Aspects of Digestive Physiology in Ruminants: Proceedings of a Satellite Symposium of the 30th International Congress of the International Union of Physiological Sciences, Held at Cornell University, Ithaca, New York, July 21-23, 1986 9781501745713

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Aspects of Digestive Physiology in Ruminants

Aspects of Digestive Physiology in Ruminants Proceedings of a Satellite Symposium of the 30th International Congress of the International Union of Physiological Sciences Held at Cornell University, Ithaca, New York

July 21-23, 1986

EDITED BY

Alan Dobson and Marjorie J. Dobson New York State College ofVeterilUlry Medicine Cornell University

Comstock Publishing Associates A division of Cornell University Press Ithaca and London

Library of Congress Cataloging-in-Publication Data Aspects of digestive physiology in ruminants.

Bibliography. Includes index. I. Digestion-Congresses. 2. Ruminants~Physiology~ Congresses. I. Dobson, A. II. Dobson, Marjorie J. III. International Union of Physiological Sciences.

Congress (30th : 1986 : Vancouver, B.C.) 599.73'504132 QP145.A78 1988 ISBN 0-8104-2027-X (alk. paper)

87-47594

Copyright© 1988 by Cornell University All rights reserved. Except for brief quotations in a review, this book, or parts thereof, must not be reproduced in any form without permission in writing from the publisher. For information, address Cornell University Press, 124 Roberts Place, Ithaca, New York 14850. First published 1988 by Cornell University Press.

The paper in this book is acid-free and meets the guidelines for permanence and durability of the Committee on Production Guidelines for Book Longevity of the Council on Library Resources.

Contents

Contributors Preface

vii Xl

1. Morphophysiological Evolutionary Adaptations of

the Ruminant Digestive System R. R. Hofmann

2. Rumen Dynamics P. J. Van Soest, C. J. Sniffen, and M.S. Allen

21

3. Particle Separation in the Forestomachs of Sheep T. M. Sutherland

43

4. Ecology of Rumen Microorganisms: Energy Use J. B. Russell

74

5. Ecology of Rumen Microorganisms: Protein Use R. J. Wallace

99

6. Afferent Vagal Traffic in Conscious Sheep M. Falempin, A. Marie, and J.P. Rousseau

123

7. Fluid and Ion Transport in the Large Intestine R. A. Argenzio

140

8. Control of Phosphorus Balance in Ruminants D. Scott

156

9. Endocrine and Metabolic Factors in Obesity J. P. McCann and E. N. Bergman

175

Contents

VI

10. Pathophysiology of Diarrhea Due to Escherichia coli

in Neonatal Calves C. L. Guard

203

11. Putative Roles of Peptides in the Genesis and Control of Parasitic Diseases D. A. Titchen and A. M. Reid

217

12. Propionate Metabolism: A New Interpretation W. D. Steinhour and D. E. Bauman

238

13. Components of Basal Energy Expenditure M. Summers, B. W. McBride, and L. P. Milligan

257

14. Modeling Metabolism R. L. Baldwin and J. L. Argyle

287

Poster Titles

299

Index

303

Contributors

Chairmen Comparative Aspects Rumen Contents Physiology Pathophysiology Metabolism

T. R. Houpt M. J. Allison A. Dobson E. N. Bergman J. M. Elliot

Speakers R. A. Argenzio R. L. Baldwin M. Falempin C. L. Guard R. R. Hofmann J.P. McCann L. P. Milligan

J. B. Russell D. Scott W. D. Steinhour T. M. Sutherland D. A. Titchen P. J. Van Soest R. J. Wallace

M.S. Allen J. L. Argyle D. E. Bauman E.-N. Bergman B. W. McBride

A. Marie A.M. Reid J. P. Rousseau C. J. Sniffen M. Summers

Co-Authors

Vll

Contributors

Vlll

Scientific Committee E. N. Bergman A. Dobson (chairman) J. M. Elliot

Addresses M.S. ALLEN, USDFRC, University of Wisconsin, 1925 Linden Drive West, Madison, Wisconsin 53706, USA M. J. ALLISON, National Animal Disease Laboratory, P. 0. Box 70, Ames, Iowa 50010, USA R. A. ARGENZIO, School of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina 27650, USA J. L. ARGYLE, Department of Animal Science, University of California, Davis, California 95616, USA R. L. BALDWIN, Department of Animal Science, University of California, Davis, California 95616, USA D. E. BAUMAN, Department of Animal Science, College of Agriculture and Life Sciences, Cornell University, Ithaca, New York 14853, USA E. N. BERGMAN, Department of Physiology, New York State College of Veterinary Medicine, Cornell University, Ithaca, New York 14853, USA A. DoBSON, Department of Physiology, New York State College of Veterinary Medicine, Cornell University, Ithaca, New York 14853, USA J. M. ELLIOT, Department of Animal Science, College of Agriculture and Life Sciences, Cornell University, Ithaca, New York 14853, USA

M. FALEMPIN, U. A. 308, CNRS, Universite des Sciences et Techniques de Lille, Batiment SN4, 59655 Villeneuve d' Ascq Cedex, France C. L. GUARD, Department of Clinical Sciences, New York State College of Veterinary Medicine, Cornell University, Ithaca, New York 14853, USA R. R. HOFMANN, Department of Comparative Anatomy of Domestic and Wild Animals, Justus Liebig-Universitat Giessen, Frankfurter Strasse 98, D-6300 Giessen, Federal Republic of Germany T. R. HouPT, Physiology Department, New York State College of Veterinary Medicine, Cornell University, Ithaca, New York 14853, USA B. W. McBRIDE, Department of Animal and Poultry Science, University of Guelph, Guelph, Ontario N1G 2W1, Canada J. P. McCANN, Department of Physiology, New York State College of Veterinary Medicine, Cornell University, Ithaca, New York 14853, USA A. MARIE, U. A. 308, CNRS, Universite des Sciences et Techniques de Lille, Batiment SN4, 59655 Villeneuve d' Ascq Cedex, France

Contributors

ix

L. P. MILLIGAN, Department of Animal and Poultry Science, University of Guelph, Guelph, Ontario N1G 2W1, Canada A. M. REID, Department of Veterinary Physiology, University of Sydney, Sydney, New South Wales 2006, Australia J.P. RoussEAU, U. A. 308, CNRS, Universite des Sciences et Techniques de Lille, Batiment SN4, 59655 Villeneuve d'Ascq Cedex, France J. B. RussELL, Agricultural Research Service, USDA, and Department of Animal Science, College of Agriculture and Life Sciences, Cornell University, Ithaca, New York 14853, USA D. ScOTT, Rowett Research Institute, Bucksburn, Aberdeen AB2 9SB, Scotland C. J. SNIFFEN, Department of Animal Science, College of Agriculture and Life Sciences, Cornell University, Ithaca, New York 14853, USA W. D. STEINHOUR, Department of Animal Science, Box 3354, University Station, Laramie, Wyoming 82071, USA M. SuMMERS, Department of Animal and Poultry Science, University of Guelph, Guelph, Ontario N1G 2W1, Canada T. M. SUTHERLAND, Department of Biochemistry, Microbiology, and Nutrition, University of New England, Armidale, New South Wales 2351, Australia D. A. TITCHEN, Department of Veterinary Physiology, University of Sydney, Sydney, New South Wales 2006, Australia P. J. VAN SOEST, Department of Animal Science, College of Agriculture and Life Sciences, Cornell University, Ithaca, New York 14853, USA R. J. WALLACE, Rowett Research Institute, Bucksburn, Aberdeen AB2 9SB, Scotland

Preface

The chapters in this book are invited papers read at a symposium at Cornell University in July 1986. This Plutonian satellite of the 30th International Congress of the International Union of Physiological Sciences held in Vancouver, Canada, was originally billed as Comparative Aspects of Physiology of Digestion in Ruminants. The committee, however, decided to make its scope more general. We tried to complement the program of the 6th International Symposium on Ruminant Physiology, held in Banff in 1984, by treating some topics that deserved a hearing but that the usual constraints on program size had precluded from the Banff gathering. This book thus will supplement the reviews from these quinquennial symposia which have proved standbys to researchers and graduate students interested in the fundamentals of microbiology, physiology, and metabolism in the ruminant gastrointestinal tract. It will also serve as a standard reference for scientists investigating the nutritional and veterinary aspects of these important food animals. This symposium was the sequel to a successful informal meeting on ruminant physiology held in conjunction with the 29th International Congress in Sydney, Australia. Papers from that meeting were published in the Quarterly Journal of Experimental Physiology in 1984. The Cornell symposium was attended by 136 registrants from five continents. Abstracts for posters were submitted and printed in advance of the meeting. The 33 titles of posters exhibited during the two poster sessions at the symposium are listed at the back of the book. The conference received financial support from the following companies: Agway Inc. American Cyanamid Company Eastman Chemicals Division, Eastman Kodak Company xi

xii

Preface

International Minerals and Chemical Corporation Norden Laboratories, Inc. Pfizer Central Research Purina Mills, Inc. SmithKline Animal Health Products and from the following units of Cornell University: Bovine Research Center College of Agriculture and Life Sciences College of Veterinary Medicine Division of Biological Sciences We thank these donors for their generous support, without which the meeting and these proceedings would not have been possible, and we are greatly indebted to the anonymous colleagues throughout the world who refereed the papers. We are also grateful to Sandra Cook for her devoted secretarial help. ALAN DOBSON MARJORIE

Ithaca, New York

J.

DOBSON

Aspects of Digestive Physiology in Ruminants

1 Morphophysiological Evolutionary Adaptations of the Ruminant Digestive System R. R. Hofmann

Not much more than 25 years ago, the first observations in wild ruminants were recorded. These observations suggested that there are ecophysiological differences in the digestion of wild ruminants compared with that in domestic cattle and sheep (Hungate 1959, Short 1963). Apart from observations on European deer species (Martin and Schauder 1938) and a report on three domesticated species, with only little detail on the goat, no systematic comparative studies of the digestive organ anatomy of ruminants were available until much later (Hofmann 1968, 1969, 1973). These studies, initially focused mainly on the complicated quadrilocular stomach of wild bovids, immediately revealed close relationships to functional variations and to differences in feeding habits and forage choice. They also soon suggested evolutionary influences of varying degrees and with physiological consequences. Subsequent comparative studies of Eurasian and North American cervids (Hofmann 1983, 1985) firmly established the three morphophysiological feeding types that were first proposed by Hofmann and Stewart (1972) and are most likely applicable to all of the approximately 150 extant ruminant species. Their digestive systems appear to reflect a gradual evolutionary adaptation that must be linked to the spread of grasses and to increasing pressure to select for improving the digestion of fibrous plant material. Selectivity plays a key role in all ruminant feeding strategies except in the most advanced group, the bovines. Classification of the ruminant feeding types in a flexible and overlapping system-instead of the use of the traditional, descriptive terms grazer and browser-has thus far been verified in 58 ruminant species from four continents: grass and roughage eaters (GR) on one end of the spectrum, concentrate selectors (CS) on the other, and a highly versatile group of intermediate opportunistic mixed feeders (IM) (Fig. 1). Although much attention has recently been paid to the ability of ruminants

RUMINANT

Africa

FEEDING TYPES

----'i'-'m"'p-=a~la'-------

Kirk's dik-dik Guenther's dik-dik suni klipspringer gray duiker red duiker bushbuck giraffe lesser kudu greater kudu gerenuk bongo

Thomson 's gazelle Grant's gazelle

II I I I I I

buffalo Uganda kob bohor reedbuck waterbuck

I oribi

eland

I

I

wildebeest I hartebeest :mountain reedbuck topi/tsessebe I oryx

steenbok

I

North America

white-tailed deer mule deer moose

mountain goat pronghorn antelope elk (wapiti) caribou

bighorn, stone, and dall sheep musk ox bison Pere David's deer

rusa

Asia

musk deer muntjac roe deer

black buck

nilgai

Chinese water deer

gaur

sika

I I I

water buffalo

axis

barasingha zebu sam bar serow

Figure 1. Ruminant species investigated morphologically to date. The farther the baseline of a species extends to the right, the greater i s the species ' ability to digest structural carbohydrates (plant cell wall); the farther to the left, the more selective the species. (Courtesy of R. R. Hofmann .)

2

Anatomical Adaptations of Digestive Tract

3

to digest fiber, that is, plant cell wall (Van Soest 1982, von Engelhardt et al. 1985), our morphological studies support the concept that many of the earlier evolved ruminant species, in an ecological adaptation to their specific habitat, prefer and use most of the plant cell contents of dicotyledonous forage plants that they specifically select. They obviously do so without losing the new benefit of ruminant forestomach fermentation of cell wall carbohydrates, although that process seems, as yet, less efficient than it is in GR. According to Van Soest (1982), most soluble carbohydrates, lipids, and proteins are either lost to the rumina! bacteria or leave the ruminoreticulum (rumina! escape) mainly unused; but this fact was established in two domesticated GR only. Several of my graduate students and I have been able to show, during broad comparative studies, the detailed results of which have been published since 1977 and will be summarized in a new monograph, that adaptive variations of typical ruminant features involve all portions of the digestive system (Hofmann 1983). Most of these variations relate to food quality and composition. Because so many morphological features differ markedly in many ruminant species from those of sheep and cattle, these latter species should no longer be considered as standard reference ruminants in physiology, nutrition, and feeding behavior texts. CS and IM, in their natural habitats, are obviously far from being inferior to the domesticated GR. Their conservation and their use require more interest and differentiation from comparative physiologists. New morphological data and functional deductions from them, or hypotheses based on them, may stimulate physiological verification. The main focus of this chapter will be on pre- and postruminal portions of the digestive tract. Several results and views advanced here are unpublished and provisional in that they are derived from current research work.

Anatomical Observations Preruminal Adaptations

Lips, tongue, lower incisor teeth, and the dental pad at the rostral end of the hard palate act as prehensile organs during food intake. All GR have short lips and a small mouth opening, whereas CS have a large mouth opening, permitting the stripping of twigs or the gnawing of inflorescences and fruit. The lips of GR contain relatively few mucous labial glands; those of IM and CS have increasingly more serous glandular lobules (extreme examples include the muntjac and dik-dik). A cutaneous mucous membrane lines the oral cavity and covers the tongue. Its stratified squamous epithelium is significantly thinner, having fewer cell layers and being less cornified, in CS and in those IM that prefer to select nonfibrous forage for as long as they are able and certainly for a much longer time than do the GR. This microstructure applies in particular to

Figure 2. Ventral aspect of palate, upper lip, and cheek structure. A. White-tailed deer (CS). B. Red deer (IM). C. Fallow deer (IM) . D. Mountain reedbuck (GR). (Courtesy ofR. R. Hofmann.)

