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Artificial Rumen Investigations. II. Correlations between in Vitro and in Vivo Measures of Digestibility and Chemical Components of Forages1, 2
ARTIFICIAL
RUMEN INVESTIGATIONS.
BETWEEN
II.
CORRELATIONS
IN VITRO AND IN VIVO MEASURES
DIGESTIBILITY
OF
AND CHEMICAL COMPONENTS O F F O R A G E S 1, 2
D . M. B O W D E N a .~.X~D D . C. CHURCH Department of Animal Science, Oregon State University, Corvallis
SUMMARY
Correlation coefficients were calculated using information on in vitro and in vivo digestibilities of 39 samples of tall fescue representing two to four cuttings per year of four genotypes harvested over a 4-yr period. Correlations between in vitro dry. matter digestibility and in vivo dry matter digestibility were highly significant. Correlations on a within-year basis were lower for the tall fescue samples which had been held in storage. Highest correlations were given by the samples harvested in 1960 and used for in vitro trials shortly after being dried and ground. Correlations using the pooled information from all fescue samples revealed a high positive relationship between the in vitro digestibilities and dry matter and cellulose with several measures of in vivo digestibility. The procedure of determining in vitro dry matter digestibility used in this study appears to be as accurate as in vitro cellulose digestibility for estimating the in vivo digestibility of forages. Crude protein content of the forages was positively correlated with both in vitro and in vivo measures of forage digestibility. Intraclass correlations revealed that in vitro digestibility measures were ineffective in predicting in vivo digestibility when only genotypes within a cutting were considered. This would indicate that it is important to consider the variation within the materials under study when in vitro methods are to be used for differentiation of nutritive values of forages. Crude protein content of the forages also showed a very low correlation when considered on a withincutting basis.
I n assessing the value of a feedstuff the ultimate objective is to determine how efficiently it will be used by the living animal. A satisfactory procedure for in vitro digestion should be highly correlated with in vivo digestion and/or animal performance. This paper will consider various correlations for some forages as a means of assessing Received for publication March 9, 1962.
the usefulness of a sinlple in vitro procedure for prediction of live animal digestion of forages. E X P E R I M E ~ T T A L PROCEDURE
Information on the rescue samples used in this study and the in vitro procedures used is given by Bowden and Church (5). Digestion trials. Live animal digestion coefficients were determined with sheep for all forages prior to in vitro digestion. Details of digestion trials with the tall fescue samples for ]957 are presented by Schubert et al. (17). The same procedure was used each year for the fescue harvested in that year. After the digestion trial with the frozen forages, samples were composited, dried at 50 C in a vacuum oven at 30 lb vacuum, ground, and stored at room temperature in screw-top jars until used for chemical analyses and in vitro digestions.
I Technicat Paper No. 1515, Oregon Agricultural Experiment Station, Corvallis, Oregon. a The data presented in this paper are taken from a thesis submitted to the Graduate School of Oregon State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy. BPresent address: Experimental Farm, Canada Department of Agriculture, Agassiz, British Columbia. 980
ARTIFICIAL RUS[EN INVESTIGATIONS. II.
981
TABLE 1 Chemical composition of tall rescue genotypcs by years of harvest" Genotype Cut 1957
Crude protein
Crude fiber
Cellulose
1958
1959
1960
]957
1958
1959
1957
1958
1959
1960
215
1 2 3 4
13.6 13.5 9.5 5.7
16.1 13.0
14.5 9.8
12.8 7.8
25.7 23.6 22.0 22.7
24.4 24.4
22.5 23.9
26.3 22.8 22.3 28.4
26.9 25.7
24.2 25.8
27.2 25.6
228
1 2 3 4
14.4 12.8 8.7
18.2 10.8 8.3 10.2
14.6 11.(,)
14.4 6.1
24.8 21.8 21.4
21.7 23.1 23.3 21.7
20.9 23.3
25.3 22.0 21.5
22.8 26.1 25.7 24.5
23.4 25.4
24.4 28.0
230
1 2
12.7 13.4
12.7 8.9
13.8 6.5
21.5 21.3
21.6 21.9
22.6 23.0
24.1 22.7
25.4 27.2
232
1 2 3 4
13.5 10.2 9.2
13,2 8.4
12.3 7.2
21.6 22.9 22.4
22.4 24.1
23.8 24.3 23.9
24.4 25.1
25.4 24.5
11,6 12.4 7,8 5.9
11.1
24.0
22.9 25.4 20.] 25.3
23.1 24.4 20.4 25.4
27.9
Information on crude protein and crude fiber for forages harvested in 1957, published by Schubert et al. (17). For other years information was supplied by Dr. P. I-I. Weswig, Department of Agricultural C:hemistry. RESULTS AND DISCUSSION The chemical composition and in vivo digestion coefficients of all rescue samples used are p r e s e n t e d in Tables 1 and 2, respectively. Correlations between in vitro a~zd in rico digestibility. Correlation coefficients were calculated s e p a r a t e l y f o r the f o r a g e s harvested in each y e a r and f r o m the pooled data f r o m 4 y r (Table 3). M e a n s f o r in vitro digestibility o f individual f o r a g e s were calculated f r o m digesti-
bility of f o u r samples of the f o r a g e in s e p a r a t e f e r m e n t a t i o n bottles. Correlations involving in vitro d r y m a t t e r digestibility did not v a r y g r e a t l y f r o m y e a r to year, but those involving in vitro cellulose digestibility vm3" markedly. Correlations f o r 1960 harvested f o r a g e s o f in vitro d r y m a t t e r digestibility with in vivo d r y m a t t e r digestibility ( r = .93), in vitro cellulose digestibility with in vivo dry m a t t e r digestibility (r---- .87), in vivo dry m a t t e r digestibility with tirade p r o -
TABLE 2 I n vivo digestion coefficients of tail rescue genotypes by ).ears of harvest "
Genotype
Dry matter
Crude fiber
Energy
Cellulose
Cut
1957
1958
1959
1960
1957
1958
1959
1957
1960
215
1 2 3 4
71.8 69.8 63.7 59.4
72.7 62.8
74.9 63.5
76.6 65.3
77.1 66.3 65.8 69.4
76.8 73.2
80.7 77.8
72.1 69.6 65.7 63.4
84.0 72.0
228
1 2 3 4
74.0 70.0 65.3
73.8 62.3 60.8 60.8
74.4 70.0
75.8 62.0
78.2 69.6 68.3
77.3 70,3 70.9 68.0
83.4 76.6
74.8 71.1 67.4
83.0 68.5
230
1 2
66.5 66.5
77.7 67.1
79.2 67.6
71.2 72.9
83.8 70.1
77.9 68.6 64.8
75.9 71.2
80.2 73.3
81.9 73,0 74,3
83.9 70.6
232
I
2 3 4
74.4 70.6 69.6 58.6
64.0
76.4 69.0 69.9 75.1
72.9
86.2 68.8 73.6 69.8 70.7 63.0
86.2 78.9
~Information on dry matter, crude fiber, and energy for forages harvested in 1957, published by Schubert et al. (17). For other years information was supplied by Dr. P. H. Weswig, Department of Agricultural Chemistry.
