Metabolic Inhibitor(s) in Orchardgrass (Dactylis glomerata L.)1

Metabolic Inhibitor(s)in Orchardgrass

(Dactylis glomerata L.)

'

K. S, SIDHU " and W. H. PFANDER.

Department of Animal Husbandry, University of Missouri, Columbia Materials and Methods

Abstract

Several solvents were used to prepare extracts and residues from orchardgrass (Dactylis glomerata L.) previously shown to cause poor animal perfornmnee. Both extracts and residues were tested for the presence of factors affecting cellulose digestion [metabolic inhibitor(s)] with the in vitro rumen fermentation technique developed at this station. Active extracts which caused an 8.5% reduction in the cellulose digestibility of a standard alfalfa were prepared from orchardgrass extracted with hot detergent (0.1% Tween 80), hot acid-detergent (0.1% Tween in 0.1 ~ HC1), and an azeotropic mixture of methanol and acetone. The results indicate the presence of more than one metabolic inhibitor in the orchardgTass used. When a cellulose digestion permitting factor soluble in dilute acid and precipitated at p H 7.0 was removed from orchardgrass, the digestibility of the residue was reduced about 50%.

Preparation of orchardgrass .fractions and extractants.

ttargus (2) fed a number of hays to lambs during a forage evaluation trial. One sample of orchardgrass (Dactylis glomerata L.) resulted in poor growth, death loss, and stiffness of the lambs. The digestibility of the hay was lower than that o£ other lots with similar contents of the proximate constituents and lignin. It was postulated that one or more unidentified compounds were present in the hay which resulted in the inhibition of rumen microorganisms, thereby lowering digestibility. Preliminary tests revealed that some direct extracts of orehardgrass inhibited cellulose digestion in the in vitro rumen fermentation teehnique (3). Investigations were initiated to find suitable solvents for extracting the metabolic inhibitor(s) from orchardgrass. Results obtained with different orchardgrass fractions prepared directly and in series are reported in this paper. Received for publication November 20, 1967. Contribution from the Missouri Agricultural Experiment Station. Journal Series no. 5301. Approved by the Director. 2 Present address: Department of Animal Science, Oklahonm State University, Stillwater.

Ground orchardgrass, the extraetants, the extracts, and residues were prepared by the procedures described by Sidhu et al. (3). The solvents are referred to as extractants. The direct or in series extracts were designated by a fraction name followed by the letter E. These were : Direct Extract 1-E. Prepared by extracting orchardgrass with a boiling solution of 0.1% Tween 80~ [polyoxyethylene- (20)-s o r b i t a n monooleate in distilled water]. Direct Extract 2-E. Prepared by extracting archardgrass with a boiling 0.1 ~ HCI solution containing 0.1% Tween 80. The filtrate obtained was neutralized to p H 7.0 with NaI-ICO~. Direct Extract 3-E. Prepared with the same extractant as in 2-E. The mixture of orchardgrass and extractant was allowed to cool and neutralized to p H 7.0 with .NaHCO:~ before filtration. I n series fractions of orchardgrass. The fractionation scheme is illustrated in Fig~are 1. The qualitative and quantitative methods used for the direct fractions were employed. To distinguish in series fractions from direct fractions, the names of the former are preceded by the letter A. A f t er the extracts were filtered, they were reduced in volume under vacuum on a water bath and the final volume measured. In the experiments to be described, the 1X level of extract represents the amount of a given extract obtained from 700 mg of orchardgrass. Acid extracts and extractants were neutralized to pill 7.0 and all extracts stored in a refrigerator until used. The residues obtained in the preparation of extracts were dried in a vacuum evaporator at 65 C, weighed, and stored in the sealed jars. Each residue is designated by a fraction name followed by the letter R. The effect of adding 1X or 2X levels of extracts and extractants on cellulose digestibility of alfalfa was studied. The cellulose digestibility of the several residues was determined and compared to the cellulose digestibility of the ground orchardgrass before extrac-

1042

Obtained from Nutritional Bioehemicals Corporation, Cleveland, Ohio.

ORCHARDGRASS

Results

ORCHAROGRASS I Extract With Hot 0.1% Tween 80 I

I

I

Hot Detergent Extract A-1-E

I

1043

INHIBITORS

Residue I

Residue I Extract With Methanol-Acetone (Azeot~opic)

!