4

5

Anatomical Adaptations of Digestive Tract

the long, slender buccal papillae, which enlarge the oral mucosal surface considerably, more conspicuously in CS than in GR (Fig. 2). Schmuck (1986), in her study of 42 ruminant species, found that CS have a denser subepithelial vascular network on their tongue than GRand, on average, a varying epithelial thickness of only 70-90% of that of GR. The GR epithelium is 10-30% thicker because of its heavier protective cornification. We can confirm this for the labial and buccal mucosae and that of the hard palate, some areas of which show very little cornification in CS and some IM. No other herbivore group has a torus linguae as pronounced as the ruminants. Starck (1982) considers its pressing interaction with the hard palate a functional compensation for the incomplete upper jaw dentition. Schmuck (1985) established that torus length, in relation to total lingual length, is the shortest in CS and the longest in GR. On the other hand, CS have, at about 33%, the longest free (mobile) portion of the tongue, GR, at about 28%, the shortest-that is, typical CS such as white-tailed deer, roe, muntjac, or dikdik have a relatively short torus with thin, piercing papillae conicae and a long mobile rostral tip of the tongue (Fig. 3). Among GR, cattle and Pere David's deer have an exceptionally short torus and a relatively long tip. Both are unselective grazers that use their tongues to tear grass bundles off the vegetation. CS, irrespective of size or body weight, have particularly long filiform papillae, but these show considerably less cornification than the filiform papillae in GR. The number of rostral fungiform papillae is, in 14 CS species, on average 188 (SD ± 72); in 13 GR species, 271 (± 72); and in 15 IM species, 340 ( ± 178). IM show a heavy seasonal swing in cornification also on the conical papillae of the torus. There is no detectable relationship between the number of circumvallate papillae on the tongue and feeding type; the number varies individually. There is a clear relationship, however, between the number of taste buds in these papillae and feeding behavior: GR with 14.2 (SD ± 10.0) have significantly more taste buds in their vallate papillae than IM (9.7 ± 4. 3) and CS ( 11.3 ± 5.4). The primarily olfactory selectivity of the two latter groups may explain Figure 3. Comparison of typical CS tongue with typical GR tongue. In CS (top) the tongue is covered by thinner, less cornified epithelium and has a shorter torus as compared with the thicker, cornified epithelium and longer torus, to the fossa linguae, of GR (bottom). The comparative ratios of the sections of each tongue on either side of the fossa linguae (f.l.) are 81:100 in CS and 95:100 in GR. (Courtesy of R. R. Hofmann.)

torus

torus

6

R. R. Hofmann

why grass-eating species need more taste receptors to test the constituents of the grass layer for palatability. The surface relief of the hard palate (see Fig. 2) shows a characteristic pattern in ruminants belonging to the same feeding type: The dental pad is significantly longer, the rugated portion is significantly wider in GR compared with that of IM orCS. CS, which have a short torus linguae, have a relatively shorter smooth portion of their palate than do GR, which have a long torus as counterpart. Generally, CS palates are less cornified, narrower, and better papillated than those of IM and GRand have a more extensively developed submucosal vascular layer with a resilient venous plexus/backflow pump. The salivary glands are considered, after the forestomachs, to be the most reliable morphological feature (Fig. 4) for distinguishing ruminant feeding types (Hofmann 1973, 1985; Kay et al. 1980). In general, CS have much more saliva-producing tissue than GR, particularly proteinaceous serous end pieces. Our examinations thus far of 17 CS species, 19 IM species, and 19 GR species reveal that CS always have very large parotid glands, from 0.18 to 0.25% of body weight, whereas GR have only one fourth or one fifth of that, 0.050.07% of body weight, and IM are, at 0.08-0.15%, truly intermediate. Head topography does not always reveal these differences; in some GR, the parotid is wide but very thin. The seromucous mandibular gland is always proportionally smaller than the parotid gland. It may amount, in GR, to 0.04%; in IM, to 0.08%; and in CS, to more than 0.10% of body weight. All ruminants also have a seromucous sublingual gland. It is proportionally smaller in GR than in CS (Saber and Hofmann 1984), and its serous proportion is higher in the latter. The dorsal, middle, and ventral buccal glands are predominantly mucous in sheep and other GR. The ventral buccal gland exceeds the sublingual in size in most ruminants, but in CS and similarly feeding IM, this gland produces mainly a serous secretion-its serous portion being bigger the more that a ruminant selects for plant cell contents. Hence, in CS, labial, buccal, and parotid saliva is predominantly serous and potentially of greater quantity, whereas in GR, much less serous parotid saliva is added by the proportionally more mucous or mixed secretions. The masticatory apparatus of ruminants is well differentiated according to feeding type (Fig. 5). As was shown by Stockmann (1979), mandibular shape, by its angles, leverage, and surface area, is adapted to food preference. Irrespective of body size, GRand predominantly grass-eating IM (Kiplel 1981) have larger surfaces for masticatory muscle attachment than CS. Both incisors and cheek teeth of CS and related IM are more delicately built than those of GR. With their sharper ridges and tubercles, especially on the lingual side, CS and CS/IM teeth are better suited for puncture crushing than for grinding of fibrous food.

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EATERS (high liber)

GRASS and ROUGHAGE

Figure 4. Diagram of the main salivary glands in ruminants of three morphophysiological feeding types. Parotid weight is given as a percentage of body weight. ~ =parotid gland,~ =mandibular gland ,~ =buccal gland. (Courtesy of R. R. Hofmann.)

~

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Roe deer .·;·..

INTERMEDIATE, • • MIXED FEEDERS

CONCENTRATE SELECTORS

8

R. R. Hofmann

Figure 5. (A) Roe deer (CS, 30 kg) and (B) cattle (GR, 500 kg) heads scaled to similar size to show relative differences in size of the masseter muscle (m) and parotid gland (p), demonstrating adaptation to low- and high-fiber forage. (Courtesy ofR. R. Hofmann.)

Forestomach Adaptations Regarding the ruminant stomach, I refer the reader to our comparative studies (Hofmann 1969, 1973; Hofmann and Schnorr 1982) and to recent review papers (Hofmann 1983, 1985). Of the three feeding types, CS have stomachs with the smallest relative weight and capacity, the least subdivision, and the largest openings, all of which result in a faster passage rate (Hoppe 1984) and shorter retention. The rumen of CS is more evenly papillated, that is, total surface enlargement is greater than in GR, particularly on the dorsal rumina! wall (Fig. 6). The rumina! pillar musculature of CS is weak, and its mucosal coat is papillated and less cornified. In GR, however, the rumina! pillar musculature is thick and powerful; it carries a heavily cornified, protective mucosa with no papillae (Fig. 7). Ingesta are not stratified in the CS

Figure 6. Homologous rumen area samples (center of dorsal wall) from five species belonging to three feeding types. A. Large absorptive papillae in the giraffe (CS), x 1.3. B. Total papillary reduction and cornified epithelium in the Uganda kob (GR), x0.8. C. Cornified rudiments in the oryx (GR, rainy season), x 0.8. D. Long, tongueshaped absorptive papillae in the roe deer (CS, summer), x2. E. Short, partly reduced papillae in the red deer (IM, winter), Xl.7. (From Hofmann and Schnorr 1982, courtesy of Ferdinand Enke Verlag.)

9

Figure 7. Relative differences in muscle fiber development of the internal oblique (longitudinal sections) in ruminants of extremely different feeding types. A. Cranial rumina! pillar of a gray duiker (14 kg), which selects fruit and forbs (CS), X3 .7. B. Cranial rumina! pillar of a nomadic fat-tailed sheep (26 kg), which forages food rich in fiber and with low digestibility (GR), X2.6. C. The caudal rumina! pillar of the duiker in (A), X4.4. D. The caudal rumina! pillar of the sheep in (B), X3.2. Note the reduction of papillae and the cornification in (B) and (D). (Courtesy of R. R. Hofmann.)

Anatomical Adaptations of Digestive Tract

11

rumen; short feeding and rumination periods alternate frequently during several diurnal periods. The omasum of CS is relatively small, or very small, with only a few laminae, but it is equipped with long clawlike papillae. The GR omasum is capacious and extensively subdivided; it offers much larger surfaces on both sides of the numerous omasallaminae of three or four sizes. Their mucosa is covered by a specialized absorptive epithelium that rests on a highly developed subepithelial vascular system, thereby facilitating fast absorption (Schnorr 1971). The reticulum is proportionally much larger in CS. The reticular crests of these animals are lower and less subdivided than those of GR but are heavily studded with sharp cornified papillae. The reticular groove is well developed in all adult ruminants. Its lips are more bulging and more muscular in GR than in CS. The distal commissure, encircling the reticulo-omasal orifice, remains above rumen-fill level in CS and in all other ruminants that have ingested easily digestible, fresh forage that is rich in cell content. The reticulo-omasal orifice, particularly that of leaf-eating species, is more effectively barred by long clawlike papillae and is relatively wider in CS than in GR. Postruminal Adaptations

The abomasum shows considerable mucosal surface enlargement in its wide initial portion, with a varying number of spiral folds. The mucosa containing the gastric glands proper, with neck, chief, and parietal cells, is thick in CS, thinner in IM, and very thin in GR (Fig. 8). This pattern results in a higher proportion of HCl-producing cells per surface area in CS and related IM (Axmacher 1987). The distribution of the functionally different cells within the abomasal mucosa points clearly to adaptive specialization to selective feeding, as Hofmann (1973) and Weyrauch and Saber (1985) have indicated. The small intestine, except for its shorter relative length in CS, does not show obvious structural differences among ruminants of different feeding types. Liver and pancreas, however, which we are still studying in the three feeding types, appear to follow the pattern of morphophysiological separation. In general, CS have a proportionally larger liver than GR; it extends well beyond the costal arch. In CS, total liver tissue amounts to 1.9-2.3% of body weight; in GR, only 1.1-1.3%; in IM, the relationship is intermediate. The large intestine exhibits such a remarkable and regular diversification of its structure, irrespective of taxonomic relationships, that both its functional importance and the evolutionary adaptation to changing food availability must be considerable. The most striking change occurs in (1) the ratio of small intestine length to large intestine length (65-73:27-35 in CS but 80-82:18-20 in GR), and (2) the ratio of the relative capacity of the ruminoreticulum to that of the wide

12

R. R. Hofmann 2.5

'E

..s

-

x concentrate Mlectorl

• lnt.rmedlot. type -- • gra• and fOU9hoge eat.,..

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1: ."'0

0.5

~

0.0

. .. . . lie.

5

10

50

100

500

1000

Weight [kg]

Figure 8. Comparison of abomasal fundic mucosa from 44 species of three ruminant feeding types (n = 84). CS: 13 species, 30 animals; IM: 16 species, 35 animals; GR: 15 species, 19 animals. (From Axmacher and Failing, unpublished.)

cecocolon portion-that is, the ratio of the proximal fermentation chamber to the distal fermentation chamber, which comprises the cecum and the almost evenly widened portion of the ansa proximalis coli. This latter ratio is 1:6-10 in CS, approximately 1:9-14 in IM, and 1:15-30 in GR (Fig. 9). At the same time, the percentage of the entire intestinal length that is spiral colon diminishes from 23-20% in CS (e.g., bushbuck and roe deer; Hoffmann 1977) and 19-16% in IM (e.g., goat and red and fallow deer; von Trott 1983) to 15-12% in GR (e.g., buffalo and mouflon; von Trott 1983). GR have correspondingly fewer colic coils. A number of observations point to considerable seasonal changes (cyclic adaptation) in length and capacity of large intestine structures, especially of the distal fermentation chamber. Such variations occur not only in wild, freeranging ruminants; sheep fed a grain concentrate with leaf silage have always had a distal fermentation chamber relatively larger than that of hay-fed animals, and CS such as roe deer and CS/IM such as chamois that feed on a fiberrich winter maintenance forage have a reduced distal fermentation chamber (Hofmann 1982, 1985). The relatively large distal fermentation chamber of forb- and foliage-selecting CS contains fewer goblet cells and more subepithelial blood vessels than the preceding ileum and the following colon ponions (Lackhoff 1983). In the cecal wall of several IM and GR, we found blood flow-regulating structures between the submucosa and the lamina propria vascular networks.

13

Anatomical Adaptations of Digestive Tract Intestine: evolutionary+ functional adaptation Concentrate selector- LOW FIBER

SS

(cell content)

Large intestine 27-35 %

Small intestine 65 -73 %

-

Volume Rum inareticulum

- DFC

0

1 : 6-10

I.L./B.L.

Intestine 12-15x body length

Grass eater- HIGH FIBER

4 mm occur in several

16

R. R. Hofmann

portions of the tract distal to the omasum and in the feces in summer. The reticulo-omasal orifice is generally wider in CS; its long papillae prevent the passage of larger leaves. It was found to be frequently widened in CS, suggesting the rapid outflow of larger undigested particles (Hofmann 1984) from a small omasum, which is still lacking the complementary absorptive function of the more advanced GR omasum. Forb, shrub, and tree foliage food particles may require more HCl, which is presumably produced in the thicker CS abomasal mucosa. It would be needed for the maceration and breakdown of their hemicellulose bonds (Ulyatt et al. 1975). We believe that such structural carbohydrates make up a much higher proportion in the food and, thus, in the ruminal escape to CS and IM than in the food and ruminal escape of GR. The distal fermentation chamber and, similarly, the spiral colon, therefore, need to be better developed in these ruminants. Hindgut fermentation has been the main digestive strategy in monogastric ungulates, which evolved before the ruminants. Clearly, this alternative method of harboring cellulolytic microbes has not been abruptly dropped during ruminant evolution. Its relatively high level of development in so many CS and IM obviously has important physiological consequences (Boornker 1981, Hoppe et al. 1983). In addition, a relatively longer spiral colon suggests delayed ingesta passage from the distal fermentation chamber and optimal absorption of fermentation products. Stevens et al. (1980) consider the surface area available for absorption of water and Na or short-chain fatty acids in the large intestine a most important factor. CS and several IM have obviously a much larger surface available than do GR. Ludwig (1986) found that on average intestinal crypts of the cecum enlarged the surface by 18-26 times, and those of the spiral colon (ansa centralis) enlarged it by 13-15 times. The highest surface enlargement was found in GR, followed by CS. The slightly lower mucosal values in CS, however, are more than compensated for by the much greater volume of their distal fermentation chamber and the excessive length of their sprial colon. These morphological differences are interpreted as adaptive expressions of a flexible, fractionized fermentation, alternating between proximal (ruminal) and distal (cecocolic) microbial systems. These adaptations must be complementary in CS, whereas the reduced distal fermentation chamber of GR appears to retain emergency safeguard functions for nutritional bottlenecks (e.g., drought, long winter) and is, therefore, a survival adaptation. Finally, the much larger liver of CS is considered to be another important link in a highly effective chain of evolutionary adaptations to toxic, self-protecting food plants on the one hand and, perhaps, to different dimensions in short-chain fatty acid influx and gluconeogenesis on the other. All too few physiological measurements and data have been established for CS or IM, but most of those established differ from data for domesticated species. Although several authors indicate that body weight, rather than diet, is the overruling factor in several digestive physiology

Anatomical Adaptations of Digestive Tract

17

variations (Clemens and Maloiy 1983, Clemens et al. 1983), comparative morphological data from 58 species ranging in body weight from 3 to 1000 kg and the feeding ecology of these species tend to confirm that diet is the primary adaptive factor.