D. M. BOWDEN AND D. c. CHURCH
982
TABLE 3 Gross correlations of in vitro dry matter and cellulose digestibilities with certain chemical components and in vivo digestion coefficients of genotypes of tall fescue Year
Factors correlated "
1957
I n vitro dry matter digestibility with: In vivo dry matter digestibility .89 ~* I n vivo aellulose digestibility I n vivo crude fiber digestibility .26 I n vivo energy digestibility .89 ~~ Crude protein content of forage .77 ~~ Cellulose content of forage --.43 Crude fiber content of forage --.04 I n vitro cellulose digestibility with: I n vivo dry matt er digestibility .31 I n vivo cellulose digestibility I n vlvo crude fiber digestibility .20 Crude protein content of forage .58 Cellulose content of forage .08 Crude fiber content of forage .19 I n vivo dry matter digestibility with : Crude protein content of forage .86 ~ P ~ 0.05. ~ P ~ 0.01. a Number of comparisons: those involving crude fiber.
of harvest
1958
1959
1960
.55
.76 ~
.93 *~ .95 ~
.54
.36
.61 ~ .37 .00 .33
Pooled .73** .30
.33 --.44 -.54
.94 ~~ --.60
.68 ~~ -.~1 ~~ -.09
.70
.87 ~* .89 ~~
.49 ~~
.36 .64 ~ .01 .04
.76 * .78 ~ --.01 --.47
.93 ~ --.37
.71 ~
.71 ~
.85 ~
.4:9** .66 ~* --.03 .03 .79 ~
1957--11; 1958--12; 1959--8; 1960--8; pooled--39, 31 for
tein of the f o r a g e ( r ---- .94), a n d in v i t r o cellulose digestibility with c r u d e p r o t e i n c o n t e n t o f the f o r a g e (r----.93) were consistently l a r g e r t h a n c o r r e s p o n d i n g correlations f o r o t h e r years. The samples h a r v e s t e d in 1960 were used in the artificial r m n e n t r i a l s s h o r t l y a f t e r dl~,ing. These differences m a y indicate some effect of storage on the in v i t r o digestibility of dried a n d g r o u n d forages. L e F e v r e a n d K a m s t r a (11) n o t e d low correlations between in vivo a n d in vitro digestibility in p r a i r i e h a y s stored f o r long periods. C l a r k a n d M o t t (7) observed a decrease in the c o r r e l a t i o n between i n vivo a n d in v i t r o digestibilities w i t h trials r u n in the fall w i t h the same f o r a g e s as in trials the p r e v i o u s s p r i n g . They suggested changes during storage as a n e x p l a n a t i o n . M a g n i t u d e s of the c o r r e l a t i o n coefficients in Table 3 indicate t h a t the in v i t r o f e r m e n t a t i o n p r o c e d u r e used in this s t u d y m a y h a v e some definite v a l u e i n e s t i m a t i n g the r e l a t i v e n u t r i t i v e value of forages. F o r p u r p o s e s of comp a r i s o n of these results with those f r o m o t h e r artificial r u m e n procedures, the correlations presented in p u b l i s h e d r e p o r t s h a v e been s u m m a r ized ( T a b l e 4) f o r those f a c t o r s correlated in the p r e s e n t study. C e r t a i n o t h e r m e a s u r e s of in v i t r o digestibility h a v e been used by o t h e r workers. P i g d e n a n d Bell (12) used a n t h r o n e c a r b o h y d r a t e digestion in v i t r o a n d o b t a i n e d estimates of t o t a l digestible n u t r i e n t s a n d digestible crude p r o t e i n which agreed closely
with those o b t a i n e d f r o m c o n v e n t i o n a l digestion trials with sheep. B a u m g a r d t et al. (3) f o u n d a closer r e l a t i o n s h i p between cellulose digestibility in v i t r o a n d in vivo t h a n f o r e i t h e r total digestible n u t r i e n t s calculated f r o m ant h r o n e c a r b o h y d r s t e i n v i t r o o r in v i t r o dry, m a t t e r digestibility. A s p l u n d et al. (1) f o u n d a close c o r r e l a t i o n between t o t a l volatile f a t t y acids p r o d u c e d in v i t r o a n d d r y m a t t e r digestibility in vivo. D o n e f e r et al. (9) d e m o n s t r a t e d a close c o r r e l a t i o n between 1 2 - h r in v i t r o cellulose digestibility of f o r a g e s a n d t h e i r N u t r i t i v e V a l u e Index, which is based u p o n v o l u n t a r y c o n s u m p t i o n a n d