Residue A-1-R

Z

I

Methanol-Acetone (Azeotropic) Residue Extract I ! I A-2-E Residue Residue i A-2-R Extract with Ethyl Ether I

I

I

Ethyl Ether Extract Residue A-3-E A-3-R FIG. 1. Scheme of preparation of in series fra,etions of orehardgrass. tion. Cellulose digestibility was d e t e r m i n e d b y the i n v i t r o t u r e e n f e r m e n t a t i o n technique developed a t this s t a t i o n (3). A s t a n d a r d alf a l f a (cellulose digestibility 4 5 % ) was i n c l u d e d i n each a s s a y set. Digestibility values of a n y day's r u n were corrected to the s t a n d a r d value. Cellulose was d e t e r m i n e d b y a modification of the method of C r a m p t o n a n d M a y n a r d (1, 3). C o m p a r i s o n s of t r e a t m e n t s w i t h the control were m a d e b y the least significant difference method (4).

The effect of a d d i n g two levels ( I X a n d 2 X ) of direct e x t r a c t s o£ o r c h a r d g r a s s on the cellulose digestibility of a l f a l f a a n d the cellulose digestibility of the residues are shown in Table 1. B o t h I X a n d 2 X levels of E x t r a c t 1-E dep r e s s e d ( P < 0.01) cellulose digestibility of alfalfa, whereas the 2 X level of hot d e t e r g e n t ext r a c t a n t e n h a n c e d ( P < 0.01) cellulose digestibility of a l f a l f a . Cellulose digestibility of Residue 1-R was n o t statistically different f r o m t h a t of orchardgrass. B o t h levels of E x t r a c t 2-E dep r e s s e d ( P < 0.01) cellulose digestibility of a l f a l f a , whereas the 2 X level of a c i d - d e t e r g e n t e x t r a c t a n t e n h a n c e d ( P < 0.05) cellulose digestibility of a l f a l f a . Cellulose digestibility of Residue 2-R was m a r k e d l y lower ( P < 0.01) t h a n t h a t of o r c h a r d g r a s s . Cellulose digestibili t y of a l f a l f a was depressed ( P < 0,01) b y the 1 X level of E x t r a c t 3-E a n d m a r k e d l y i n h i b i t e d ( P < 0.01) b y the 2 X level. B o t h levels of a c i d - d e t e r g e n t e x t r a c t a n t e n h a n c e d ( P < 0.01) cellulose digestibility of a l f a l f a . The digestibility of cellulose of Residue 3-R was h i g h e r ( P < 0.01) t h a n t h a t of o r c h a r d grass. The effect of a d d i n g in series extracts on cellulose digestibility of a l f a l f a , a n d the cellulose digestibility of t h e residues are shown in Table 2. B o t h levels of h o t d e t e r g e n t e x t r a c t ( A - l - E ) depressed ( P < 0.01) cellulose digestibility of

TABLE 1. Effect of direct extracts of orchardgrass on cellulose digestibility of alfalfa, and the cellulose digestibility of residues2 Cellulose digestibility %b Extract Fraction no.

1 2 3

Name

Hot detergent Hot aciddetergent ~ Hot aciddetergent h

Alfalfa ¢ Level

1X e 2X ]X 2X IX 2X

+ Extract

42.0 41.6 41.7 41.3 40.9 29.7

-4- 1.2 ~~ ~ 1.2 ~ ~ 0.9 ~ _+ 0.1 ~ _+ 1.4 ~* _+ 1.5 ~

+ Extractant

47.2 48.7 46.2 47.7 51.1 50.2

___ 0.4 _+ 0.3 ~ ___3.0 +_ 0.3 ~ -4- 1.0 *~: _ 0.1 '~;¢

i % s i d u e '~

30.1 ± 0.9 16.4 ___ 1.1 ~ 35.8 ~ 1.2 ~

Significant at the .05 level of probability. ~ Significant at the .01 level of probability. " I n vitro. b Mean of cellulose digestibility values in three reaction tubes. c Average cellulose digestibility of alfalfa in control reaction tubes was 45.4%. a Cellulose digestibility of orchardgrass was 31.7 -4- 1.7. X equivalent to 700 rag. Standard deviation. g Neutralized to p t I 7.0 after filtration. ~' Neutralized to p i t 7.0 before filtration. L.S.D. extracts and extraetants vs. control (.05) = 2.3. L.S.D. extracts and extraeta~ts vs. control (.01) = 3.0. L.S.D. residues vs. orchardgrass (.05) = 2.5. L.S.D. residues vs. orchardgrass (.01) = 3.4. J-. DAI]~Y SCIENCE VOh. 51, NO. 7