Conclusions The structural design of the ruminant digestive system shows adaptive modifications of the "original" morphophysiological concept. These adaptations appear to be developed to the most favorable degree in each evolutionary stage, resulting in three overlapping ruminant feeding types, CS, IM, and GR, that are distributed over highly varied climatic and vegetational zones of the earth. Each group has retained a wide range of adaptive tolerance, especially in response to photoperiod and seasonal changes in forage quality. During evolution, the ruminant digestive tract appears to have achieved a gradual switch-over from being adapted to the digestion of dicotyledonous plant cell contents, combined with a fractionized fore- and hindgut fermentation of plant cell wall, to a highly efficient digestion of the fiber of low-quality monocotyledonous forage (grass, hay), performed mainly in the ruminoreticulum. As a consequence, several structures along the digestive tract have regressed (e.g., salivary glands, rumina! papillae, abomasal mucosa, liver tissue, distal fermentation chamber, and spiral colon), whereas others have progressed. In advanced ruminants-that is, OR-structures that are retained, even though their functional need appears to have decreased, can be considered as alternative safeguards for survival under extreme conditions of habitat, climate, and season; human management (e.g., feedlots, winter grain feeding), however, has frequently not made best use of the evolutionary adaptations of the ruminant.

Acknowledgments I am grateful to Monika Wehner and J utta Perschbacher for technical help and to my graduate students Heike Axmacher and Jeanette Ludwig for allowing me to use provisional results. Klaus Failing's statistical help is much appreciated. This work is supported by the German Research Community (Deutsche ForschungsGemeinschaft grant HO 273/6-1 +2, "Digestive Tract of Wild Ruminants"). References Axmacher, H. Vergleichend-histologische und morphometrische Untersuchungen an der Mucosa abomasi von 40 Haus- und Wildwiederkiiuem (Ruminantia). Giessen, Germany: Univ. Giessen; 1987. Dissertation.

18

R. R. Hofmann

Boomker, E. A. A study of the digestive processes of the common duiker, Sylvicapra grimmia (L.). Pretoria: Univ. Pretoria; 1981. Thesis. Clemens, E. T.; Maloiy, G. M. 0. Digestive physiology of East African wild ruminants. Comparative Biochemistry and Physiology A 76:319-333; 1983. Clemens, E. T.; Maloiy, G. M. 0.; Sutton, J. D. Molar proportions of volatile fatty acids in the gastrointestinal tract of East African wild ruminants. Comparative Biochemistry and Physiology A 76:217-224; 1983. Cooper, S. M.; Owen-Smith, N. Condensed tannins deter feeding by browsing ruminants in a South African savanna. Oecologia, Berlin 67:142-146; 1985. Hoffmann, R. Morphologische Untersuchungen am Darm des Rehes (Capreolus capreolus L.) einschliesslich der assoziierten Strukturen. Schriftenreihe des AKWJ an der Univ. Giessen. Heft 2. Giessen, Germany: Univ. Giessen; 1977. Hofmann, R. R. Comparisons of rumen and omasum structure in East African game ruminants in relation to their feeding habits. In: Crawford, M. A., ed. Comparative Nutrition of Wild Animals. Symposium of the Zoological Society of London. No. 21. London: Academic Press; 1968:p. 179-194. Hofmann, R. R. Zur Topographie und Morphologie des Wiederkauermagens im Hinblick auf seine Funktion. Zentralblatt fiir Veteriniirmedizin. Beiheft 10. Berlin and Hamburg: Paul Parey Verlag; 1969:p. 1-180. Hofmann, R. R. The Ruminant Stomach: Stomach Structure and Feeding Habits of East African Game Ruminants. East African Monographs in Biology. Vol. 2. Nairobi: East African Literature Bureau, Box 30022; 1973:p. 1-354. Hofmann, R. R. Zyklische Umbauvorgange am Verdauungsapparat des Gamswildes als Ausdruck evolutionarer Anpassung an extreme Lebensraume. Allgemeine Forst Zeitschrift 37:1562-1564; 1982. Hofmann, R. R. Adaptive changes of gastric and intestinal morphology in response to different fibre content in ruminant diets. In: Wallace, G.; Bell, L., eds. Fibre in Human and Animal Nutrition. The Royal Society of New Zealand Bulletin 20:5158; 1983. Hofmann, R. R. Comparative anatomical studies imply adaptive variations of ruminant digestive physiology. Canadian Journal of Animal Science 64(Supplement):203205; 1984. Hofmann, R. R. Digestive physiology of the deer-Their morphophysiological specialisation and adaptation. In: Fennessy, P.; Drew, K., eds. Biology of Deer Production. The Royal Society of New Zealand Bulletin 22:393-407; 1985. Hofmann, R. R.; Schnorr, B. Die funktionelle Morphologie des Wiederkauer-Magens. Stuttgart: Ferdinand Enke Verlag; 1982. Hofmann, R. R.; Stewart, D. R. M. Grazer or browser: A classification based on stomach structure and feeding habits of East African ruminants. Mammalia, Paris 36:226-240; 1972. Hoppe, P. P. Strategies of digestion in African herbivores. In: Gilchrist, F.; Mackie, R., eds. Herbivore Nutrition in the Tropics and Subtropics. Johannesburg: The Science Press; 1984:p. 222-243. Hoppe, P. P.; van Hoven, W.; von Engelhardt, W.; Prins, R. A.; Lankhurst, A.; Gwynne, M. D. Pregastric and caecal fermentation in dikdik (Madoqua kirki) and suni (Nesotragus moschatus). Comparative Biochemistry and Physiology A 75: 517-524; 1983. Hungate, R. E. Microbial fermentation in certain mammals. American Association for the Advancement of Science 130: 1192-1194; 1959. Kay, R. N. B. Comparative studies of food propulsion in ruminants. In: Ooms, L.A. A.; Degryse, A.-D.; Marsboom, R., eds. The Ruminant Stomach: Proceed-

Anatomical Adaptations of Digestive Tract

19

ings of an International Workshop. Vol. 1; 1985 March 17-20; Antwerp, Belgium. Antwerp: Janssen Research Foundation; 1985:p. 159-173. Kay, R. N. B.; von Engelhardt, W.; White, R. G. The digestive physiology of wild ruminants. In: Ruckebush, Y.; Thivend, P., eds. Digestive Physiology and Metabolism in Ruminants. Lancaster, England: MTP Press; 1980:p. 743-761. Kiplel, M. N. Comparative study of the functional anatomy of the masticatory muscles in ruminants of the intermediate feeding type. Nairobi: Univ. Nairobi; 1981. Thesis. Lackhoff, M. Vergleichende histologische und morphometrische Untersuchungen am Darm von Rehwild und Buschschliefer. Giessen, Germany: Univ. Giessen; 1983. Dissertation. Ludwig, J. Vergleichende-histologische und morphometrische Untersuchungen am Dickdarm von Haus- und Wildwiederkauern (Ruminantia). Giessen, Germany: Univ. Giessen; 1986. Dissertation. Martin, P.; Schauder, W. Anatomie der Hauswiederkauer (Band III). Stuttgart: Schickhardt und Ebner; 1938:p. 165. Prins, R. A.; Lankhorst, A.; van Hoven, W. Gastrointestinal fermentation in herbivores and the extent of plant cell wall digestion. In: Gilchrist, F.; Mackie, R., eds. Herbivore Nutrition in the Tropics and Subtropics. Johannesburg: The Science Press; 1984:p. 408-434. Provenza, F. D.; Malechek, J. C. Diet selection by domestic goats in relation to blackbrush twig chemistry. Journal of Applied Ecology 21:831-841; 1984. Renecker, L. A. Quality of forage used by moose. In Annual Report-Wildlife Productivity and Management Programme, Department of Animal Science, University of Alberta; 1985:p. 19-20c. Saber, A. S.; Hofmann, R. R. Comparative anatomical and topographic studies of the salivary glands of red deer, fallow deer, and mouflon-Ruminantia: Cervidae, Bovidae. Gegenbaurs morphologisches Jahrbuch, Leipzig 130:273-286; 1984. Schmuck, U. Die Zunge der Wiederkauer (Vergleichend-anatomische und -histologische Untersuchungen an 42 Haus- und Wildwiederkauer-Arten, Ruminantia SCOPOLI 1777). Schnorr, B. Das BlutgefiiBsystem des Netzmagens und Blattermagens der Ziege. Zentralblatt fi.ir Veterinarmedizin A, Berlin 18:738-766; 1971. Short, H. L. Rumen fermentation and energy relationships in white-tailed deer. Journal of Wildlife Management27:184-195; 1963. Starck, D. Vergleichende Anatomie der Wirbeltiere auf evolutionsbiologischer Grundlage, Band 3. Berlin, Heidelberg, and New York: Springer-Verlag; 1982. Stevens, C. E.; Argenzio, R. A.; Clemens, E. T. Microbial digestion: Rumen versus large intestine. In: Ruckebusch, Y.; Thivend, P., eds. Digestive Physiology and Metabolism in Ruminants. Lancaster, England: MTP Press; 1980:p.685-706. Stockmann, W. Differences in the shape of the mandibles of African Bovidae (Mammalia) in relation to food composition. Zoologisches Jahrbuch fi.ir Systematik 106: 344-373; 1979. Ulyatt, M. J.; Dellow, D. W.; Reid, C. S. W.; Bauchop, T. Structure and function of the large intestine of ruminants. In: McDonald, I. W.; Warner, A C. I., eds. Digestion and Metabolism in the Ruminant. Armidale, Australia: Univ. of New England Publishing Unit; 1975:p. 119-133. Van Soest, P. J. Nutritional Ecology of the Ruminant. Corvallis, Oregon: 0 & B Books; 1982. von Engelhardt, W.; Dellow, W.; Hoeller, H. The potential of ruminants for the utilization of fibrous low-quality diets. Proceedings of the Nutrition Society 44:3743; 1985.

20

R. R. Hofmann

von Trott, F. W. Morphologische Untersuchungen am Darm von Rotwild (Cervus elaphus), Damwild (Cervus dama), und Muffelwild (Ovis ammon musimon) und der assoziierten Strukturen. Giessen, Germany: Univ. Giessen; 1983. Dissertation. Weyrauch, K. D.; Saber, A. S. Fine structure of the fundic stomach epithelium of some East African game ruminants. Anatomischer Anzeiger, Jena 158:437-451; 1985.

2 Rumen Dynamics P. J. Van Soest, C. J. Sniffen, and M. S. Allen

The adaptation of ruminants to pregastric digestion has involved a system of retention of digesta, which is an essential part of the mechanism for maximal extraction of energy in the predigestive fermentation. The retentive process involves the complex structure of the ruminoreticulum and the omasum, which collectively sort the slower digesting fiber from the more easily digestible portions of the diet. Because there is a limit on the size of fiber that can be passed from the ruminoreticulum and omasum, rumination is required toreduce the refractory ingesta to pass that limit. This retention requires some sacrifices in food intake, which becomes more limited on highly fibrous or poorer quality diets, because the coarser ingesta must be retained longer to achieve efficient extraction of energy. Fill, then, becomes a limiting factor in food intake, which might be alleviated by introducing either a more rapidly digesting feed or a more efficient process of disposal of the less digestible lignified portions of the diet that occupy space in the ruminoreticulum. Thus, rumina} function imposes certain problems of energy and protein efficiencies and encourages a sensitivity to diet quality that affects both rumen microbes and host. Interest in this function lies in the ability to manipulate its dynamics and to predict, from the composition and nature of the diet, the animal's responses. There is a need for predictive and integrative information that can be used in models of digestion and animal responses. These models transcend the rumen, dealing also with absorption, lower tract phenomena, and efficiency of use of absorbed nutrients. The rumen model portion of this system, however, is relevant to the energy extraction in the rumen, the contribution of microbial fermentation to protein synthesis, and the extent of "bypass," that is, the escaping of potentially digestible protein to the postruminal digestion. Rumen dynamics have been modeled by Baldwin et al. (1977), Mertens and 21

22

P. J. Van Soest, C. J. Sniffen, and M. S. Allen

Ely (1979), Black et al. (1980), and France et al. (1982). The models are limited in various ways related in part to the lack of information about the processes that dispose of particulate matter and in part to the understanding and interpretation of existing data. There is a considerable divergence between the concepts of Australian models as outlined by Faichney (1986) and the American model being developed collaboratively by Mertens and Cornell associates. This chapter attempts to provide insights on the disagreements and a perspective for future research that might resolve problems. D. R. Mertens, C. J. Sniffen, and J. D. O'Connor (unpublished) have developed a mass action model that addresses many of the weaknesses not dealt with in other published models. The submodel for particulate matter is shown in Fig. 1. The model attempts to account for particle size reduction (Mertens and Ely 1979, Mertens et al. 1984) and allows three particle pools: large, medium, and small. These pools contain both unavailable and available fractions. Availability is defined as the potentially digestible matter in the diet, some of which is liable to digestive escape depending on the competition between speeds of digestion and passage. Availability will be an inverse

PARTICLES

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OIFT

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8]-"--{ Q·

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~~

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Figure 1. Model showing the conceptual degradation and fate of ingested particulate matter. Unavailable portions (U) and available portions (A) are shown in relative proportion. Finer fractions of diet are less lignified and contain less U. All fractions are subject to process H (hydration and microbial attachment). Hydration capacity is greater for larger particles (Van Soest 1982), but the process is slower. Large particles are above the relative size limit for passage. The rumen retains larger particles that are enriched there; the smaller ones are lost in inverse proportion to their size in passage. Large particles can disappear only through rumination and digestion (R and D), whereas medium and small particles disappear by means of rumination, digestion, and passage-passage becoming more important the smaller the particle.

