by the ruminant. Bioehem. J., 36: 392. (12) Reid, R. L., and G. A. Jung. 1965. Influence of fertilized treatment on the intake, digestibility, and palatability of tall rescue hay. J. Animal Sci., 24: 615. (13) Smith, L. W., H. K. Goering, and C. H. Gordon. 1969. Influence of chemical treatments upon digestibility of r u m i n a n t feces. Proc. Conf. Animal Waste Management, Cornell Univ., Ithaca, N.Y., pp. 88. (14) Tomlin, D. C. 1960. Crystallinity of cellulose and digestibility of fecdstuff cellulose in the bovine tureen. Ph.D. Thesis, Univ. of Florida, University Microfilms 60-5148. (15) Van Soest, P. J. 1965. Symposium on factors influencing the voluntary intake of herbage by ruminants: Voluntary intake in relation to chemical composition and digestibility. J. Animal Sci., 24: 834. (16) Van Soest, P. J. 1968. Chemical estimates of the nutritional value of feeds. Proc. Cornell Nutr. Conf., p. 38. (17) Waldo, D. R., L. W. Smith, and E. L. Cox. 1969. Developments on a theore*ical model of fiber digestion in relation to lignification and unresolved problems. Presented at the Tenth Conference on Rumen Function, Chicago, Illinois, DCRB 69-10. (18) Wilkens, R. J. 1969. The potential digestibility of cellulose in forage and feces. J . Agr. Sci., 73: 57.