digestible e n e r g y of a forage. C o n s i d e r a t i o n of the results f r o m o t h e r workers would indicate t h a t the simplified a r t i ficial t u r e e n p r o c e d u r e used in this s t u d y is capable of p r o v i d i n g estimates of in vivo digestibility as a c c u r a t e as those f r o m m u c h more complex procedures. This finding adds some s u p p o r t to the conclusion r e a c h e d b y W a l k e r (20), t h a t complexity of in v i t r o trials is n o t a criterion f o r o b t a i n i n g digestibility values which a g r e e closely w i t h in vivo values. One p o i n t to consider in r e g a r d to obtaining r e a s o n a b l e estimates of digestibility is the diet consumed b y the a n i m a l s u p p l y i n g r u m e n liquor f o r the inoculum. W o r k of T a y l o r et al. (19) indicates some difficulties which can arise when a diet differs f r o m t h a t b e i n g digested in vitro. W i t h r u m e n liquor f r o m a w e t h e r on a n a l f a l f a h a y diet they were able to get in
983
ARTIFICIAL RU~IEN INVESTI(4ATIONS. II. vitro cellulose digestibilities for a l f a l f a not significantly different f r o m the digestibilities of cellulose in vivo. Blue g r a m a and sudan grass digested by the stone inoculum, however, gave cellulose digestibilities significantly different f r o m in vivo values. Reid et al. (14) demonstrated with data on 124 forages f r o m seven stations that the relationships between in vitro and in vivo digestibility at the different staions varied with the diet fed to the animal providing the inocula for in vitro digestion. Reid et al. (15) noted differences in cellulose digestibility of mixed pasture forage with inocula f r o m lambs fed a standard grass hay and those fed the mixed pasture forage. E a r l i e r reports by Asplund et al. (1) and Church and Petersen (6) noted a p p a r e n t differences in the ability of tureen liquor f r o m animals on different diets to digest substrates. Despite the failure of Stewart and Schultz (18), Salsbury et aL (16), and Quicke et al. (13) to demon-
strafe large differences in cellulose digestion by inocula f r o m different sources, it seems advisable f o r most accurate estimates to use iI~ocula f r o m an animal which is on a diet of nutritional ingredients as close as possible to that behlg digested. There is an indication that differences between grasses and legumes may be particularly marked in this respect. Correlations between digestibility and chemical co~stituents. Pooled correlations (Table 3) of in vitro dry matter digestibility with crude protein in the forage ( r - = .68), in vitro cellulose digestibility with crude protein content (r = .66), and in vivo dry m a t t e r digestibility with crude protein content (r = .79) are all high, indicating a close relationship between digestibility of a f o r a g e either in vivo or in vitro and its crude protein content. Magnitude of the correlation of in vivo dry matter digestibility with crude protein content of the forage indicates that the crude protein content o f a
TABLE 4 Summary of correlations between in vitro and in vivo digestibilities given in the literature Source of data
Asplund etal. (1) Reid et al. (15)
Clark and Mott (7)
Substrates digested
11 Hays 6 Hays Mixed pasture grasses 11 Dried forages
Factors correlated
Correlations
In vitro, DM digestibility with in vivo : D.5{. digestibility
.71"
D.M. digestibility D.M. digestibility
.82* .98*
D.M. digestibility
.77"
In vitro cellulose dig. with in vivo : Baumgardt
et
at. (3)
3 Alfalfa and 8 grass foragcs 8 Grass forages only
D.M. digestibility Energy digestibility Cellulose digestibility Cellulose digestibility
.81"* .80 *4 .50 .90**
Baumgardt et al. (4)
31 Hays
Digestible energy
.85**
tIershberger et al. (10)
35 Forages of 6 spee.
Cellulose digestibility
.97**
Digestible energy
.92" *
Digestible energy
.97 ~*
Cellulose digestibility
.84 ~
Donefer et al. (8) LeFevre and Kamstra (11) Reid et aI. (14)
Barnett (2) * P % 0.05. ~* P % 0.01.