Rumen Dynamics

23

function of lignification; the ratio of lignin to cell wall determines the maximum.extent of digestion of cell wall components (Mertens 1973, Chandler et al. 1980). Factors of particle size, hydration rate, initial and hydrated density, and substrate type will modify the passage process and affect the escape of potentially digestible matter from the rumen (Welch 1982, Hooper et al. 1986). Factors such as salivary flow, rumination, and microbial pools (3 bacterial plus 1 protozoal) are also included in the overall model. Although this model is quite sensitive to diurnal events it lacks two major components: First, it does not take into account the ''mat effect'' on the medium and small particle pool. This model permits too extensive a washout of the small particle pool and an overall overestimation of depression in digestibility of organic matter. The fibrous mat is important in trapping a fraction of these smaller denser particles (Sniffen et al. 1986). Second, the effects of pH on the changes in microbial ecology are underestimated and need to be adjusted. Successful dynamic mass action models for the rumen will require, above all, an understanding of the complexity of the physical rumen (Van Soest 1982, Czerkawski 1986), including rumen motility, osmolarity, particulate hydration, movement of material into the rumination pool, and the mat/liquid interchange. Hydration and waterlogging of the pore space increases the effective density of solid digesta and promotes passage of particles of density greater than one (Hooper et al. 1986). The maximum passage occurred at a density of 1.2 in the studies of desBordes and Welch (1984). Rumination time is increased by consumption of coarse, neutral-detergent fiber (Welch 1982). The modeling of the interchange between liquid and particulate pools has been attempted in studies involving continuous fermentation in vitro (Czerkawski 1986, Jeraci 1984). Submodels are needed that more effectively describe the frequency of feeding and eating, and consequent rumination processes, which include sorting, reduction, and hydration of particles, as well as salivary flow under the various conditions. The data of Robinson (1983) indicate a considerable effect of frequency of feeding upon rumen escape. He compared feedings of one, two, four, and eight times a day. Infrequent feeding tended to induce pulsative flow at the duodenum and to promote passage of potentially digestible matter from the rumen (Robinson 1983). Slowness of feed to hydrate and come into equilibrium with microbes, and rumination could interact with this phenomenon. Another problem is methodological. There is agreement that preformed dietary fine particles have faster passage than do larger insoluble components. They are also more digestible because they are less lignified (Van Soest 1982). Lignified plant materials are more resistant to grinding and shattering than are less lignified tissues; such resistance lends to accumulation of lignin from heavier and coarser matter (Van Soest 1966, 1982). This factor is common

24

P. J. Van Soest, C. J. Sniffen, and M. S. Allen

knowledge and is made use of in the forage dehydration industry in the separation of leaf and stem meals. It is possible to estimate fiber content of forage from the leaf-stem distribution (Pick and Sniffen 1985). Lignin is the major factor reducing digestibility of plant cell walls (Van Soest 1964, 1967). The passage of fines produced by rumination and digestive disintegration is slower than that of the larger particles from which they originated (Fig. 2). This finding is also corroborated by data of Smith ( 1968), Lascano ( 1979), and Smith et al. (1983). The limiting rate appears to be the diminution in size due to rumination and digestion. The passage of fine particles, once formed by rumination, is probably similar to that of those particles of similar size already existing in the feed. Nevertheless, fine particulate pools are treated as entities whereas they are not uniform and contain both particles of preformed feed and daughter products from larger particles that have disparate apparent rates of passage and different composition (Lascano 1979, Van Soest 1982). The difficulty in further developing and applying this concept is the lack of sufficient quantitative data, the obtaining of which is related to the adequacy of particulate markers (see later discussion). Initial work (O'Connor et al. 1984) would suggest that rumen flow is not ideal and needs to be subdivided into more uniform subcomponents. The importance of this concept was partially delineated in a recent experiment (Hooper et al. 1986, Sniffen et al. 1986) in which forages of three particle sizes were fed (long, 1.9 mm, 0.3 mm) twice a day to steers. Mat consistency (Welch 1982), eating, and chewing decreased as the particle size did. Observations of the rumination pattern on the 0.3-mrn forage showed abnormal and ill-defined eating patterns as compared with the other two forages. The particle size and hydrated densities of the floating rumen mat were affected by dietary particle size of the hay. These data emphasize the need for better description of the factors affecting zoned regions in the rumen. The particulate compartments of the rumen need to be characterized according to particle size distribution and density. Unfortunately, too many conclusions have been drawn from studies on the rumen in which the diets were continuously fed, finely chopped, or pelleted and fed at less than ad libitum intake. These conditions promote a more stabilized rumen that is easier to study because they avoid the nonideal mechanics that influence practical situations. Most ruminants exhibit significant diurnal variation in feeding and ruminating behavior (Lofgreen et al. 1957, McDowell1972).

Technical Problems of Measurement Measurement of the factors needed for adequate models describing rumen dynamics is limited by the inadequacy of existing methodology. Two great problems are the measurements of particle sizes and the adequacy of markers.

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300 - 600 -() 150 - 300 75 - 150 -- ·-+

Figure 2. Example of particulate passage in a sheep dosed with sized (300-600 J.Lm) chromium-mordanted cell wall prepared from the timothy forage of the diet. Particles in feces smaller than those dosed arise from rumination and digestion and have slower rates of appearance in feces (Uden 1978). k 1 rates probably reflect rumina) passage and k 2 lower tract passage (Grovum and Williams 1977). (Data from Uden 1978.)

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FECAL FRACTION (fl.m)

26

P. J. Van Soest, C. J. Sniffen, and M. S. Allen

Measurement of Particle Distribution

Particle sizing and physics have a long history. Most of the sizing of feeds has followed the recommendations of the American Society of Agricultural Engineers. Using this methodology, Waldo et al. (1971) applied regression statistics to obtain the median value. These calculations of modulus of fineness assume logarithmic distribution of particle size relative to weight-a hotly discussed topic at the precongress symposium at Banff. Everyone will agree that ideal particles have a logarithmic distribution, but feedstuffs contain nonuniform components, such as leaf and stem fractions of forages, that will form their own overlapping, nonideal distributions (Allen et al. 1984). A subsidiary problem concerns the length-width distribution of the solid particles and the tendency of some forages, particularly grasses, to shatter into needlelike matter. A further concern is whether sieving methods sort particles by length or cross section. Wet sieving may more appropriately reflect rumen factors than the old dry sieving procedure. Van Soest (1975) suggested that wet sieving tended to mat fibers through filtration effects, and, therefore, to measure the length as opposed to the cross section. Further results, shown in Table 1, indicate that wet sieving does have this bias, which has been noted by others (Ehle 1984b). There are considerable differences between feedstuffs; for example, wheat bran, which is flaky and not linear, shows no important difference between the methods, whereas alfalfa and timothy show successively greater increase in median size with wet sieving but not with dry. Grasses tend to shatter into longer particles than do legumes. This selective effect of filtration might be diminished by greater agitation in the wet sieving process and could well be a reason for some of the differences between results of various researchers. Another problem is the underestimation, due to clumping, of very fine particles during the dry-sieving of feces. This clumping can be due to electrostatic attraction after drying. We believe that selective filtration in the rumen is likely a major effect of the rumination and passage processes and that the limit in particle size that can Table 1.

Median particle size of three feeds Particle size (!Lm)

Substrate

Wet

Dry

Difference

Wheat bran Alfalfa Timothy

858 530 645

434 478

895

-37 96 167

Source: Allen (1986). Note: Data ate the result of eight replicated experiments. Median values were determined by fitting gamma distributions (Allen et al. 1984).

Rumen Dynamics

27

be passed is influenced by the dietary distribution in particle size, as originally advanced by Van Soest (1966, 1982) and supported by Smith et al. (1983). Faichney argues that particle size limits are constant and not influenced by feed preparation. The original data of Van Soest ( 1966) indicated an increase of fecal particle size as a result of (1) ingestion of pelleted forage and (2) increased feed intake of a single forage. These observations led to the suggestion that pelleting forage eliminated the mat filtration in the ventral rumen and the omasum, thus allowing coarser feed particles to pass. It is not an uncommon observation that highly productive dairy cattle, when fed adequate forage that will raise milk fat and rumen pH, show a reduced appearance of com kernels in feces, probably due to the increased rumination. The concept presented here is that a limiting particle size for passage is not a constant, but is affected by feeding conditions, particularly by level of intake of feed and grinding of feedstuffs (Van Soest 1966, 1982; Cardoza and Mertens 1986). Welch (1982) has shown reduction in the number of chews per gram of forage when the same feed is fed at higher intake. This observation is consistent with the expectation that more of the larger particles will leave the rumen, having escaped rumination, because of the higher rate of passage associated with higher feed intake. Hofmann (1985) has suggested an "opening of the pipes" concept that might account for the ability of small African ruminants and perhaps other ruminants to cope with low feed quality. The problem expresses the need for more critical data and refined concepts.

Markers The rumen system is characterized by separate kinetics for liquids and solids. For this reason, much attention has been directed toward effective markers that might measure the individual dynamics of liquids and solids. The gastrointestinal behavior of the respective markers has a great deal to do with the integrity ofthe passage measurements, the problems being generally greater in the case of the solid markers. Accepted criteria for adequate markers generally include inertness, with no effects on passage and digestion, an ability to remain with the labeled fraction and not to transfer to other fractions, and to be recoverable (Kotb and Luckey 1972).

Liquid Markers The most common liquid markers in use are polyethylene glycol (PEG) and the EDTA or diethyltriaminepentaacetic acid complexes of chromium, cobalt, and rare earth elements. PEG has a comparatively large molecular weight and

28

P. J. Van Soest, C. J. Sniffen, and M.S. Allen

is a soluble polymer with ether linkages. Surface adsorption to particulate matter should be limited to nonionic forces, and for most feedstuffs this is not a problem. PEG is precipitated by tannins, however, and is thus precluded with tanniniferous forages and grains (Warner 1981). Soluble coordinated complexes of Cr, Co, and rare earth elements are of lower molecular weight than PEG and therefore might have some osmotic effect on liquid passage, if overdosed. EDTA complexes of trivalent elements are monovalent anions (divalent anion in the case of divalent Co) and could conceivably interact with the anion exchange of solid feedstuffs (Warner 1981). The anion exchange of plant materials is very low, although it could be involved in the adsorption of bile acids by fiber as reported in human dietary fiber studies (Story and Kritchevsky 1978). A low adsorption of EDTA complexes to particulate surfaces could induce aberrant behavior at low marker concentrations, but should be obliterated by concentrations that are high relative to the exchange. Small amounts (1-5%) of EDTA complexes are absorbed and excreted in the urine (Uden 1978, Teeter 1981), an effect that can be enhanced by increasing the osmotic pressure of the rumen fluid (Dobson et al. 1976). Faichney (1986) emphasizes the need for correction for absorption and urinary losses to avoid overestimation ofthe passage rate. Increased osmotic pressure of rumen liquid promotes washout and decreases water absorbed across the rumen wall. Greater washout is associated with a higher microbial yield (Harrison et al. 1975) (see later section on rumen efficiency).

Particle Markers The problems of satisfying marker criteria are considerably more difficult with particulate markers than with liquid ones because the requirements of associated movement generally mean coordinated attachment, which necessitates an indelible and indigestible linkage with the particulate matter, thus violating the condition that the marker have no effect upon the substrate. Most of the well-attached particle markers reduce digestibility, probably because they block sites for microbial attachment. Markers that are insufficiently attached will migrate during the digestion process and consequently have no effect on digestibility. There are, thus, two aspects of validation studies: those that examine particle markers for inhibitory effects on digestion and passage and those concerned with migration and separation of the marker from the fraction intended to be labeled. Particle markers may be classified into several categories: first, insoluble, identifiable materials added to feed such as plastic, rubber, and the like; and, second, heavy metal complexes. Whereas plastic particles may disassociate from the feed because of different physical properties, colored or radiopaque plastic markers have been useful in ruminant and nonruminant studies, be-

Rumen Dynamics

29

cause properties such as their specific gravity and size can be closely controlled and the associated behavior determined. Feed stained with dye may have the same physicochemical properties of the particulate fraction of the diets, but the stained particles are difficult, if not impossible, to quantify. Lack of recovery can represent apparent digestion and promotes overestimation of passage rates. (The bias will depend on the mathematical procedure. Different bias arises if fecal (or postruminal) recovery is low, or if the marker is expressed as a proportion of net marker recovered. Rate of disappearance from the rumen is the sum of digestion and passage, so that recoverable indigestible components like lignin will show slower turnover than the digestible feed components with which they are associated.) The use of feed stained with dyes is largely historical. Measurement was based on the proportion of fecal particles observed and was perturbed by low recovery. One has to ask what happened to the dye on digested particles. The current emphasis is on metallic ion derivatives that are more quantifiable. An alternative that has seen limited use because of measurement difficulties is 14C-labeled plant material-plants grown in light chambers or plastic tents in an atmosphere containing labeled C02 (Smith 1968, Alexander et al. 1969, Smith et al. 1983). This technique requires careful separation of undigested feed from digestion products and is essentially limited to indigestible cell wall components. Heavy metal ions such as those of Cr and the rare earth elements can be attached directly to feed. Although quantifiable, they generally cause alterations of the marked substrate. Various metal ions, or their coordination compounds, have been prepared for attachment to feed particles. If such combinations remained indelibly attached, they could become a means of assaying particulate disintegration and rumination effects. Such markers have included forage or feed material treated with ruthenium phenanthroline, Cr mordant, and rare earth adsorption complexes. The practicality of these markers depends on the degree of their attachment (i.e., no movement during digestion) and their ease of measurement. Radioactive isotopes have been popular in some countries, but not highly so in others because of potential radioactive hazards. Neutron activation is a solution, but may have a high sample cost. Cold marker analysis generally means atomic absorption or other specific chemical characterization. Ruthenium Phenanthroline

Ruthenium is added to feed as a coordinated complex that has a high affinity for particulate matter, but it appears to migrate during digestion (Faichney 1986). This behavior, although allowing Ru to be used as a general particle marker, precludes its use for particulate subfractions. Ru is a very expensive element and appears limited to assay as the radioactive species.