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SIDHU AND PFANDEl~

TABLE 2. Effect of in series extracts of orchardgrass on cellulose digestibility of alfalfa, and the cellulose digestibility of residues.~

Cellulose digestibility %b Fraction no. A-] A-2 A-3

Extract Name Hot detergent Methanolacetone (azeotropic) Ethyl ether

Alfalfa ¢ Level 1X ~

÷ Extract

2X

39.5 35.4 42.4 40.3

1X 2X

46.4 ± 0.5 48.4 -~ 2.1 ~

2X

1X

+ 1.7 ~~ ___4.4 ~ ___ 2.0 ~ -4- 0.2 ~

÷ Extractant

Residue a

48.6 g'~ 45.1 ___ 1.2 45.4 ~ 2.1 45.4 ± 2.8

29.8g

46.8 ± 0.1 48.1 +__ 1.8 ~

27.4 +_ 0.8 ~

26.6 ± 1.0 ~

Significant at the .05 level of probability. ~ Significant at the .01 level of probability. In vitro. b Mean of cellulose digestibility values in three reaction tubes. ¢ Average cellulose digestibility of a.]falfa in control reactiol~ tubes was 45.4c~. d Cellulose digestibility of orchardgrass was 31.7 ~ 1.7. X equivalent to 700 mg. Standard deviation. Represent only one tube. L.S.D. extracts and extraetants vs. control (.05) = 2.3. L.S.D. extracts and extraetants vs. control (.01) = 3.0. =2.5. L.S.D. residues vs. orehardgrass (.05) =3.4. L.S.D. residues vs. orchardgrass (.01) alfalfa. The 1 X level of e x t r a c t a n t e n h a n c e d ( P < 0.01) cellulose digestibility of alfalfa. Cellulose digestibility of Residue A - 1 - R was not statistically different f r o m t h a t of o r c h a r d gra.ss. Both the levels of m e t h a n o l - a c e t o n e E x t r a c t ( A - 2 - E ) depressed ( P < 0 . 0 1 ) cellulose digestibility of alfafa. N e i t h e r the I X n o r 2 X level of e x t r a c t a n t showed a significant effect on cellulose digestibility of a l f a l f a . Digestibility of cellulose of Residue A - 2 - R was lower ( P < 0.01) t h a n t h a t of o r c h a r d g r a s s . I t was also lower t h a n t h a t of its p a r e n t Residue A - 1 - R (26.6 vs. 29.8). The 2 X level of ether E x t r a c t ( A - 3 - E ) e n h a n c e d ( P < 0 . 0 1 ) cellulose digestibility of alfalfa. The 2 X level of e x t r a c t a n t also e n h a n c e d ( P < 0.05) it. Digestibility of cellulose of Residue A - 3 - R was lower ( P < 0.01) t h a n t h a t of o r c h a r d grass, b u t was a p p r o x i m a t e l y equal to t h a t of its p a r e n t Residue A-2-R. Discussion

The lower cellulose digestibility of a l f a l f a a f t e r the a d d i t i o n of E x t r a c t s 1-E to 3-E ( T a b l e 1), A - 1 - E a n d A - 2 - E ( T a b l e 2), shows t h a t the metabolic i n h i b i t o r ( s ) p r e s e n t in o r c h a r d g r a s s is e x t r a c t a b l e with hot detergent, hot a c i d - d e t e r g e n t , a n d m e t h a n o l - a c e t o n e (azeotropic) solutions. The results o b t a i n e d with hot d e t e r g e n t E x t r a c t s I - E a n d A - 1 - E were in a g r e e m e n t with the previously r e p o r t e d results (3). The s t i n m l a t o r y effect of E x t r a c t A - 3 - E ( T a b l e 2) is similar to the r e s p o n s e f r o m the e x t r a c t a n t a n d m a y be due to the s e p a r a t i o n s J. DAIRY SCIE=qCE ~OL. 51, NO. 7