9 Forages of 5 spec. 16 Rations of various roughage levels 124 Forages from 7 stations 10 Oat siIages and 17 grass silages
D.M. digestibility
Highly significant
Energy digestibility
Highly significant
Crude fiber digestibility
High
D. ~L BOWDEN AND D. C. CHURCI~
984
TABLE 5 Effect of reducing variance among forage samples used for calculation of correlations Factors correlated
Within genotype
In vitro dry matter digestibility with In vivo dry nmtter digestibili~" Crude protein content of forage Cellulose content of forage In vitro cellulose digestibility with: In vivo dry matter digestibility Crude protein content of forage Cellulose content of forage In vivo dry matter digestibility with: Crude protein content of forage Cellulose content of forage
Within cuts b
Single trial ~
.76 ~ .76 ~ --.28
.3-9 .36 --.41
.37 --.43 .18
.81 ~ .84 ~ -.08
--.11 .59 --.09
.51 .21 --.1.5
.94 ~ --.31
--.28 --.51 ~
--.26 .40
P < 0.05. ~ P < 0.01. Correlations calculated from data including two cuts per year of each of three genotypes for 4 yr and using sums of squares and sums of products for within genotypes. b Same data as in ~ shove, but correlations calculated using sums of squares and sums of products for within cuts. ° Correlations calculated from data from a single in vitro trial which included 12 first-cut forages representing four genotypes in each of 3 yr. forage may be as good an indicator of the dry matter digestibility of forage as the in vitro digestibility of dry m a t t e r or cellulose. This agrees with Asplund et al. (1), who found that volatile f a t t y acid yields, dry matter digestibility in vitro, and dry nmtter digestibility in vivo were closely correlated with the crude protein content of hays. Contrary to this result, H e r s h b e r g e r et al. (10) observed that digestible energies of forages were more closely associated with their in vitro cellulose digestibilities than with either their crude protein or crude fiber contents. Correlations in Table 3 do not indicate any appreciable association between digestibility of a f o r s g e and its content of cellulose (except f o r the pooled analysis) or crude fiber. Correlations within classifications. Gross correlations presented in Table 3 indicate the value of in vitro digestibility in differentiating between forages of widely variable digestibility. A more precise measure of the accuracy of in vitro digestibility in determining differences in nutritive values within a group of forages is necessary if its usefulness in selecting forages is to be f u r t h e r considered. To gain some f u r t h e r information on this point certain correlations within forage classifications have been calculated (Table 5). Correlations within genotypes were determined f r o m data including two cuts p e r y e a r of each of three genotypes for 4 yr. These correlations are comparable in magnitude to the gross correlations reported earlier. These high correlations may reflect the large varia-
bility in protein content between cuts of a genotype. The second classification of correlations (Table 5) is that within cuts. These were cMculated from the same data as the within genotypes correlations, but the variability within the classes is less, since the differences ill protein content between genotypes within the same cut are much less than the differences hetween cuts of a given genotype. I t is evident that the correlations have been markedly reduced by classification in this manner, indicating little, if any, relationship between in vitro and in vivo digestibi]ity. The significant correlation of in vitro cellulose digestibility with crude protein content may reflect the low protein content of some of the second-cut forages. These forages ranged from 6.1 to 13.5% crude protein and perhaps at the lower protein levels the function of the eetlulolytic microorganisms is limited. The third set of correlations are those calculated f r o m a single in vitro trial involving 12 ilrst-cut forages representing f o u r genotypes in each of 3 yr. H e r e again, the correlations are low. The correlation between in vitro cellulose digestibility and crude protein content of the forage is markedly lower thau the same correlation mentioned above, suggesting that the low protein levels of the second-cut forages were responsible f o r increasing the correlation. I n the ilrst-cut forages crude protein contents ranged from 11.6 to 18.2%. I t appears that these levels would be sufficiently high that proteiu would not be a limiting factor to the microorganisms digesting these forages.
ARTIFICIAL RUMEN INVESTIGATIONS. The reduced degree o f correlation n o t e d would indicate a lack of association between in vitro and in vivo digestibility when the variability between f o r a g e s is low. U n d e r these circumstances the i n h e r e n t errors associated with d e t e r m i n a t i o n s of in vitro a n d in vivo digestibilities would nmke i n t e r p r e t a t i o n o f results difficult, i f n o t impossible. These results t e n d to indicate that in vitro digestibilities a n d crude p r o t e i n contents of f o r a g e s are useful tools only when the differences between f o r a g e s being exanfined are marked. A t least in using the in vitro p r o c e d u r e one should consider the size o f difference to be estimated b e f o r e using the procedure. F u r t h e r results with other f o r a g e s would be helpful in assessing the m a g n i t u d e of differences which can be estimated.
(9)
(10)
(11)
(12)
(13)
REFERElqCES (1) ASPLI_-ND~ J. M., BEI~G, R. T., MCELROY, L. W., ANn PmDEN, W. J. Dry Matter Loss and Volatile F a t t y Acid Production in the Artificial Rumen as Indices of Forage Quality. Canadian J. Animal Sci., 38: 171. 1958. (2) BARNETT, A. J. G. Studies on the Digestibilities of the Cellulose Fraction of Grassland Products. P a r t 1. The Relation Between the Digestibility of Silage Cellulose as Determined in Vitro and Silage Crude Fiber Digestibility Determined by Feeding Trial. J. Agr. Sci., 49: 467. 1957. (3) BALr}.IGAR.DT,B. R., CASON, J. L., AND MARKLEY, R. A. Comparisons of Several Laboratory Methods as Used in Estimating the Nutritive Value of Forages. J. Animal Sci., 17: 1205. 1958. (4) ~BAu~iGARJ)T, B. R., TAYLOI%, iA¢[.W., AND CASON, J. L. A Simplified Artificial R u m e n Procedure for Estimating the Digestible Energy Content of Hays. J. Animal Sci., 18: 15'38. 1959. (5) BOV/DE~ x:, D. M., AND C~IURClt, D. C. Artificial Rumen Investigations. I. Variability of Dry Matter and Cellulose Digestibility and Production of Volatile F a t t y Acids. J. Dairy Sci., 45: 972. 1962. (6) CH~O~, D. C., AN]) P~rFmSEN, R. G. Effect of Several Variables on in Vitro Rumen Fermentation. J. Dairy Sei., 43: 81. 1960. (7) ChaRK, K. W., AND MOT-r, G. D. The Dry Matter Digestion in Vitro of Forage Crops. Canadian J. Plant Sci., 40: 123. 1960. (8) Doh'ErEa, E., CRAMPTON, E. W., A*'D LLOYD, L. E. Prediction of the Nutritive Value
(14)
(15)
(16)
(17)
(]8)
(19)
(90)
n.