P_. J. Van Soest, C. J. Sniffen, and M. S. Allen

30 Chromium Mordant

Mordants are metal complexes used in dying textiles. They were originally devised as a means of increasing fastness of dyes. Cr, iron, and titanium salts are among the compounds used. Feed profiles are mordanted with the objective of forming a permanently attached marker. The trivalent Cr complex of feed organic matter is formed by the reduction of hexavalent complexes of Cr by ascorbic acid. Because Cr can form stable complexes with practically any compound containing free alcohol groups, feed must be free of starch, sugar, and other potentially soluble substances that can react with Cr to avoid producing soluble complexes. The Cr complexes of plant cell wall and protein are highly insoluble and totally stable in the rumen (Uden et al. 1980, Teeter 1981, Colucci 1984). Because of the tightness of the attachment, Cr reduces digestibility of the feed fraction to which it is attached. About 8-10% will reduce digestibility to zero. These higher levels also increase specific gravity, so that the material may not behave in the rumen in a manner comparable to the unattached feed that it is intended to label (Ehle 1984a). This characteristic restricts the use of Cr to lower levels of complexing (ca. 2%) that affect both digestibility and density to a lesser degree. Nevertheless, Cr mordant is the only marker at present, other than natural compounds such as lignin, that has complete integrity relative to permanent attachment. Rare Earth Complexes

Complexes of rare earth elements have been popular markers for some time under the general supposition that all these elements are biologically inert and behave similarly. There are 16 or 17 elements that can be called rare earths, including the 14 lanthanides, lanthanum, yttrium, and sometimes scandium. Element 61 (promethium) has no stable isotopes, does not occur naturally, and has not been used as a marker. All rare earths exhibit trivalence as the dominant valence, although some show other valences (2 or 4). Rare earths have larger ionic radii than do other trivalent elements and are stronger bases and weaker in liganding ability than Cr, although stronger than any of the common divalent or monovalent ions (Sinha 1966). The opinion that the biological behavior of rare earths is uniform is not a safe generalization. Much has been written about their active metabolism in certain plants, especially those that contain oxalates and soluble phenolics, which can have extraordinary chemical affinity for rare earths. Plants known to metabolize large amounts of rare earths include hickories, pecans, and certain water plants (Thomas 1975), and we have found that levels in excess of 100 ppm of net rare earth may occur in legumes grown on certain soils. Up to 8000 ppm of Y, La, cerium, neodymium, and praseodymium have been reported in bone from cattle (Robinson and Edgington 1938). Most of the hal-

31

Rumen Dynamics

ance studies, however, have indicated negligible absorption of the compounds used as markers (Kotb and Luckey 1972). Biological activity among the rare earths is greater for the lighter elements but may vary irregularly with atomic numbers. Attachment to plant cell wall is lowest in the light rare earths (La, Ce, etc.), increases to a maximum with samarium, decreases to a minimum with dysprosium and holmium, and increases again toward the end of the lanthanide series (Allen and Van Soest 1984). This variation might account for the poor performance of Ce (Uden et al. 1980) and, perhaps, of La (Hartnell and Satter 1979). Lack of rare earth in a centrifuged supernatant is not good evidence for particulate attachment because of the colloidal nature of the interferences. Mader et al. (1984) found passage rates with rare earths differed depending on the method of marking. Other results indicate that the more tightly bound elements may have a greater effect on reducing digestibility of labeled particles (Allen 1982). Allen (1982) also examined La, Sm, and ytterbium and found that they did not migrate between particles during in vitro rumen digestion, but that subsequent gastric digestion removed and randomized rare earths as pH decreased (Allen 1986). This should happen in the abomasum and could vitiate results of particle markers in duodenal and fecal collections. Comparative data on the respective markers are relatively rare. Some comparisons between rumen turnover of Cr mordant, Yb, and lignin are shown in Table 2. These data, from the thesis of Robinson (1983), are from animals fed the same diet at different levels of intake. The lignin and neutral detergent fiber (NDF) results are based on rumen emptying, whereas the turnover for Cr and Yb is calculated from k1 output rates in feces. As would be expected from the model (see Fig. 1), the selective retention of lignin causes its turnover to exceed that of Cr and Yb, which are more likely to be uniformly distributed over the particulate matter. The turnover of the latter is compared with that of

Table 2. Comparative rumen turnovers and calculated digestibility of NDF based on the marker turnovers for partict.~late markers Chromium mordant T

Digestibility

(%)

T (hr)

Digestibility

Cow

(%)

(hr)

(%)

2896 2811 2880 1939

3.67 3.58 3.04 1.30

25 26 30 45

0 -15 30 27

24 20 22 48

-4 -50

Intake*

Source: Data from Robinson ( 1983). *Feed intake as percentage of body weight.

Lignin

Ytterbium

5

31

NDF

T (hr)

Digestibility (%)

T (hr)

33 40 29 56

25 25 28 41

25 30 21 33

32

P. J. Van Soest, C. J. Sniffen, and M. S. Allen

NDF, which is disappearing through both passage and digestion. The NDF, however, includes the lignified fraction that is selectively retained. Theoretically, the ratio of the NDF turnover to that of indigestible marker represents the indigestibility coefficient (P. J. Van Soest, unpublished). On the basis of lignin ratios, reasonable estimates of digestibility are obtained, whereas the corresponding estimates from Cr and Yb are underestimated and even negative. The point is that the nonideality of the rumen equilibrium renders the use of the best particulate markers problematic. Note that the NDF digestibilities based on lignin show a consistent lowered digestibility with higher feed intake. Problems in Preparation and Administration of Rare Earth Markers

Poor experiences with particle rare earth markers could arise from the manner and method of preparation. Like chromium in the mordanting technique, rare earths have a wide reactivity toward feed ingredients and other substances in the rumen environment. The greatest reaction is with substances with free carboxyl groups, oxalates, phosphates, rumen microbial cell walls, and saliva (Allen 1986). The rare earth complexes with phosphate and saliva are not associated with feed particles, are generally insoluble and colloidal, and are likely to move independently of feed particles. It is not advisable to place rare earth solutions, as such, into a rumen, because this will promote unspecific labeling. As in the case of the chromium mordant, the rare earth solutions should be applied to washed feed particles, as free as possible from soluble phosphates. Washing with EDTA solution helps to remove soluble matter; however, all EDTA needs to be removed, and the feed should be washed after the rare earth application, because the rare earth will react with EDT A to form a stable soluble marker. Application in the form of acetate salts is better than application as chlorides or nitrates because of the greater stability of neutral solutions of rare earth acetates. This stability of acetate solutions is related to the chelated nature of rare earth acetates, whereas nitrates and chlorides are comparatively uncoordinated (Sinha 1966). Solutions of rare earth nitrates and chlorides run the risk of hydrolysis with precipitation of colloidal hydroxides or carbonates. Particulate Flow

The Cornell model postulates the sizing of particulate matter by entrapment in the rumen mat or, perhaps, also by filtration in the omasum. The limiting size appears to be 1-2 mm for sheep and 3-4 mm for cattle (Kennedy and Pappi 1984). There is disagreement over the constancy of these values. Faichney (1986) argues for constancy, whereas, in our view, the limiting size rises in the case of pelleted feeds and in that of increased feed intake of a given diet.

Rumen Dynamics

33

This view appears to be consistent with the decreased rumination for each gram of NDF consumed (Bae et al. 1979) and the larger mean fecal particle size associated with pelleting or increased feed intake (Van Soest 1966). The Cornell model is designed for highly productive dairy cattle, not the smaller intake differences associated with nonlactating animals. The more extensive Australian conditions of animal feeding would perhaps diminish these effects on particle size and account for the divergence in opinion.

Dynamics of Rumen Microbes The growth and efficiency of rumen organisms is promoted by the quality of their substrate, in much the same way as feed quality promotes animal responses. There is a tendency in rumen modeling to estimate microbial production from organic matter fermented. Microbial yield per unit of fermented matter, however, is not constant but varies with the substrate, type of microbe, dilution rate, and the amount of energy available to the microbe, above that needed for its maintenance (see Figs. 3 and 4). The most important factor influencing microbial efficiency is its rate of metabolism. One boundary of metabolism can be set by the dilution rate, or washout rate, which effectively eliminates organisms with a growth rate slower than the dilution rate. Another factor limiting growth rate is the quality of substrate, which, being set by the physicochemical limitations of its composition, establishes the maximum possible rate of digestion. A third factor is the supply of required growth factors. The determined growth rate (the result of the combined effects of the various limiting factors) is in competition with washout and passage. Fig. 3 predicts that increased turnover of rumen contents will increase microbial efficiency because of the decrease in apparent maintenance, which is probably a function of the elimination of slow-growing organisms, reduction in predation and death rates, and the promotion of a population of younger mean age and greater growth potential. Chemostatic studies have verified these relationships and the validity of the prediction (Isaacson et al. 197 5, Van Nevel and Demeyer 1979). Although these relationships have been established in the laboratory, there has been reluctance to apply them to the rumen; however, a summary of the available world literature up to 1982, in Fig. 3, shows a significant relationship but with a rather wide standard error. Robinson and Sniffen (see Owens and Goetsch 1986) found an R 2 of 0.22 in their data from dairy cattle fed between one and four times maintenance. In the same data, net microbial nitrogen is correlated at +0.9 with potentially digestible organic matter (PDOM) escaping the rumen (see Fig. 5). The escaping PDOM, however, is not correlated well with particulate and liquid passage (r values 0.2-0.4). This situation might arise if markers were imper-

P. J. Van Soest, C. J. Sniffen, and M . S. Allen

34

5 :E

0 4 0

.....

•

(.:J

0

;: 3

-(.:J

z

...J

:=; 2

MARKERS

cr w

o • •

.....

u

-

u c o:s

>-

0

::::J

co

J ll ~ J rJ ~

n rtl

Sieve 4.0

r:J

~

c

E E

.s (I)

co

a: c 0

~

'E (I) E

'5 (I) (/)

- 0.~[

'+'

_] lfJ _] w 3

4J

4J

B Sieve 2.0 rb

c

Sieve 1.0

LiJ D

Sieve 0.5

4J

4J

4J

4J

......

6

12

24

4J

E Sieve 0.25

Time after Feed (hr)

Figure 4. The mean direction and velocity of movement of particles of different sizes at different times after feeding in undiluted ventral rumen samples incubated in vitro at 40°C. Values shown are the means(± SE) of eight determinations for particles of (A) 4-mm, (B) 2-mm, (C) 1-mm, (D) 0.5-mm, and (E) 0.25-mm sieve sizes. Bars above lines (A) and (B) represent flotation rates; bars below lines (C), (D), and (E) represent sedimentation rates.

Particle Separation in Sheep

57

on 4.0- and 2.0-mm sieves, and the smaller particles, which migrate downward and are collectable on 0.5-1.0-mm and 0.25-0.5-mm sieves. There is little apparent movement of the intermediate particles, which can be collected between 1.0 and 2.0 mm. As noted previously, a transition in density from below to above that of the liquid is obviously taking place as particles are reduced in size through the critical 1.0-2.0-mm range. In the experiments in which the contents are diluted, I found that the 1.0-2.0-mm range contained two essentially equal populations: one above and one below the density of the liquid. The velocities recorded for this range are therefore the result of effects in which migration is taking place in two opposite directions. The stage ofthe feeding cycle has considerable effect on the migration rates, with diminished velocities occurring later in the cycle. At least part of this effect could be due to an increase in viscosity during the period, as DM concentration rose, especially at the end of the cycle. An attempt was made to measure velocities in reticular contents, but, because of the need to work with small samples, errors were high. This was especially true for the large particles because these small samples contained so few of them. Owing to the general similarities between ventral rumen and reticular contents and the poorer accuracy of the observations on the reticular material, only the rumen values are presented in detail. The velocities observed may appear rather small, but it should be remembered that, in the ruminoreticulum, the periodicity of the mixing cycle is of the order of a minute. It should also be noted that, because of the nature of the determination, these are necessarily minimum values. In the dilution experiments reported in Table 3, the populations of dorsal and ventral rumen particles seemed to be similar with respect to the proportions of floating and sedimenting particles. This finding suggests that particles are continuously interchanging between the dorsal and ventral regions. The migration velocities of the predominant populations of the various sized particles are in directions favorable to creating a high distribution coefficient for the large particles, with diminished coefficients for the smaller particles. For the large particles, upward migration and ease of mechanical entanglement will facilitate their remaining with the raft. For the small particles, ease of escape in response to dorsal contractions and a tendency to move away from the raft once freed will increase their transfer into the ventral phase; however, even for the smaller particles, the concentration per unit wet weight is higher in the dorsal regions than in the ventral regions. Two factors are important here. First, small particles are, to a considerable degree, the result of mastication during eating or rumination (Ulyatt et al. 1985) and the return of such particles is to the dorsal rumen. Second, the currents of liquid movement (Waghorn and Reid 1977, Wyburn 1980) must bring ventral material into the raft where entrapment can occur. At present, there is a great need for some simple

58

T. M. Sutherland

experiments in which small particles suitably marked are introduced below the raft and followed in time at various sampling points within the rumen and reticulum. Welch (1982) reports results of an experiment of this type but, unfortunately, only for one particle size and specific gravity. The relationship between the contents of the ventral rumen and those of the reticulum is interesting. They are quite similar, but the reticular contents appear to be depleted of their lighter particles to some degree (see Table 3). The reticular contractions are very rapid; thus, for most of a mixing cycle, the reticular walls move little, if at all. Upward movement of low-density particles would leave the lowest liquid layer in the reticulum depleted of these particles. The nature of the reticular sampling was that the material sampled came in a gush at the end of the approximately 1-minute quiescent period. It seems likely that part of the sample taken was from the depleted layer. The amount of material expelled through the reticulo-omasal orifice at the end of the second reticular contraction is probably only about 6-8 ml, whereas the volume of the reticular contents on this type of diet and regimen is approximately 550 ml (Waghom et al. 1986). The upward movement of particles during the time the reticulum is at rest would be expected to clear a volume considerably in excess of 8 ml of particles of low density. The remaining question is whether this volume would remain cleared of these particles during the partial emptying and filling of the second reticular contraction. Hofmann (1973) observed that, during contraction, the honeycomb cells of the reticular mucosa shrink greatly and their walls become elevated. Such change would provide precisely the kind of protection from movement needed to retain the separated layer at the bottom of the reticulum until the system of walls and gutters, formed as contraction continues, could convey this material to the reticulo-omasal orifice. A mechanism of this sort has been suggested by Reid (1984). A very simple test of this hypothesis would be to apply the dilution separation method described here to the omasal contents. If the mechanism described is functioning, the omasal contents should be essentially free of particles of specific gravity less than that of clarified reticular fluid and the suspension fluid. If this is the unique mechanism between reticulum and omasum leading to particle separation, the size distribution within omasal content should resemble that of the sedimented fraction of reticular contents taken simultaneously, slightly enriched, with regard to the particle sizes of highest sedimentation velocity. If this mechanism is supplemented by an additional mechanism of filtration within the omasum followed by backflow, the omasal contents will still be free of low-density particles but will show a distribution different from that of the sedimented reticular material. If no such sedimentation/flotation mechanism exists within the reticulum, the low-density particles should occur in the omasal contents in the same proportions for the various particle sizes as are found in the samples from the ventral reticulum.

59

Particle Separation in Sheep

Behavior of Stalk and Leaf The mechanisms examined in the previous section for the differential separation of particles basically depend on the predominating population of large particles within the ruminoreticulum having a specific gravity less than that of the liquid and the small particles generally having densities greater than that of the liquid. The chaffed lucerne fed had approximately equal proportions of stalk (53%) and leaf. These components are so different that one must question the simplistic nature of analyses in which the basis is purely one of particle size. In a dilution experiment carried out on raft material obtained 3 hours after the start of feeding, the top and bottom layers were fractionated by the usual method of wet sieving and were then separated further into material of leaf and of stalk origin. The results of this experiment are shown in Fig. 5 and are expressed as 2

Buoyant Particles

.E

Ol

"Q)

$: Qj $: -,.R.

~

05

~

~

~

0

0 Sedimenting Particles

.E Ol

2

W}j Leaf

Q)

s:

Qj $: -,.R.

Ostalk

~

05

~

~

>-

0

0

4.0

1.0

0.5

0.25

Retaining Sieve Size

Figure 5. The distribution of DM between populations of buoyant and sedimenting particles of different size and origin in dorsal rumen contents at 3 hours after the start of feeding.

