Preparation and Digestibility of Alfalfa Leaves and Stems Marked with Gold or Chromium A. Y. Robles, F. A. Martz, R. L.

Belyea and W. P. Warren J Anim Sci 1981. 52:1417-1420.

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PREPARATION AND DIGESTIBILITY OF ALFALFA LEAVES AND STEMS MARKED WITH GOLD OR CHROMIUM 1
A. Y. Robles 2, F. A. Martz 3, R. L. Belyea 3 and W. P. Warren 3
University o f Missouri, Columbia 65211

Summary
Alfalfa leaves and stems were prepared four ways - unprocessed, water-boiled, acid-boiled and neutral detergent fiber (NDF). Au and Cr were each attached to one set of preparations. Less Au or Cr was attached to NDF (2.53 and 12.6 mg/g dry residue, respectively) than to the other preparations (4.6 to 5.3 mg Au/g dry residue and 17.5 to 27.7 mg Cr/g dry residue). Highest recovery of marker was found with NDF; some marker may have attached to cell solubles in the unprocessed, water-boiled and acid-boiled preparations. Marked leaves and stems had lower in vitro digestibilities than unmarked; attachment of Cr reduced in vitro digestion more than did attachment of Au, except in NDF. Marker concentrations appeared adequate for detection in ingesta or feces when added to the diet in small amounts. (Key Words: Gold, Chromium, Markers, Alfalfa, Forages.)

I ntroduction
Dietary markers can be used for determining absorption, secretion, passage rate, retention time and digestibility (Kotb and Luckey, 1972; Engelhardt, 1974). In ruminants, the ideal marker should be (1) unabsorbed, (2) noninteractive with gastrointestinal tract or microbial population, (3) similar to and(or) intimately associated with the diets and (4) simple and easy to analyze (Faichney, 1975). Martz et al. (1973) indicated that markers that do not attach to food particles travel through the digestive tract at different rates than markers that are attached. Stained forage particles are

reduced in size during rumination and digestion; this affects precision of recovery. On the other hand, unattached markers like chromic oxide are unsatisfactory for determining flow of digesta in ruminants because they primarily follow the liquid phase (Faichney, 1972). Feeds marked with inert elements that can be analyzed by neutron activation have been used successfully in ruminants for studying digestion, rate of passage and effects of particle size of diet (Ellis, 1968; Martz et al., 1971; Olbrich et aL, 1971). Martz et al. (1973) attached Cr, Ag and Au to plant cell walls and used neutron activation analysis to study forage digestion in ruminants. There are many approaches for marking dietary components; e.g., marking the entire ruminal contents through a cannula, marking a single meal, marking successive meals, et cetera. Marking a meal(s) usually means that some forage similar to that being fed must be prepared with a marker before being fed. Objectives of this experiment were to determine (1) effects of different forage preparations upon marker attachment and (2) effects of the presence of markers upon in vitro digestibility of alfalfa.

Materials and Methods

Alfalfa (Medicago sativa L) hay was separated mechanically into leaves and stems; each was prepared in one of four ways: (1) unprocessed, control; (2) 1.0 g refluxed in 200 ml water for 30 min, water-boiled; (3) 1.0 g refluxed in 200 ml concentrated H2 SO4 for 1 hr, acid-boiled, and (4) neutral detergent fiber (NDF) (Goering and Van Soest, 1970). One set of the preparations was soaked in an Au solution for 72 hr (1.0 g of forage + 12.3 mg of HAuCL4 9 3H20 in 50 ml distilled water) and filtered. Cr was attached to another set of preparations by refluxing for 3 hr 1.0 g of forage with 1.0 g Na2Cr207 9 2 H 2 0 added to 100 ml of distilled H 2 0 ; after reflv Jng, samples

Contribution from Missouri Agr. Exp. Sta. Journal Ser. No. 8630. Approved by the Director. 2Present address: Dairy Training and Research Institute, UPLB, Philippines. 3 Dept. of Dairy Sci.