985
of a Forage from in Vitro Rumen Fermentation Data. J. Animal Sei., 19:545. 1960. DON-EFER, E., LLOYD, L. E., AND C:KA~IP'I~ON, E. W. Prediction of the Nutritive Value Index of Chopped and Ground Forages by an in Vitro Method. J. Animal Sci., 19: 1304. 1960. ]-IERSHBERDER, T. V., LONG, T. A., HART° SOCK, E. W., AND Swl'l~l'~ R. W. Use of the Artificial Rumen Technique to Estimate the Nutritive Value of Forages. J. Animal Sci., 18: 770. 1959. LEFEvRE, C. F., AND KA~STRA, L. D. A Comparison of Cellulose Digestion in Vitro and in Vivo. J. Animal Sci., 19: 867. 1960. PIGDE.',-, W. J., AN]) BELL, J. M. The Artificial Rumen as a Procedure for Evaluating Forage Quality. J. Animal Sci., 14: 1239. 1955. QUICKE, G. V., BE.~'FLEY, O. G., SCOTT, H. W., A:,-D MOXON, A. L. Cellulose Digestion of Forage Cellulose in Ruminants. J. Animal Sci., 18: 275. 1959. REID, R. L., CLARK, B., WELCH, J. A., JUNO, G. A., .~ND SHELTON, D. C. Relationship of Forage Digestibility and Intake Data to in Vitro and in Vivo Fermentation Indices. J. Animal Sci., 19: 1312. 1960. REID, R. L., SH~LTON, D. C., WEbclZ, J. A., AND JUNO, G. A. Pasture Quality as Determined by in Vitro and in Vivo Techniques. J. Animal Sci., 18: 1537. 1959. SALSBURY,R. L., VANDERKOLK, A. L., B.tLTZER, B. V., AND LUF~CKE, R. W. The Rates of Digestion of the Cellulose of Some Plant Fractions by Rumen Microorganisms in Vitro. J. Animal Sci., 17: 293. 1958. SCHUBERT, J. R., CHURCq=I, D. C., COWAN, J. P., :XND OLD,toLD, g. E. Agronomic and Biochemical Evaluation of Genotypes of Tall Fescue (Festuca arundinacea). II. Digestibility, Energy, and Nitrogen Balance Among Three Selections with Sheep. Proc. West. Sec. ASAP, 9 : L V - 1 . 1958. STE%VART, W. E., AN]) SOHULTZ, L. H. I n Vitro "Volatile F a t t y Acid Production from Various Feeds by Bovine Rumen Microorganisms. J. Animal Sci., 17: 737. 1958. TAYLOR, B. G., R]~P, W. H., AND WATKINS, \V. E. An Artificial Rumen Technique versns Conventional Digestion Trials for Determining Digestibility of Blue Grass, Sudan, and Alfalfa Itays. Proc. West. Sec. ASAP, I I : X L V I I I . 1960. W.~-LKER, D. M. The in Vitro Digestion of Roughage Dry Matter. J. Agr. Sci., 53: 192. 1959.
RUMEN INVESTIGATIONS.
BETWEEN
II.
CORRELATIONS
IN VITRO AND IN VIVO MEASURES
DIGESTIBILITY
OF
AND CHEMICAL COMPONENTS O F F O R A G E S 1, 2
D . M. B O W D E N a .~.X~D D . C. CHURCH Department of Animal Science, Oregon State University, Corvallis
SUMMARY
Correlation coefficients were calculated using information on in vitro and in vivo digestibilities of 39 samples of tall fescue representing two to four cuttings per year of four genotypes harvested over a 4-yr period. Correlations between in vitro dry. matter digestibility and in vivo dry matter digestibility were highly significant. Correlations on a within-year basis were lower for the tall fescue samples which had been held in storage. Highest correlations were given by the samples harvested in 1960 and used for in vitro trials shortly after being dried and ground. Correlations using the pooled information from all fescue samples revealed a high positive relationship between the in vitro digestibilities and dry matter and cellulose with several measures of in vivo digestibility. The procedure of determining in vitro dry matter digestibility used in this study appears to be as accurate as in vitro cellulose digestibility for estimating the in vivo digestibility of forages. Crude protein content of the forages was positively correlated with both in vitro and in vivo measures of forage digestibility. Intraclass correlations revealed that in vitro digestibility measures were ineffective in predicting in vivo digestibility when only genotypes within a cutting were considered. This would indicate that it is important to consider the variation within the materials under study when in vitro methods are to be used for differentiation of nutritive values of forages. Crude protein content of the forages also showed a very low correlation when considered on a withincutting basis.
I n assessing the value of a feedstuff the ultimate objective is to determine how efficiently it will be used by the living animal. A satisfactory procedure for in vitro digestion should be highly correlated with in vivo digestion and/or animal performance. This paper will consider various correlations for some forages as a means of assessing Received for publication March 9, 1962.
the usefulness of a sinlple in vitro procedure for prediction of live animal digestion of forages. E X P E R I M E ~ T T A L PROCEDURE
Information on the rescue samples used in this study and the in vitro procedures used is given by Bowden and Church (5). Digestion trials. Live animal digestion coefficients were determined with sheep for all forages prior to in vitro digestion. Details of digestion trials with the tall fescue samples for ]957 are presented by Schubert et al. (17). The same procedure was used each year for the fescue harvested in that year. After the digestion trial with the frozen forages, samples were composited, dried at 50 C in a vacuum oven at 30 lb vacuum, ground, and stored at room temperature in screw-top jars until used for chemical analyses and in vitro digestions.
I Technicat Paper No. 1515, Oregon Agricultural Experiment Station, Corvallis, Oregon. a The data presented in this paper are taken from a thesis submitted to the Graduate School of Oregon State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy. BPresent address: Experimental Farm, Canada Department of Agriculture, Agassiz, British Columbia. 980
ARTIFICIAL RUS[EN INVESTIGATIONS. II.