of lipid f r a c t i o n s f r o m cellulose b y ethyl ether, r e s u l t i n g in a more effective a t t a c k on cellulose b y r u m e n microorganisms. The absence of a n y d e p r e s s i n g effect on cellulose digestion b y the e x p e r i m e n t a l levels of a n y e x t r a c t a n t used indicates t h a t the depressing activity in various extracts c a n n o t be att r i b u t e d to e x t r a c t a n t s themselves. E x r a c t a n t s c o n t a i n i n g d e t e r g e n t h a d a s t i n m l a t i n g effect on cellulose digestion of a l f a l f a . This finding is in agTeement with the results r e p o r t e d earlier (3). I t m a y be due either to a n effect of the d e t e r g e n t on m i c r o o r g a n i s m s or to a n i n d i r e c t effect because cellulose was m a d e more available f o r the microbial attack. The d e p r e s s i n g effect of e x t r a c t s on cellulose digestion is n o t a t t r i b u t e d to soluble s u g a r s in the e x t r a c t s because both cold w a t e r a n d steam extracts of o r c h a r d g r a s s n o t only failed to s h o w n a n y d e p r e s s i n g effect b u t t e n d e d to i m p r o v e cellulose digestion of a l f a l f a (3). The presence of activity in series E x t r a c t s A - 1 - E a n d A-2-E suggests th~,t more t h a n one metabolic i n h i b i t o r is p r e s e n t in o r c h a r d g r a s s . A similar stepwise e x t r a c t i o n p r o c e d u r e could aid in s e p a r a t i o n a n d isolation of different inhibitor(s). The m a r k e d l y reduced cellulose digestion of Residue 2-R ( T a b l e 1) m a y be a t t r i b u t e d to the p r i o r e x t r a c t i o n of some cellulose digestion p e r m i t t i n g factor. The h i g h e r cellulose digestibility of Residue 3-R ( T a b l e 1) as comp a r e d to o r e h a r d g r a s s indicates t h a t either the i n h i b i t o r ( s ) is r e m o v e d or the digestion per-

ORCttARDGRASS INI-IIBITORS mitring factor is precipitated back to the residue on neutralization to pH 7.0. Alternatively, both events could occur sinmltaneously. The marked inhibition of cellulose digestibility of alfalfa by the 2X level of Extract 3-E (Table 1) lends support to the view that both the cellulose digestion permitting and the inhibiting factor(s) are soluble in hot acid-detergent, but the former is precipitated at p H 7.0. The low cellulose digestibility of Residue A-2-R (Table 2) agrees with previously reported findings (3). I t may be due to the partial extraction of some cellulose digestion aiding factor along with the cellulose digestion inhibiting factor(s). As Extract A-2-E (Table 2) showed a depressing effect on the cellulose digestion of alfalfa, it appears that the cellulose digestion inhibiting factor(s) is more completely extracted than is the digestion aiding factor. The lower cellulose digestibility of Residue A-3-R (Table 2) as compared to that of orchardgrass is because it was derived from low cellulose-digestibility Residue A-2-R. Conclusions

Cellulose digestion inhibitor(s) present in a sample of orchardgrass was extracted with hot detergent, hot acid-detergent, and azeotropic

]045

mixture of methanol and acetone. The results of extracts prepared in series indicate the presence of more than one inhibitor. Hot acid-detergent ~lso extracts a cellulose digestion permitting factor from orchardgrass. This factor is precipitated at p H 7.0. Acknowledg ment

The senior author acknowledges appreciation to Dr. R. L. Preston of the University of Missouri for advice with statistical analysis. References (1) Cra~pton, E. W., and L. A. Maynard. 1938. T h e relation of cellulose a n d ]ignin contempt

to the nutritive values of animal feeds. J. Nutrition, 15: 383. (2) ttargus, W. A. 1962. Ovilae bioenergetics and nutritional efficiency with special reference to forage uti]izatioll. Ph.D. dissertation, University of Missouri, Columbia. (3) Sidhu, K. S., W. A. Ha.rgus, and W. H. Pfander. 1967. Metabolic inhibitor(s) in fractions of orchardgrass (Dactylis glomerata L.) detected by in vitro rumell fermentation technique. Proc. Soc. Exptl. Biol. Ned., 124: 1038. (4) Snedecor, G. W. 1965. Sta~istlcal Methods. 5th cd. The Iowa State University Press, Ames.

J. DAIRY SCIENCE ~70r~.51, NO. 7