1417 JOURNAL OF ANIMAL Downloaded from jas.fass.org by on February 3, 2010.SCIENCE, Vol. 52, No. 6, 1981

1418

ROBLES ET AL.

were filtered and rinsed with hot distilled water until filtrates were clear. Marked leaves and stems were dried (100 C for 48 hr) and ground through a 1.0-ram screen ; in vitro dry matter digestibility ( I V D M D ) w a s determined by 48 hr incubation and 15 min neutral detergent extraction (Goering and Van Soest, 1970). Protein was determined as N x 6.25. Concentrations of markers before and after in vitro digestion were determined by neutron activation analysis, for Au, and by atomic absorption spectrophotometry (AAS), for Cr. Marker concentration was expressed as amount of marker remaining on residue after filtering. Means were compared by Least Significant Difference (Snedecor and Cochran, 1967).
Results and Discussion

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Protein and detergent fiber contents of the leaves and stems before processing are listed in table 1. Effectiveness of the mechanical separation technique in separating leaves from stems is evident by the lower detergent fiber and high protein of the leaves. The amount of each marker that attached to leaves and stems is given in table 2. The smallest amounts of Au were attached to NDF. Leaves and stems apparently had similar affinities for the Au marker (4.31 and 4.49 mg/g forage, respectively). Attachment of Cr to leaves and stems was more variable than was that of Au, indicating that preparation affected degree of attachment (table 2). The largest amounts of Cr were attached to the acid-boiled forages; less was attached to NDF and waterboiled preparations. Somewhat more Cr was attached to leaves than to stems, but means

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T A B L E 1. C R U D E PROTEIN AND D E T E R G E N T FIBER CONTENTS a OF A L F A L F A LEAVES A N D STEMS (PERCENTAGE OF DRY MATTER) Component Crude protein Neutral detergent fiber Acid detergent fiber Hemicellulose Cellulose Lignin b Leaves 21 47 36 11 24 12 Stems 13 64 52 12 34 18

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PREPARATION OF GOLD- AND CHROMIUM-MARKED FORAGES

1419

TABLE 3. IN VITRO DRY MATTER DIGESTIBILITY OF ALFALFA LEAVES AND STEMS WITH AND WITHOUT ATTACHMENT OF GOLD AND CHROMIUM No marker Treatment Leaf Stem Leaf % Unprocessed Water boiled Acid treated Neutral detergent fiber 71.2 56.5 47.2 24.8 57.1 45.6 32.9 18.8 53.2 45.5 30.5 15.8 42.9 39.0 15.8 10.6 32.8 J3.7 10.8 34.7 27.9 26.4 11.3 22.7 Au marker Stem Leaf Cr marker Stem

were not statistically different. Although there was variability in the extent to which Au and Cr were attached to unprocessed, water-boiled and acid-boiled preparations, attachment of each marker to NDF was much lower than that of other preparations. The reason for this phenomenon was not obvious, but, apparently, there were limited attachment sites in the NDF compared to the unprocessed, water-boiled and acid-boiled preparations. We determined the IVDMD of leaves and stems with and without markers attached to find out whether the markers affected digestibility (table 3). Among the unmarked forages, digestibility was lowest for NDF and highest for unprocessed preparations. This difference probably was due to complete extraction of solubles in the NDF preparation, which in effect concentrated fiber and reduced dry matter digestibility. Unprocessed leaves and stems contained solubles and had the highest IVDMD. IVDMD was lower for leaves and stems marked with Au than for unmarked leaves and stems; except for NDF, preparations marked with Cr had lower IVDMD than either Au-marked or unmarked

preparations. For reasons unknown, NDF marked with Cr had higher IVDMD than Aumarked or unmarked NDF. Within the two markers, IVDMD was depressed the most in acid-boiled and the least in water-boiled preparations. The decrease in IVDMD raises questions about the extent to which digestion and passage characteristics are affected in marked forages. The IVDMD o f fescue forages marked with Cr was considerably lower than that of fescue marked with Ce or of unmarked fescue (M. F. Weiss, u n p u b l i s h e d data); those marked with Cr also had longer retention times and slower rates of passage than Ce-marked forages when fed to sheep (Weiss, 1979). However, differences in retention time between Cemarked and Cr-marked forage were predictable and the discrepancies did not seem serious (Weiss, 1979). Amounts of marker remaining on the leaves and stems after 48 hr of in vitro digestion and extraction with neutral detergent are presented in table 4. Across forages and treatments, Cr had a higher recovery (75.3%) than Au (60.4%).

TABLE 4. PERCENTAGE RECOVERY OF GOLD AND CHROMIUM MARKERS AFTER IN VITRO DIGESTION OF ALFALFA LEAVES AND STEMS Au recovery Treatment Unprocessed Water boiled Acid boiled Neutral detergent fiber Mean Alfalfa leaf 48.4 67.5 53.7 71.4 60.2 Alfalfa stem 45.3 67.3 62.4 67.3
60.6

Cr recovery Mean 46.8 c 67.4 ab 58.0 b 69.4 a 60.4 Alfalfa leaf 67.2 66.3 89.2
65.9

Alfalfa stem 72.1 73.6 88.7

79.2

Mean
69.6

72.2

78.4

70.0 89.0 72.6 75.3

a'b'CMeans in same column with different superscripts differ (P<.05).