981
TABLE 1 Chemical composition of tall rescue genotypcs by years of harvest" Genotype Cut 1957
Crude protein
Crude fiber
Cellulose
1958
1959
1960
]957
1958
1959
1957
1958
1959
1960
215
1 2 3 4
13.6 13.5 9.5 5.7
16.1 13.0
14.5 9.8
12.8 7.8
25.7 23.6 22.0 22.7
24.4 24.4
22.5 23.9
26.3 22.8 22.3 28.4
26.9 25.7
24.2 25.8
27.2 25.6
228
1 2 3 4
14.4 12.8 8.7
18.2 10.8 8.3 10.2
14.6 11.(,)
14.4 6.1
24.8 21.8 21.4
21.7 23.1 23.3 21.7
20.9 23.3
25.3 22.0 21.5
22.8 26.1 25.7 24.5
23.4 25.4
24.4 28.0
230
1 2
12.7 13.4
12.7 8.9
13.8 6.5
21.5 21.3
21.6 21.9
22.6 23.0
24.1 22.7
25.4 27.2
232
1 2 3 4
13.5 10.2 9.2
13,2 8.4
12.3 7.2
21.6 22.9 22.4
22.4 24.1
23.8 24.3 23.9
24.4 25.1
25.4 24.5
11,6 12.4 7,8 5.9
11.1
24.0
22.9 25.4 20.] 25.3
23.1 24.4 20.4 25.4
27.9
Information on crude protein and crude fiber for forages harvested in 1957, published by Schubert et al. (17). For other years information was supplied by Dr. P. I-I. Weswig, Department of Agricultural C:hemistry. RESULTS AND DISCUSSION The chemical composition and in vivo digestion coefficients of all rescue samples used are p r e s e n t e d in Tables 1 and 2, respectively. Correlations between in vitro a~zd in rico digestibility. Correlation coefficients were calculated s e p a r a t e l y f o r the f o r a g e s harvested in each y e a r and f r o m the pooled data f r o m 4 y r (Table 3). M e a n s f o r in vitro digestibility o f individual f o r a g e s were calculated f r o m digesti-
bility of f o u r samples of the f o r a g e in s e p a r a t e f e r m e n t a t i o n bottles. Correlations involving in vitro d r y m a t t e r digestibility did not v a r y g r e a t l y f r o m y e a r to year, but those involving in vitro cellulose digestibility vm3" markedly. Correlations f o r 1960 harvested f o r a g e s o f in vitro d r y m a t t e r digestibility with in vivo d r y m a t t e r digestibility ( r = .93), in vitro cellulose digestibility with in vivo dry m a t t e r digestibility (r---- .87), in vivo dry m a t t e r digestibility with tirade p r o -
TABLE 2 I n vivo digestion coefficients of tail rescue genotypes by ).ears of harvest "
Genotype
Dry matter
Crude fiber
Energy
Cellulose
Cut
1957
1958
1959
1960
1957
1958
1959
1957
1960
215
1 2 3 4
71.8 69.8 63.7 59.4
72.7 62.8
74.9 63.5
76.6 65.3
77.1 66.3 65.8 69.4
76.8 73.2
80.7 77.8
72.1 69.6 65.7 63.4
84.0 72.0
228
1 2 3 4
74.0 70.0 65.3
73.8 62.3 60.8 60.8
74.4 70.0
75.8 62.0
78.2 69.6 68.3
77.3 70,3 70.9 68.0
83.4 76.6
74.8 71.1 67.4
83.0 68.5
230
1 2
66.5 66.5
77.7 67.1
79.2 67.6
71.2 72.9
83.8 70.1
77.9 68.6 64.8
75.9 71.2
80.2 73.3
81.9 73,0 74,3
83.9 70.6
232
I
2 3 4
74.4 70.6 69.6 58.6
64.0
76.4 69.0 69.9 75.1
72.9
86.2 68.8 73.6 69.8 70.7 63.0
86.2 78.9
~Information on dry matter, crude fiber, and energy for forages harvested in 1957, published by Schubert et al. (17). For other years information was supplied by Dr. P. H. Weswig, Department of Agricultural Chemistry.
D. M. BOWDEN AND D. c. CHURCH
982
TABLE 3 Gross correlations of in vitro dry matter and cellulose digestibilities with certain chemical components and in vivo digestion coefficients of genotypes of tall fescue Year
Factors correlated "
1957
I n vitro dry matter digestibility with: In vivo dry matter digestibility .89 ~* I n vivo aellulose digestibility I n vivo crude fiber digestibility .26 I n vivo energy digestibility .89 ~~ Crude protein content of forage .77 ~~ Cellulose content of forage --.43 Crude fiber content of forage --.04 I n vitro cellulose digestibility with: I n vivo dry matt er digestibility .31 I n vivo cellulose digestibility I n vlvo crude fiber digestibility .20 Crude protein content of forage .58 Cellulose content of forage .08 Crude fiber content of forage .19 I n vivo dry matter digestibility with : Crude protein content of forage .86 ~ P ~ 0.05. ~ P ~ 0.01. a Number of comparisons: those involving crude fiber.