60

T. M. Sutherland

percentage of DM for the various particle sizes and for the two origins. The histogram above the line represents the weight of the material found in the upper layer, that is, material of density above that of the medium, whereas the histogram below the line represents the material of higher density found in the bottom layer at the end of the 2-minute separation period. The transition from a predominantly floating population for large particles to a predominantly sedimenting population for small particles is extremely sharp for the stalk particles, with the transition occurring in the fraction collected between the land 2-mm sieves. For the leaf-derived particles, the larger particles were almost equally distributed above and below the specific gravity of the medium, but most of the smaller particles were found in the bottom layer. The material responsible for the raft formation comes almost entirely from the stalk. Despite the fact that the sample was drawn at the end of eating and about 47% of the feed was leaf, only slightly more than 20% of the material examined was leaf derived. Only 20% of the leaf-derived material had a density below that of the medium, whereas 54% of the stalk-derived material was found in the upper layer. That lucerne leaf is more rapidly attacked in the rumen than is stalk is well recognized, and this fact was confirmed in some porous polyester bag experiments in which samples of the manually separated leaf and stalk were placed in the rumen for various periods. A preliminary experiment with stalk showed digestion to be complete by 24 hours, with no further change in digestibility or particle distribution visible at 48 or 72 hours. The remaining experiments with stalk were conducted with bags withdrawn at 3, 6, 12, and 24 hours, whereas corresponding experiments with leaf used bags withdrawn at 1, 2, 4, and 8 hours after insertion in the rumen. Insertion was always immediately before feeding. Experiments were conducted with relatively intact material-the stalk was cut transversely with scissors and collected in dry form on a 2-mm sieve. Other samples were sieved after hammer milling and were collected dry between 1.0- and 2.0-mm sieves. Leaves were examined with no further treatment after manual separation from the stalk and also after milling and collection between 1.0- and 2.0-mm sieves in dry form. There were marked differences between the rates of disappearance of DM from stalk and from leaf. For large particles, the rate constants (hr- 1 ) were 0.077 and 0.26 for stalk and leaf, respectively. For the 1.0-mm particles, the corresponding values were 0.13 and 1.65. The much more rapid attack on the smaller particles was presumably because of improved microbial access. The incubated bags were washed rapidly in warm artificial saliva (McDougall 1948) and submitted to a 2-minute dilution separation in a polyethylene bag before sieve analysis. The original large stalk material had 88% of the DM present as particles retained on a 2-mm screen in wet sieve analysis. After 24 hours, 80% of the DM of the final residue was still in this same fraction. Although they had lost more than 50% of their original weight, the particles

Particle Separation in Sheep

61

were obviously retaining more or less their original dimensions (Van Soest 1975, Welch 1982). In contrast, stalk milled and dry sieved to between 1.0 and 2.0 mm had originally about 83%·of 1.0-mm material on wet sieve analysis, but only 50% of the final residue was present in that fraction after 24 hours in the rumen. There was, however, a considerable increase in 0.5-mm particles-an increase that closely corresponded to the deficit in the 1.0-mm fraction. It appears that once stalk particles are broken longitudinally, some transfer to smaller fractions occurs through microbial action, but when an undamaged segment exists with only exposed ends, the stalk tends to retain its original outer form. In fact, much of the residue from the large stalk fraction consisted of hollow tubes. These observations on the behavior of the stalk are similar to those made by Welch (1982), whose paper shows illustrations of the residual material taken from porous bags after prolonged rumen exposure. After 8 hours in the rumen, particle digestibilities for whole and small lucerne leaf were 48% and 78%, respectively, with 60% of the residue from the larger particles still collectable on a 2.0-mm sieve. In contrast, less than 15% of the original particulate weight from the small leaf remained collectable on a 1.0-mm sieve after 1 hour and only 5% after 8 hours. Despite presoaking overnight before insertion in the rumen, the large stalk had high percentages of buoyant particles at all times. Percentages observed at 3, 6, 12, and 24 hours were 89%, 94%, 81%, and 81% (SE 4.4), respectively. The digests produced from the 1.0- and 2.0-mm stalk had negligible buoyant populations at 3 hours, but had some 35 ± 2.5% of the DM as buoyant particles at the end of 24 hours. Buoyancy distributions for leaf materials were highly variable, and although the percentage of buoyant material from the large leaf ranged between 30 and 40% in the first 2 hours, this percentage fell below 5% for the later periods. The small leaf started at 1 hour with about 10% of the material buoyant, but the proportion of buoyant material became quite low thereafter. The main aim of this small series of experiments was not so much to study the rates of digestion as to explore the effects of rumen exposure on buoyancy. Brazle and Harbers ( 1977) presented rates of digestion for whole leaf and stem and also the results of scanning electron microscopy on the residues from rumen digestion. The residue from leaf consisted of unhydrolyzed adaxial cuticle, abaxial cuticle, and hair and of a small amount of partially degraded vascular tissue. The undigested residue from the stem consisted of incompletely degraded vascular tissue. The very high potential for gas retention of the highly structured stem residue compared with the more opened out residue from the leaf is clearly visible in their photomicrographs. Hooper and Welch ( 1985) reported specific gravities of rumen-exposed early cut and mature alfalfa. They used chilled, ethanol-water-washed samples, a procedure that is likely to measure the densities of the particles after the loss of readily ex-

62

T. M. Sutherland

changed gas. Few of the particles measured in this way had specific gravities below 1 after a 1-hour exposure to the rumen despite the fact that more than 30% of the early cut feed and about 80% of the mature alfalfa had specific gravities below 1. These results seem to indicate a rapid loss of the nonexchangeable gas space when feed is introduced into the rumen. Particle Density Density is the sole determinant of the directionality and a very important factor in the magnitude of sedimentation/flotation velocities. The density of digesta particles depends on the relative volumes occupied by solid components, liquid, and gas. Simple calculation shows that, if one assumes that particles of 16% DM are being digested at a first-order rate, with K = 0.025hc 1 , and that 1.5 moles of short chain fatty acid in the molar proportions 70% acetic:20% propionic and 10% butyric are being produced for each mole of carbohydrate monomer digested, the gas produced per minute directly, or from acid action on bicarbonate, amounts to about 3.6% of the fluid space. Gas production rates and, consequently, gas escape rates are thus quite high. It appears likely that the volumes occupied by gas and liquid could change rather rapidly, depending on the conditions in which the particles were placed for density determination, and that estimating what the density of the particles might be in vivo could prove extremely difficult. Simple observations of the ratios of wet weight to dry weight of particles in which gas production has ceased and the gas space has been replaced by liquid can yield some useful information on maximum densities and critical gas volumes. The term critical gas volume is defined here as the proportion of the fluid space within the particle that has to be occupied by gas to make the density of the particle equal to that of the surrounding medium. DM Density and Critical Gas Volumes Unit volume of a collection of similar digesta particles is made up of the relative volumes occupied by structural components (a), liquid ([3), and gas (X.) so that

a+J3+X.=l Density is thus

where dP, d 8 , d1, and dg are the densities of the particles, the solid components, the liquid, and the gas, respectively.

63

Particle Separation in Sheep

For degassed particles, the density becomes free density. d~

=a

· d5

d~,

+ (1

which we will call the gas-

- a)d1

_a_= d~- d 1

and in particular

1- a

and

d5

-

d~

!3'=1-a

It should be noted that if the particle is porous in the sense that part of the contained liquid may exchange with an external medium and the density is measured in a density gradient, the particle will settle to a density determined by the volume of the nonexchangeable contained liquid and the skeletal solid material. If all the fluid is exchangeable, the particle will settle to the density of the skeletal solid. If xis the fraction of DM of the gas-free particles, then 1 - x would be the weight of liquid in 1 g of particles. The specific volume of the particles is X

1d,

X

V=-+-P

ds

and the particle gas-free density is d' = _!_ p

vp

At the critical point between flotation and sedimentation, the density of the particle must equal that of the fluid medium. If A.c is the relative gas volume at this critical point, then dp = a · d 5

and Eliminating

a

+ !3c + Ac = 1

+ !3c · di + 'A-c · dg =

d,

13c

The critical gas volume, the fraction of space available to liquid but occupied by gas, to achieve critical buoyancy is given by Ac

!3'

a

(d5

-

d,)

(1 - a) (d1 - dg)

64

T. M. Sutherland

Results of Wet Weight to Dry Weight Ratio Determinations

The percentages of DM were determined on sieve-analyzed residues from porous bag experiments. This material had been stored overnight in dilute sodium fluoride. The DM% rose as particle size decreased. For stalk-derived particles after 6 hours incubation in the rumen, the relationship was Y

=

32.14- 13.55logS

r2

=

0.91

where Y equals the percentage of DM and S equals 10 times the sieve size (in mm). For stalk particles incubated for 24 hours or longer and the digestion of which was complete, the regression was Y

= 26.42 - 6.95log S

r2

= 0.70

For rumen samples taken 6 hours after feeding started, the relationship resembled that for the 6-hour bag samples Y = 32.77- 11.77logS

r 2 = 0.94

A relationship between particle size and specific gravity was shown by Evans et al. (1973), and an extensive study of functional specific gravities for various sizes of feed particles was made by Hooper and Welch (1985). Waghorn et al. ( 1986) examined the chemical composition of particles from lucerne-fed sheep at various times during a 24-hour feeding cycle. Particles down to those collected on a 0.25-mm sieve were examined and were shown to contain 8595% cell wall materials and 1-2% nitrogen. Lange (1966) lists a range of 1.31.4 for the specific gravity of cellulose. This range is also likely to cover the hemicelluloses. The small quantity of other non-cell wall components present (to an appreciable extent protein) would not be expected to shift the density of the particles outside this range. The relationship derived above between DM fraction, gas-free density, and critical gas volume was used to calculate the lines drawn in Fig. 6 for critical gas volumes. For the top line, a density of the skeletal material of 1.4 was assumed; for the bottom line, a value of 1.3 was used. The arrows above the band intercept the lines at the DM% found for the stalk-derived particles of the 6-hour period of the porous bag experiment. The arrows below the bottom line are at the DM% for the rumen samples. It is clear from this diagram that to achieve buoyancy, the smaller particles must retain much more gas than the larger particles. The seminal ideas in the relationship between particle size and volume are

65

Particle Separation in Sheep 15

4.0

Dry Matter (%)

Figure 6. Relationship between critical gas volumes and DM for particles whose skeletal material has densities of 1.3 (lower line) or 1.4 (upper line). The arrows above the lines intercept the Jines at the DM% found, at each sieve size (mm), for stalkderived particles exposed to the rumen for 6 hours in a polyester bag. The arrows below the line are at the DM% found, at the same sieve sizes, for rumen particles.

contained in the "hotel theory" of Van Soest (1975). As the particle becomes smaller, the ratio of enclosed "room" volume to structural wall decreases and the maximum density increases. To attain buoyancy, particles have to retain gas at least to the critical volumes just described. The rate at which gas is lost is a surface area phenomenon, that is, a function of the square of the linear dimensions, whereas the amount of gas to be retained to achieve buoyancy is a fraction of the volume and thus depends upon the third power of the linear dimensions. This point is nicely illustrated in the observations of Hooper and Welch ( 1985), who demonstrated that the rate of change of functional specific gravity with time was dependent on particle size. The rate of change of specific gravity in these experiments is largely the rate at which internal gas is exchanged for liquid and is thus a measure of the rate of gas escape. The rates of change of specific gravity were for comparable periods 0.090, 0.074, 0.060, and 0.053 hr- 1 for milled hay particles of 1.0-, 2.0-, 4 .0-, and 6.0-mm screen size, respectively. Thus, because smaller particles have higher intrinsic maximum densities, they need a greater proportion of their fluid volumes to be gas-filled to achieve buoyancy; but they have poorer architecture for gas entrapment and an increased ratio of surface area to volume that facilitates gas loss. The effective specific gravity within the rumen is closely determined by particle size, which thus becomes a fundamental property on which mechanisms of particle separation may be based.

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General Discussion The Rumen The results reported here suggest that, on dried lucerne diets, at least two mechanisms operate in the differential flow of particles from the ruminoreticulum. Both of these mechanisms are dependent upon the differences of flotation/ sedimentation properties of large and small particles. The first mechanism is concerned with selective retention within the raft in the rumen; the second is the reticular settling mechanism, which is discussed in the following paragraphs. The distribution of materials within the rumen is the result of the mixing action of the cycles of ruminoreticular contractions, the unmixing tendencies of the spontaneous movements of the rumen particles themselves, and the effects of particle-particle interactions. The separation velocities of the particles are the measure of their unmixing tendencies. The forces acting to maintain these velocities against the resistance to flow or apparent viscosity are the differences between the weights of the particles and that of the medium or, more strictly, the contents displaced; thus, they depend markedly on particle density. The relationship between particle size and effective density has already been discussed here. But density and velocity are not the only factors that determine velocity. Size and shape are also important. For a spherical particle moving in a medium with Newtonian viscosity, it is readily shown by Stokes' law that velocity is proportional to the density difference between particle and medium, inversely proportional to the viscosity, and proportional to the square of the radius. For an equal deviation between particle density and liquid density, larger particles would be expected to migrate more rapidly than smaller particles-hence, the formation of a relatively stable raft that is based on large particles in the rumen and reticulum. Size itself is thus a factor in increasing upward velocity; as shown earlier, it is associated with low intrinsic densities and low critical gas volumes for buoyancy and with a greater tendency for gas retention. All these factors force the larger particles into the dorsal rumen and away from the infranatant ventral layers available for outflow. In the rumen, the low densities of large feed particles and the slowness of their gas exchange (Hooper and Welch 1985) presumably maintain buoyancy until fermentation begins within the larger particles to produce a steady supply of gas. Even when digestion has been completed, spaces in these particles may be large enough to accommodate unattached bacteria, which can produce enough gas to keep the large particle buoyant. The distribution coefficients of the large particles at 24 hours after feeding and the results of the porous bag experiments with large stalk suggest that such particles remain buoyant even when they are no longer capable of producing gas. This buoyancy prolongs the

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duration of the raft. The raft enhances size discrimination, so that even when they are sufficiently reduced in size as to have specific gravities below that of the liquid medium, particles of intermediate size are still held preferentially, compared with those of smallest size. The raft thus constitutes a very effective first-stage separator while any appreciable quantity of large material remains. The Reticulum

The nature of the reticular contractile cycle allows a second-stage separation to occur in which remaining light particles in the ventral fluid may rise out of the bottom reticular layer and escape passage. Sedimented reticular material, observed in the experiments reported previously, was remarkably free of large particles, and, if this material can be conveyed to the reticulo-omasal orifice by the mechanism proposed by Reid (1984), a combination of raft entrapment and reticular buoyancy separation would provide an effective means to ensure a particulate outflow essentially free of large material, at least for diets in which stalk is a major component. A small proportion of dried grass was present in the lucerne chaff feed. This dried grass, with some lucerne leaf, constituted most of the small amount of nonbuoyant longer fragments found in the reticulum. If materials generating considerable quantities of large dense particles were to constitute a major part of the diet, mechanisms not dependent on density would be necessary to prevent the passage of these particles. There would be a tendency for the ventral rumen to retain large dense fragments, but the reticular separation would operate in the wrong direction. Filtration and return by the unguliform papillae (which, with the lucerne diets, need only act as a minor back-up mechanism) would now have to play a more important role unless a major mechanism exists in the omasum (Ehrlein 1980). McBride et al. (1984), from their endoscopic studies, maintain very strongly that the reticulo-omasal orifice does not constitute a barrier to the passage of large particles and that the low passage of such material is through its failure to reach that point. Separation within the Omasum