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1420

ROBLES ET AL. marker and its determination by radioactive analysis. J. Agr. Food Chem. 16:220. Engelhardt, W. V. 1974. Radioactive reference substances in gastro-intestinal studies. P. 112. In Tracer Techniques in Tropical Animal Production. International Atomic Energy Agency, Vienna. Faichney, G. J. 1972. An assessment of chromic oxide as an indigestible marker for digestion studies in sheep. J. Agr. Sci. (Camb.) 79: 493. Faichney, G. J. 1975. The use of markers to partition digestion within the gastro-intestinal tract of ruminants. P. 277. L. W. McDonald and A.C.1. Warner (Ed.). In Digestion and Metabolism in the Ruminant. University of New England Press, Australia. Goering, H. K. and P. J. Van Soest. 1970. Forage fiber analysis. ARS, USDA Agr. Handbook No. 379. Koth, A. R. and T. D. Luckey. 1972. Markers in nutrition. Nutr. Abstr. Rev. 42:28. Martz, F. A., K. H. Asay, G. W. Leddicotte, G. Krause and L. B. Daniels. 1971. Activation analysis of gold-197 for measuring rate of ingesta passage in ruminants. J. Dairy Sci. 54:926. Martz, F. A., P. J. Van Soest, J. R. Vogt and E. S. Hilderbrand. 1973. Use of elemental tracers and activation analysis in digestion rate of ingesta flow and food particle tracking studies in cattle. European Assoc. for Anita. Prod. 14:111. Olbrich, S. E., F. A. Martz, J. R. Vogt and E. S. Hilderbrand. 1971. Use of neutron activation analysis in the determination of digestibility with cerium as an inert marker. J. Anim. Sci. 33: 899. Snedecor, G. W. and W. G. Cochran. 1967. Statistical Methods (6th Ed.). Iowa State University Press, Ames. Weiss, M. F. 1979. Intake, digestibility, fiber composition and marker passage of three tall fescue varieties fed ad libitum to sheep. M.S. Thesis. Univ. of Missouri, Columbia.

F o r p r e p a r a t i o n s m a r k e d w i t h Au, b e s t rec o v e r y was o b t a i n e d f r o m N D F (69.4%), w i t h a similar r e c o v e r y f r o m w a t e r - b o i l e d p r e p a r a t i o n s ( 6 7 . 4 % ) ; leaves a n d s t e m s were similar in rec o v e r y o f Au m a r k e r (60.2 a n d 60.4%, respectively). A m o n g t h e p r e p a r a t i o n s m a r k e d w i t h Cr, r e c o v e r y was g r e a t e s t f r o m acid-boiled ( 8 9 . 9 % ) ; recoveries f r o m o t h e r p r e p a r a t i o n s were l o w e r (69.6 to 72.6%). R e c o v e r y of Cr f r o m s t e m s (78.4%) was g r e a t e r t h a n t h a t f r o m leaves (72.2%). L o w e r recoveries o f A u occ u r r e d w i t h u n p r o c e s s e d (46.8%) a n d acidboiled ( 4 8 . 0 % ) p r e p a r a t i o n s t h a n w i t h t h e w a t e r - b o i l e d (67.4%) a n d N D F ( 6 9 . 4 % ) ; this i n d i c a t e d t h a t some Au m a y have a t t a c h e d t o cell solubles a n d ( o r ) p r o t e i n t h a t were r e m o v e d d u r i n g digestion or e x t r a c t i o n in t h e I V D M D procedure. C o n c e n t r a t i o n s o f A u a n d Cr in leaves a n d stems p r e p a r e d a n y o f t h e f o u r ways s h o u l d be s u f f i c i e n t t o p e r m i t precise d e t e c t i o n in ingesta a n d feces (Martz e t al., 1971). Data in t a b l e s 2, 3 a n d 4 suggest t h a t A u has s o m e a d v a n t a g e s over Cr: (1) t h e r e was less variability in a m o u n t a t t a c h e d ; (2) in vitro digestibility was a f f e c t e d less adversely, a n d (3) r e c o v e r y a f t e r in vitro digestion was a d e q u a t e . In a d d i t i o n , Au p r o b ably is less i n h i b i t o r y t o m i c r o f l o r a t h a n Cr, a n d Au m a r k i n g is a s o m e w h a t easier p r o c e d u r e t h a n Cr m a r k i n g . H o w e v e r , large-scale use o f Au m a y n o t b e e c o n o m i c a l , a n d Au is m o r e d i f f i c u l t to m e a s u r e if n e u t r o n a c t i v a t i o n is n o t available.

Litoraturo Citotl Ellis, W. C. 1968. Dysprosium as an indigestible

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