of harvest
1958
1959
1960
.55
.76 ~
.93 *~ .95 ~
.54
.36
.61 ~ .37 .00 .33
Pooled .73** .30
.33 --.44 -.54
.94 ~~ --.60
.68 ~~ -.~1 ~~ -.09
.70
.87 ~* .89 ~~
.49 ~~
.36 .64 ~ .01 .04
.76 * .78 ~ --.01 --.47
.93 ~ --.37
.71 ~
.71 ~
.85 ~
.4:9** .66 ~* --.03 .03 .79 ~
1957--11; 1958--12; 1959--8; 1960--8; pooled--39, 31 for
tein of the f o r a g e ( r ---- .94), a n d in v i t r o cellulose digestibility with c r u d e p r o t e i n c o n t e n t o f the f o r a g e (r----.93) were consistently l a r g e r t h a n c o r r e s p o n d i n g correlations f o r o t h e r years. The samples h a r v e s t e d in 1960 were used in the artificial r m n e n t r i a l s s h o r t l y a f t e r dl~,ing. These differences m a y indicate some effect of storage on the in v i t r o digestibility of dried a n d g r o u n d forages. L e F e v r e a n d K a m s t r a (11) n o t e d low correlations between in vivo a n d in vitro digestibility in p r a i r i e h a y s stored f o r long periods. C l a r k a n d M o t t (7) observed a decrease in the c o r r e l a t i o n between i n vivo a n d in v i t r o digestibilities w i t h trials r u n in the fall w i t h the same f o r a g e s as in trials the p r e v i o u s s p r i n g . They suggested changes during storage as a n e x p l a n a t i o n . M a g n i t u d e s of the c o r r e l a t i o n coefficients in Table 3 indicate t h a t the in v i t r o f e r m e n t a t i o n p r o c e d u r e used in this s t u d y m a y h a v e some definite v a l u e i n e s t i m a t i n g the r e l a t i v e n u t r i t i v e value of forages. F o r p u r p o s e s of comp a r i s o n of these results with those f r o m o t h e r artificial r u m e n procedures, the correlations presented in p u b l i s h e d r e p o r t s h a v e been s u m m a r ized ( T a b l e 4) f o r those f a c t o r s correlated in the p r e s e n t study. C e r t a i n o t h e r m e a s u r e s of in v i t r o digestibility h a v e been used by o t h e r workers. P i g d e n a n d Bell (12) used a n t h r o n e c a r b o h y d r a t e digestion in v i t r o a n d o b t a i n e d estimates of t o t a l digestible n u t r i e n t s a n d digestible crude p r o t e i n which agreed closely
with those o b t a i n e d f r o m c o n v e n t i o n a l digestion trials with sheep. B a u m g a r d t et al. (3) f o u n d a closer r e l a t i o n s h i p between cellulose digestibility in v i t r o a n d in vivo t h a n f o r e i t h e r total digestible n u t r i e n t s calculated f r o m ant h r o n e c a r b o h y d r s t e i n v i t r o o r in v i t r o dry, m a t t e r digestibility. A s p l u n d et al. (1) f o u n d a close c o r r e l a t i o n between t o t a l volatile f a t t y acids p r o d u c e d in v i t r o a n d d r y m a t t e r digestibility in vivo. D o n e f e r et al. (9) d e m o n s t r a t e d a close c o r r e l a t i o n between 1 2 - h r in v i t r o cellulose digestibility of f o r a g e s a n d t h e i r N u t r i t i v e V a l u e Index, which is based u p o n v o l u n t a r y c o n s u m p t i o n a n d digestible e n e r g y of a forage. C o n s i d e r a t i o n of the results f r o m o t h e r workers would indicate t h a t the simplified a r t i ficial t u r e e n p r o c e d u r e used in this s t u d y is capable of p r o v i d i n g estimates of in vivo digestibility as a c c u r a t e as those f r o m m u c h more complex procedures. This finding adds some s u p p o r t to the conclusion r e a c h e d b y W a l k e r (20), t h a t complexity of in v i t r o trials is n o t a criterion f o r o b t a i n i n g digestibility values which a g r e e closely w i t h in vivo values. One p o i n t to consider in r e g a r d to obtaining r e a s o n a b l e estimates of digestibility is the diet consumed b y the a n i m a l s u p p l y i n g r u m e n liquor f o r the inoculum. W o r k of T a y l o r et al. (19) indicates some difficulties which can arise when a diet differs f r o m t h a t b e i n g digested in vitro. W i t h r u m e n liquor f r o m a w e t h e r on a n a l f a l f a h a y diet they were able to get in
983
ARTIFICIAL RU~IEN INVESTI(4ATIONS. II. vitro cellulose digestibilities for a l f a l f a not significantly different f r o m the digestibilities of cellulose in vivo. Blue g r a m a and sudan grass digested by the stone inoculum, however, gave cellulose digestibilities significantly different f r o m in vivo values. Reid et al. (14) demonstrated with data on 124 forages f r o m seven stations that the relationships between in vitro and in vivo digestibility at the different staions varied with the diet fed to the animal providing the inocula for in vitro digestion. Reid et al. (15) noted differences in cellulose digestibility of mixed pasture forage with inocula f r o m lambs fed a standard grass hay and those fed the mixed pasture forage. E a r l i e r reports by Asplund et al. (1) and Church and Petersen (6) noted a p p a r e n t differences in the ability of tureen liquor f r o m animals on different diets to digest substrates. Despite the failure of Stewart and Schultz (18), Salsbury et aL (16), and Quicke et al. (13) to demon-
strafe large differences in cellulose digestion by inocula f r o m different sources, it seems advisable f o r most accurate estimates to use iI~ocula f r o m an animal which is on a diet of nutritional ingredients as close as possible to that behlg digested. There is an indication that differences between grasses and legumes may be particularly marked in this respect. Correlations between digestibility and chemical co~stituents. Pooled correlations (Table 3) of in vitro dry matter digestibility with crude protein in the forage ( r - = .68), in vitro cellulose digestibility with crude protein content (r = .66), and in vivo dry m a t t e r digestibility with crude protein content (r = .79) are all high, indicating a close relationship between digestibility of a f o r a g e either in vivo or in vitro and its crude protein content. Magnitude of the correlation of in vivo dry matter digestibility with crude protein content of the forage indicates that the crude protein content o f a
TABLE 4 Summary of correlations between in vitro and in vivo digestibilities given in the literature Source of data
Asplund etal. (1) Reid et al. (15)
Clark and Mott (7)
Substrates digested
11 Hays 6 Hays Mixed pasture grasses 11 Dried forages
Factors correlated
Correlations
In vitro, DM digestibility with in vivo : D.5{. digestibility
.71"
D.M. digestibility D.M. digestibility
.82* .98*
D.M. digestibility
.77"
In vitro cellulose dig. with in vivo : Baumgardt
et
at. (3)
3 Alfalfa and 8 grass foragcs 8 Grass forages only
D.M. digestibility Energy digestibility Cellulose digestibility Cellulose digestibility
.81"* .80 *4 .50 .90**
Baumgardt et al. (4)
31 Hays
Digestible energy
.85**
tIershberger et al. (10)