It is now well established that liquid flows in both directions through the reticulo-omasal orifice (Stevens et al. 1960, McBride et al. 1984), with a normal pulse of digesta leaving the reticulum with the second reticular contraction and intermittent return of material from omasum to reticulum. Such a twoway flow opens up the possibility of an active sorting operation within the omasum in which large particles are strained off and held within the omasum while small particles in suspension are pumped into the abomasum. Later, the backflow could gather the accumulated large particles and return them to the

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reticulum. One would expect to see some differences in particle size distribution between the omasum and abomasum if such a mechanism were operating. No such differences are apparent in the data of Waghorn et al. (1986), obtained from the examination of slaughtered sheep. Weston and Cantle (1984), however, have provided evidence for the first part of the mechanism in demonstrating differential passage of particles through the omasum, with larger particles having lower fractional clearances. Thus, the backflow comes from an omasum enriched with respect to larger particles; whether it indeed carries an increased proportion of such particles compared with the inflow remains to be proved. Weston and Cantle also found differential passage rates from the abomasum, with the larger material being again preferentially retained. The occurrence of differential passage in both organs would tend to diminish omasal-to-abomasal differences and may explain why these two organs appeared to have similar distributions in the experiments of W aghorn et al. (1986). The Control of Particle Outflow

The manner by which the outflow of particles is controlled continues to attract interest, especially in terms of the possible effects on voluntary intake (Balch and Campling 1962, Conrad et al. 1964, Campling 1970, Ellis 1978, Weston and Kennedy 1984). Because the considerable space that the particles occupy is a potential limitation on intake, the optimal strategy for the animal might be to pass the digested particles as rapidly as possible. The fact that these particles must leave in liquid suspension is a major constraint on achieving this rapid clearance; high concentrations or rapid liquid flows or both are required. In the early stages of a feeding cycle when enough liquid must be present to allow a raft to form and effect particle separation, particle concentrations are low in the ventral regions. The high fluid volumes needed for raft formation and function probably follow naturally from the increased salivary secretion induced by stimuli from the fibrous material in the mouth and from the ruminoreticulum. In the ruminant, the main supply of energy to the host animal is in the form of the short-chain fatty acids. The supply of other nutrients such as amino acids and vitamins is quite distinct. The short-chain fatty acids come mainly through the rumen wall, whereas the amino acids and vitamins are produced by the digestion in the small intestine of the microbiota and feed materials that have escaped from the ruminoreticulum (Hungate 1966). To match the supply of energy with that of the other nutrients, outflow from the reticulo-omasal orifice has to anticipate the movement of the fatty acids across the rumen wall. The rate of flow of digesta from the rumen is highest shortly after feeding (Thompson 1973), whereas there is some delay before fatty acid concentrations in the rumen reach their maximum. The increased rumen volume must have a feed-forward effect on outflow. The low particle concentrations in the

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outflow probably minimize postruminal feedback effects on outflow (Phillipson and Ash 1965). Although the majority of rumen microbes appear to be associated with feed particles (Forsberg and Lam 1977), there is little or no adherence to the cuticle, sclerenchyma, or lignified vascular tissue, which are the materials constituting the indigestible residues (Akin 1979). The small particles that are the end products of the physical and chemical breakdown of the larger particles must, as they approach the limit of digestibility, carry very small microbial burdens. The majority of the rumen microorganisms must be attached to the larger particles, which have suitable substrate but low probabilities of escape from the rumen. The microbial supply to the small intestine would therefore seem to be determined to a large extent by the product of the microbial concentration in the free liquid phase and the liquid outflow. The later part of the feeding cycle is characterized by a considerable decline in the total volume of rumen contents (Ulyatt et al. 1984) presumably due to a decreased salivary secretion and despite a diminished liquid outflow (Thompson 1973). As the raft forming elements become fewer and less effective, the dorsal and ventral contents become increasingly similar to each other with a gradual rise in the ventral concentration of particulate DM. In the experiments of Ulyatt et al. (1984), sheep were fed lucerne chaff once daily at the levels used here and at a lower rate. At both levels, the DM losses from the rumen from 4 to 14 hours after the start of feeding were very similar to those occurring from 14 to 24 hours, despite a markedly lower rate of fermentation reflected by diminished volatile fatty acid concentrations in the latter period. The passage of DM is obviously greater in the later period presumably as a result of the increased concentration of particles available in the ventral reticulum. The need for liquid outflow for the passage of solids means that the factors controlling the former must largely determine the latter, but variations in the escape coefficients during the feeding cycle modify the effects on passage of particulate matter. A complex series of interactions between factors controlling rumen volume and rumen movements must continually be integrated to provide that compromise in ventral particular DM concentrations that allows efficient particle separation yet maintains adequate clearance rates from the rumen.

Summary The results shown on the distribution of particles within the rumen (see Table 1) indicate clearly that the raft can function very effectively as a differential retaining mechanism with high selectivity for large particles. The analyses of Evans et al. (1973) for the cow show an essentially similar distribution pattern within the rumen, and the same inferences can be drawn.

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An assessment of the potential of the raft-associated mechanisms can be made from Fig. 2, which illustrates the effect of raft proportions on the escape coefficients of particles of various sizes. The finding that small particles, which have natural tendencies to sediment, are found in greater concentration in the dorsal than in the ventral rumen indicates the importance of filtration or mechanical entanglement as part of the raft mechanism. Particles of stalk origin predominate in the raft, and the fact that most of the large stalk particles remain buoyant even when their microbial digestion is complete points clearly to their role as the principal raft-forming components. By the time the raft is no longer discernible in the feeding cycle, few large particles remain, and these are still concentrated to some degree in the dorsal region. While the raft is in place, the solid material found in the ventral rumen contains appreciable but diminished proportions of large particles compared with the raft and the whole rumen. The proportions in the ventral rumen are higher than normally found in feces, thus suggesting the existence of additional mechanisms beyond the rumen itself. The finding that particles do sediment or float at appreciable rates (see Fig. 4) means that, given a period free of mixing activity, a bottom layer can be produced that is without buoyant particles. Evidence indicates that this depletion occurs in the reticulum during the long period that exists between reticular contractions. Increased proportions of sedimenting particles were found in reticular samples that appeared to be delivered with the reticular contraction. These observations support the type of mechanism proposed by Reid (1984), which attributes an important role to the walls of the honeycomb cells in conducting material to the reticulo~omasal orifice. The general resemblance of the distribution of particle sizes in the sedimentable reticular particles to that found in the omasum and abomasum by Waghom et al. (1986) is additional support for the existence of a reticular sedimentation mechanism. A potential role for the omasum in particle separation was discussed in connection with some recent experimental findings that point toward this possibility. The two major mechanisms explored in this chapter are both dependent on the relationship between particle size and particle density. A relationship of this kind, in which low density is associated with large particle size, was demonstrated previously for rumen digesta by Evans et al. (1973) and for milled hay samples by Hooper and Welch (1985). The theoretical basis for this relationship has been discussed here in terms of the origin, structure, digestion, and intrinsic and effective densities of the particles.

Acknowledgments The experiments reported here were done during a study leave spent at the Applied Biochemistry Division of the Department of Scientific and Industrial

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Research (DSIR) New Zealand. My thanks to C. S. W. Reid and M. J. Ulyatt for permission to work there and to D. W. Dellow, A. John, and G. C. Waghom for generously sharing their ideas, experience, and unpublished work. Dr. Waghom had earlier examined the relationship between particle size and wet weight to dry weight ratios and kindly allowed me to see his extensive data in the area before they were published. The skilled technical assistance of I. D. Shelton throughout the experiments and K. Kelly in the differentiation of leaf and stalk fragments is gratefully acknowledged. References Akin, D. E. Microscopic evaluation of forage digestion by rumen microorganisms. Journal of Animal Science 48:701-710; 1979. Balch, C. C.; Campling, R. C. Regulation of voluntary food intake in ruminants. Nutrition Abstracts and Reviews 32:669-686; 1962. Brazle, F. K.; Harbers, L. H. Digestion of alfalfa hay observed by scanning electron microscopy. Journal of Animal Science 46:506-512; 1977. Campling, R. C. Physical regulation of voluntary intake. In: Phillipson, A. T., ed. Physiology of Digestion and Metabolism in the Ruminant. Newcastle-upon-Tyne, England: Oriel Press; 1970:p. 226-234. Campling, R. C.; Freer, M. The effect of specific gravity and size on the mean time of retention of inert particles in the alimentary tract of the cow. British Journal of Nutrition 16:507-518; 1962. Conrad, H. R.; Pratt, A. D.; Hibbs, J. W. Regulation of feed intake in dairy cows. 1. Changes in importance of physical and physiological factors with increasing digestibility. Journal of Dairy Science 47:54-62; 1964. desBordes, C. K.; Welch, J. G. Influence of specific gravity on rumination and passage of indigestible particles. Journal of Animal Science 59:470-475; 1984. Egan, J. K.; Doyle, P. T. A comparison of particulate markers for the estimation of digesta flow from the abomasum of sheep offered chopped oaten hay. Australian Journal of Agricultural Research 35:279-291; 1984. Ehle, F. R. Influence of feed particle density on particulate passage from rumen of Holstein cow. Journal of Dairy Science 67:693-697; 1984. Ehrlein, H. J. Forestomach motility in ruminants. Publikationen zu Wissenschaftlichen Filmen, Sektion Medizin, Series 5, Nummer 9, Film C1328. Gottingen, Germany: Institi.it fi.ir den Wissenschaftlichen Film; 1980:p. 1-29. Ellis, W. C. Determinants of grazed forage intake and digestibility. Journal of Dairy Science 61: 1828-1840; 1978. Evans, E. W.; Pearce, G. R.; Burnett, J.; Pillinger, S. L. Changes in some physical characteristics of the digesta in the reticula-rumen of cows fed once daily. British Journal of Nutrition 29:357-376; 1973. Faichney, G. J. The use of markers to partition digestion within the gastro-intestinal tract of ruminants. In: McDonald, I. W.; Warner, A. C. 1., eds. Digestion and Metabolism in the Ruminant. Armidale, Australia: Univ. of New England Publishing Unit; 1975:p. 277-291. Forsberg, C. W.; Lam, K. Use of adenosine-5'-triphosphate as an indicator of the microbiota biomass in the sheep rumen. Applied Environmental Microbiology 33: 528-537; 1977.

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Hofmann, R. R. The ruminant stomach. East African Monographs in Biology, 2. Nairobi: East African Literature Bureau; 1973. Hooper, A. P.; Welch, J. G. Effects of particle size and forage composition on functional specific gravity. Journal of Dairy Science 68:1181-1188; 1985. Hungate, R. E. The Rumen and Its Microbes. New York and London: Academic Press; 1966. Kennedy, P. M. Techniques and Quantitative Analysis of Food and Digesta Particle Size. Occasional publication no. 1. Edmonton: Canadian Society of Animal Science; 1984. King, K. W.; Moore, W. E. C. Density and size as factors affecting passage of ingesta in the bovine and human digestive tracts. Journal of Dairy Science 40:528-536; 1957. Lange, N. A. Handbook of Chemistry. lOth ed. New York: McGraw-Hill Book Company; 1966. McBride, B. W.; Milligan, L. P.; Turner, B. V. Endoscopic observations of digesta transfer from the reticulo-rumen to omasum of cattle. Canadian Journal of Animal Science 64(Supplement):84-85; 1984. McDougall, E. I. Studies of ruminant saliva. 1. The composition and output of sheep's saliva. Biochemical Journal43:99-109; 1948. Pearce, G. R. Changes in particle size in the reticulo-rumen of sheep. Australian Journal of Agricultural Research 18:119-125; 1967. Phillipson, A. T.; Ash, R. W. Physiological mechanisms affecting the flow of digesta in ruminants. In: Dougherty, R. W., ed. Physiology of Digestion in the Ruminant. Washington, D.C.: Butterworth; 1965:p. 97-107. Poppi, D.P.; Norton, B. W.; Minson, D. J.; Hendricksen, R. E. The validity of the critical size theory for particles leaving the rumen. Journal of Agricultural Science, Cambridge 94:275-280; 1980. Poppi, D. P.; Hendricksen, R. E.; Minson, D. J. The relative resistance to escape of leaf and stem particles from the rumen of cattle and sheep. Journal of Agricultural Science, Cambridge 105:9-14; 1985. Reid, C. S. W. The progress of solid feed residues through the rumino-reticulum: The ins and outs of particles. In: Baker, S. K., ed. Ruminant Physiology: Concepts and Consequences. Perth: Univ. of Western Australia Press; 1984:p. 79-84. Reid, C. S. W.; Ulyatt, M. J.; Monro, J. A. The physical breakdown of feed during digestion in the rumen. Proceedings of the New Zealand Society of Animal Production 37:173-175; 1977. Reid, C. S. W.; John, A.; Ulyatt, M. J.; Waghorn, G. C.; Milligan, L. P. Chewing and the physical breakdown of feed in sheep. Annales de Recherches Veterinaires 10:205-207; 1979. Stevens, C. E.; Sellars, A. F.; Spurrell, F. A. Function of the bovine omasum in ingesta transfer. American Journal of Physiology 198:449-455; 1960. Thompson, F. The effect of frequency of feeding on the flow and composition of duodenal digesta in sheep given straw-based diets. British Journal of Nutrition 30:87-94; 1973. Uden, P.; Van Soest, P. J. The determination of particle size in some herbivores. Animal Feed Science and Technology 7:35-44; 1982. Ulyatt, M. J. Plant fibre and regulation of digestion in the ruminant. In: Wallace, G.; Bell, L., eds. Fibre in Human and Animal Nutrition. Wellington: The Royal Society of New Zealand; 1983:p. 103-107. Ulyatt, M. J.; Waghorn, G. C.; John, A.; Reid, C. S. W.; Monro, J. Effect of intake and feeding frequency on feeding behavior and quantitative aspects of digestion in

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sheep fed chaffed lucerne hay. Journal of Agricultural Science, Cambridge 102: 645-657; 1984. Ulyatt, M. J.; Dellow, D. W.; John, A.; Reid, C. S. W.; Waghorn, G. C. The contribution of chewing during eating and rumination to the clearance of digesta from the rumina-reticulum. In: Milligan, L. P.; Grovum, W. L.; Dobson, A., eds. Control of Digestion and Metabolism in Ruminants. Englewood Cliffs, N.J.: Prentice-Hall; 1985:p. 498-515. Van Soest, P. J. Physico-chemical aspects of fibre digestion. In: McDonald, I. W.; Warner, A. C. 1., eds. Digestion and Metabolism in the Ruminant. Armidale, Australia: Univ. of New England Publishing Unit; 1975:p. 351-365. Waghorn, G. C.; Reid, C. S. W. Rumen motility in sheep and cattle as affected by feeds and feeding. Proceedings of the New Zealand Society of Animal Production 37:176-181; 1977. Waghorn, G. D.; Reid, C. S. W.; Ulyatt, M. J.; John, A. The feed comminution, particle composition, and distribution between the four compartments of the stomach in sheep fed chaffed lucerne hay at two feeding frequencies and feeding levels. Journal of Agricultural Science, Cambridge. 106:287-296; 1986. Welch, J. G. Rumination, particle size and passage from the rumen. Journal of Animal Science 54:885-894; 1982. Welch, J. G.; Smith, A. M. Particle sizes passed from rumen. Journal of Animal Science 46:309-312; 1978. Weston, R. H.; Cantle, J. A. The movement of undigested plant particle fractions through the stomach of roughage-fed young sheep. Canadian Journal of Animal Science 64(Supplement):322-323; 1984. Weston, R. H.; Kennedy, P. M. Techniques in Particle Size Analysis of Feed and Digesta in Ruminants. Occasional publication no. 1. Edmonton: Canadian Society of Animal Science; 1984:p. 1-17. Wyburn, R. S. The mixing and propulsion of the stomach contents of ruminants. In: Ruckebusch, Y; Thivend, P., eds. Digestive Physiology and Metabolism in Ruminants. Lancaster, England: MTP Press; 1980:p. 35-54.