35 Forages of 6 spee.
Cellulose digestibility
.97**
Digestible energy
.92" *
Digestible energy
.97 ~*
Cellulose digestibility
.84 ~
Donefer et al. (8) LeFevre and Kamstra (11) Reid et aI. (14)
Barnett (2) * P % 0.05. ~* P % 0.01.
9 Forages of 5 spec. 16 Rations of various roughage levels 124 Forages from 7 stations 10 Oat siIages and 17 grass silages
D.M. digestibility
Highly significant
Energy digestibility
Highly significant
Crude fiber digestibility
High
D. ~L BOWDEN AND D. C. CHURCI~
984
TABLE 5 Effect of reducing variance among forage samples used for calculation of correlations Factors correlated
Within genotype
In vitro dry matter digestibility with In vivo dry nmtter digestibili~" Crude protein content of forage Cellulose content of forage In vitro cellulose digestibility with: In vivo dry matter digestibility Crude protein content of forage Cellulose content of forage In vivo dry matter digestibility with: Crude protein content of forage Cellulose content of forage
Within cuts b
Single trial ~
.76 ~ .76 ~ --.28
.3-9 .36 --.41
.37 --.43 .18
.81 ~ .84 ~ -.08
--.11 .59 --.09
.51 .21 --.1.5
.94 ~ --.31
--.28 --.51 ~
--.26 .40
P < 0.05. ~ P < 0.01. Correlations calculated from data including two cuts per year of each of three genotypes for 4 yr and using sums of squares and sums of products for within genotypes. b Same data as in ~ shove, but correlations calculated using sums of squares and sums of products for within cuts. ° Correlations calculated from data from a single in vitro trial which included 12 first-cut forages representing four genotypes in each of 3 yr. forage may be as good an indicator of the dry matter digestibility of forage as the in vitro digestibility of dry m a t t e r or cellulose. This agrees with Asplund et al. (1), who found that volatile f a t t y acid yields, dry matter digestibility in vitro, and dry nmtter digestibility in vivo were closely correlated with the crude protein content of hays. Contrary to this result, H e r s h b e r g e r et al. (10) observed that digestible energies of forages were more closely associated with their in vitro cellulose digestibilities than with either their crude protein or crude fiber contents. Correlations in Table 3 do not indicate any appreciable association between digestibility of a f o r s g e and its content of cellulose (except f o r the pooled analysis) or crude fiber. Correlations within classifications. Gross correlations presented in Table 3 indicate the value of in vitro digestibility in differentiating between forages of widely variable digestibility. A more precise measure of the accuracy of in vitro digestibility in determining differences in nutritive values within a group of forages is necessary if its usefulness in selecting forages is to be f u r t h e r considered. To gain some f u r t h e r information on this point certain correlations within forage classifications have been calculated (Table 5). Correlations within genotypes were determined f r o m data including two cuts p e r y e a r of each of three genotypes for 4 yr. These correlations are comparable in magnitude to the gross correlations reported earlier. These high correlations may reflect the large varia-
bility in protein content between cuts of a genotype. The second classification of correlations (Table 5) is that within cuts. These were cMculated from the same data as the within genotypes correlations, but the variability within the classes is less, since the differences ill protein content between genotypes within the same cut are much less than the differences hetween cuts of a given genotype. I t is evident that the correlations have been markedly reduced by classification in this manner, indicating little, if any, relationship between in vitro and in vivo digestibi]ity. The significant correlation of in vitro cellulose digestibility with crude protein content may reflect the low protein content of some of the second-cut forages. These forages ranged from 6.1 to 13.5% crude protein and perhaps at the lower protein levels the function of the eetlulolytic microorganisms is limited. The third set of correlations are those calculated f r o m a single in vitro trial involving 12 ilrst-cut forages representing f o u r genotypes in each of 3 yr. H e r e again, the correlations are low. The correlation between in vitro cellulose digestibility and crude protein content of the forage is markedly lower thau the same correlation mentioned above, suggesting that the low protein levels of the second-cut forages were responsible f o r increasing the correlation. I n the ilrst-cut forages crude protein contents ranged from 11.6 to 18.2%. I t appears that these levels would be sufficiently high that proteiu would not be a limiting factor to the microorganisms digesting these forages.
ARTIFICIAL RUMEN INVESTIGATIONS. The reduced degree o f correlation n o t e d would indicate a lack of association between in vitro and in vivo digestibility when the variability between f o r a g e s is low. U n d e r these circumstances the i n h e r e n t errors associated with d e t e r m i n a t i o n s of in vitro a n d in vivo digestibilities would nmke i n t e r p r e t a t i o n o f results difficult, i f n o t impossible. These results t e n d to indicate that in vitro digestibilities a n d crude p r o t e i n contents of f o r a g e s are useful tools only when the differences between f o r a g e s being exanfined are marked. A t least in using the in vitro p r o c e d u r e one should consider the size o f difference to be estimated b e f o r e using the procedure. F u r t h e r results with other f o r a g e s would be helpful in assessing the m a g n i t u d e of differences which can be estimated.
(9)
(10)
(11)
(12)
(13)
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