4 Ecology of Rumen Microorganisms: Energy Use J. B. Russell

The term Oekologie was coined in 1866 by Ernest Haeckel, who used the word to relate differences in animal morphology to Charles Darwin's newly described theory of evolution and natural selection (Mcintosh 1980). The word ecology comes from the Greek words oikos, meaning "home," and logos, which is taken to mean "science" (Colinvaux 1973). More modem definitions range from the study of ''interrelationships between living organisms and their environment" (Buchsbaum and Buchsbaum 1957) to "man's best attempt to understand the living world" (Damelll973). The biosphere is composed of many billions of individual organisms, which, for convenience, can be organized into different biological levels (Fig. 1). In this scheme, individual organisms are organized into single species groups or populations, which, in tum, live and interact with other populations in a community of living organisms. A community and its physical environment constitute an ecosystem, and all ecosystems together constitute the biosphere (Darnell 1973). The rumen is a microbial ecosystem (Hungate 1975) that rumen microbiologists have sought to organize and understand from an ecological point of view (Hungate 1960, Hungate 1979, Wolin, 1979, Hobson and Wallace 1982, Wolin and Miller 1983). In 1960, Hungate pointed out that a complete ecological analysis of a natural habitat requires an elaboration of (1) the kinds of organisms present, (2) the activities of these organisms, and (3) the extent to which the activities are expressed (Hungate 1960). Our analysis of the rumen is not complete, but we have made much progress since the first isolations of strictly anaerobic bacteria some 40 years ago. Most, if not all, of the predominant rumen bacteria have been identified, and pure culture studies have given us a good indication of many of their potential activities. The extent to which their activities are expressed in vivo is still unknown (Russell and Hespell 1981). 74

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i i i i i i

ECOSYSTEM

COMMUNITY

Traditional Realm of Ecology

POPULATION

ORGANISM

MACROMOLECULES

Figure 1. Levels of biological organization in the biosphere. (Modified from Darnell 1973.)

MOLECULES

Rumen as a Habitat The rumen is an ideal habitat for the growth of anaerobic microorganisms. Ingested food provides nutrients needed for microbial growth, and the temperature remains relatively constant (36-40°C). Water and saliva create a well-buffered, neutral, aqueous environment. The relatively small amounts of air taken in with the food are used by a small number of facultative organisms or are washed out with fermentation gases. The low partial pressure of oxygen selects for fermentative organisms, and the end products of the fermentationvolatile fatty acids, which can be toxic to microbial metabolism-are removed through the mucosa into the blood. Autoregulatory processes keep food moving through the rumen at a pace slow enough for digestion and fast enough to provide nutrients needed by the microorganisms and the host. Fermentation of feedstuffs in the ruminoreticulum yields short-chain volatile fatty acids (primarily acetic, propionic, and butyric acids), carbon dioxide, methane, ammonia, microbial cells, heat, and, occasionally, lactic acid. Ruminants use the organic acids and microbial protein as a source of energy and amino acids, respectively, but methane, heat, and ammonia represent a loss of energy and nitrogen to the animal. The quality and quantity of rumen fermentation products depend on the types and activities of microorganisms in the rumen.

Rumen Microbial Diversity Bacterial diversity within the rumen ecosystem is very great, with more than 20 species present at counts greater than 107 • g- 1 . Because total numbers are

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more than 10 10 • g- 1 , organisms with counts less than 107 • g- 1 make up a minor fraction of the population. One must be careful, however, in assessing the significance of less numerous species. Even a minor part of the population can be important if it exerts an ecological impact on others (Hungate 1966). The vast majority of microorganisms inhabiting the ruminoreticulum are found in the lumen, but scanning electron microscopy has revealed a distinct population of bacteria attached to the mucosal lining. These bacteria have little direct influence on feed digestion, but adherent ureolytic bacteria may be important in recycling nitrogen to the rumen as ammonia (McCowan et al. 1980). The bacteria have a dominant role in the overall process of feed digestion and fermentation. Protozoa are found in much lower numbers than the bacteria in the rumen (0-1 06 · g- 1 ), but, because of their size, they sometimes account for as much as one half of the microbial mass. Nearly all rumen protozoa have cilia, whose location can be used in classification. The holotrichs have cilia over the entire body, whereas the entodiniomorphs have cilia in distinct areas or whorls. Holotrichs are found in higher numbers in ruminants fed lush forage. They swim rapidly and ingest soluble sugars. The entodiniomorphs, which take up small particles, including fed grains, increase when animals are fed cereal grains. There are at least 13 genera of rumen protozoa (Coleman 1980). The rumen flagellates, Neocallimastrixfrontalis, Piromonas cummunis, and Sphaeromonas cummunis, were originally classified as rumen protozoa, but Orpin (1977) questioned this classification in the 1970s. He found that these organisms had morphological characteristics and a means of reproduction that were typical of fungi. It is now apparent that the flagellates are actually zoospores of phycomycetes. Sporangia and extensive rhizoid development have been observed on surfaces of plant particles from the rumen (Bauchop 1979). Knowledge of rumen fungi has been based largely on microscopic studies. Scanning electron microscopy has shown widespread attachment to, and penetration of, plant materials. The most fibrous and least digestible diets supported the greatest populations of fungi, which may be significant as initial colonizers of lignocellulose.

Ecological Relationships Microorganisms have been inhabiting the rumen for some 70 million years, and they exhibit many interrelationships. These interactions fall within several broad definitions common in ecological literature. When two species have no effect on each other, a state of neutralism exists. There are a few examples of neutralism in the rumen, but usually the degree of interaction is stronger. In commensalism, the growth of one species is promoted by the presence of a second and the growth of the second is unaffected by the first. In mutualism,

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both species benefit. Competition arises when two species depend on the same limiting nutrient or physical niche. Toxicities can lead to amensal relationships, in which one species is adversely affected by the relationship and the other is unaffected, and predation also occurs. Any understanding of the rumen as an ecosystem is confounded by the fact that a single species may be involved in several types of interactions.

Mechanistic Models Traditionally, ecology has been concerned with levels of biological organization at or above that of the individual organism (see Fig. 1). At these levels, it is often difficult, or even impossible, to perform controlled experiments. Most ecological hypotheses have been based on changes in the number of individual organisms and recognition of patterns in these changes. Theoretical population ecology has provided information on the types of organisms that will occupy a particular habitat, but it has rarely told us how. In recent years, it has become more apparent that an organism's distribution in nature can depend on subtle differences at subcellular levels of organization (see Fig. 1). The literature is rife with reports about the relative numbers of a particular rumen microorganism increasing or decreasing dramatically after a specific diet was fed. These observations have been explained by the increased amount of a specific feed component that was used by the organism. Such explanations do not indicate why other organisms capable of using the same substrate did not proliferate as much. The biological fitness of an individual species in the rumen is controlled by a broad range of metabolic and physiological characteristics. The rumen ecosystem as a whole represents a pattern of such frightening complexity, involving many species and their potential interactions, that it cannot be comprehended all at once. A mechanistic approach to understanding such a system entails a systematic analysis of the parts that work together. First, relationships of particular importance are identified, whereas those of lesser importance are either laid aside or discarded. Second, the relationships are expressed in quantitative terms or equations. Third, the relationships are organized in a model that can simulate the system or part of the system. Finally, predictability, the true test of any scientific concept, is examined.

General Strategies of Growth Numerous relationships and general strategies for the growth of rumen microorganisms have been identified. Some progress has been made at defining these factors quantitatively, but relatively few attempts have been made at simulating and validating the system.

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Energy and Carbon Sources

In most natural habitats, the supply of suitable energy sources is a key factor limiting the growth of individual species. Because energy sources are often limiting, survival depends on an effective strategy for procuring them (Table 1). One strategy is analogous to the economic strategy practiced on diversified farms. By using a variety of energy sources, the organism can survive even if the competition for a particular one is intense. Some rumen microorganisms (e.g., Butyrivibrio fibrisolvens) practice a diversified approach, but an even greater number have selected particular energy sources with a strategy comparable to specialized or intensive farming. This group of organisms includes Bacteroides amylophilus and many of the ruminococci, which can use starch and cellulose, respectively, but not glucose or most other sugars. Specialization can be applied to the type of work that is performed as well as to the resources that are selected. Rumen cellulolytic bacteria have lost the ability to synthesize de novo, or even take up, branched-chain amino acids (Bryant 1973). They depend on other rumen bacteria for a supply of branchedchain volatile fatty acids that they use in making the branched-chain amino acids. The use of branched-chain volatile fatty acids decreases the need for carbon and energy, but it creates a dependency on other organisms as "suppliers.'' Growth Rates and Efficiency

The rate and efficiency of substrate use can also affect survival (Russell and Respell 1981). When nutrients are plentiful soon after feeding, a fast growth rate is desirable because it enables a more rapid conversion of nutrients into cell material than a slower growth rate would. Later, survival depends more on the ability of organisms to scavenge, and efficiently use, limiting nutrients for growth or maintenance. We can once again use an economic analogy: In the United States, natural resources are plentiful, and a relatively small population (at least in density) produces goods from these resources at a very rapid rate. Examination of U.S. garbage dumps and junk yards shows much waste. The situation in developing countries is often quite different. Natural resources are in short supply, and the population density is very high. Per capita productivity is low, but little is wasted. In the rumen, Streptococcus bovis is well suited to an abundance of starch but uses the starch inefficiently in the production of cell material. B. fibrisolvens grows much more slowly than S. bovis, but it takes up and uses substrates very efficiently. It proliferates on straw diets (Hungate 1966). Cross-Feeding

Theft sometimes results in economic well-being, and there are examples of biological theft in the rumen. Bacteria possess transport systems capable of

Energy Use by Rumen Microorganisms

79

taking up low-molecular-weight nutrients, but most feeds are composed of large, relatively insoluble, and sometimes complex polymers. These polymers must be degraded by extracellular enzymes to products that are available to all organisms, not just to the individual that did the work of synthesizing the degradative enzyme. When Bacteroides succinogenes and Selenomonas ruminantium were grown in cultures containing cellulose as an energy source, S. ruminantium grew even though it was unable to degrade cellulose (Scheifinger and Wolin 1973). It, and a variety of other noncellulolytic rumen bacteria (see Table 1), are able to use cellodextrins that they most likely "steal" from the cellulolytic bacteria (Russell 1985). Attachment to food particles may be a means the rumen cellulolytic bacteria use to decrease their losses. By being close to the site of extracellular hydrolysis, they should hypothetically receive a larger share of released nutrients. Many of the rumen microorganisms in pure culture produce substances that are not observed in vivo: lactate, succinate, ethanol, formate, and hydrogen gas (Table 2). The absence of these products in vivo can be directly or indirectly explained by cross-feeding. Lactate is used by organisms that convert it to propionate and acetate. Because the lactate-using organisms generate ATP for growth, this process is analogous to the economic process of recycling. Counotte et al. (1981) indicated that Megasphaera elsdenii is the predominant lactate user in grain-fed cattle. Succinate is converted to propionate and S. ruminantium appears to be the only species present in high enough numbers to account for this conversion (Scheifinger and Wolin 1973). Because the pathway of succinate to propionate contains no traditional sites for ATP formation, the advantage to S. ruminantium is not obvious (Bryant and Wolin 1975). S. ruminantium is unable to grow with succinate as the sole energy source, but Schink and Pfennig (1982) recently isolated an anaerobic bacterium, Propionigenium modestum, that can. This isolate did not contain cytochromes, but it had a very high sodium requirement. It may use the free energy of succinate decarboxylation to generate a sodium gradient, which subsequently drives ATP formation. A similar but less efficient system might provide S. ruminantium with some energy in much the same way that amino acid fermentation provides energy for maintenance but not enough for growth (Russell 1983). Allison et al. (1979) showed that ct-ketoglutarate could be formed by the reductive carboxylation of succinate inS. ruminantium. Succinate could thus serve as a carbon source for protein synthesis. Most of the rumen formate is converted to hydrogen, which is, in tum, used by methanogenic bacteria (Hungate et al. 1970), but stoichiometric relationships showed that much more methane was produced in vivo than could originate from formate (Hungate 1966). Several of the rumen bacteria produce hydrogen in pure culture, but the amounts are generally small because the oxidation of reduced nucleotides by hydrogenase is not thermodynamically

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Lipids Hydrolyzes triglycerides Ferments glycerol Soluble sugars Ferments sucrose Ferments fructose Ferments glucose Ferments galactose Organic acids Ferments lactate Ferments malate Ferments fumarate Ferments succinate Proteins Hydrolyzes protein Ferments peptides Ferments amino acids Incorporates amino acids Fermentation products Utilizes H 2 Ferments methanol

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Caproate

Source: Russell (1984), courtesy of The Science Press. Note: + = most strains produce this substance; - = most strains do not produce this substance; blank = not studied.

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Ruminococcus albus Ruminococcus flavefaciens Bacteroides succinogenes Butyrivibrio fibrisolvens Bacteroides ruminicola Bacteroides amylophilus Selenomonas ruminantium Streptococcus bovis Megasphaera elsdenii Succinomonas amylolytica Eubacterium ruminantium Succinivibrio dextrinosolvens Lachnospira multiparus Anaerovibrio lipolytica Veillonella alcalescens Methanobrevibacter ruminantium Vibrio succinogenes Eubacterium limosum

+ + +

Acetate

Formate

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Species

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Energy Use by Rumen Microorganisms

83

favorable. The methanogens, however, are able to keep the partial pressure of hydrogen low enough that hydrogenase can oxidize NADH. Interspecies hydrogen transfer to the methanogens provides an additional substrate for methane production as well as an alternative means of oxidation. When methanogens were co-cultured with hydrogen-producing bacteria, ethanol, propionate, succinate, and lactate decreased, whereas acetate increased (Wolin 1979). Recent work has likewise indicated that fermentation of highly reduced, branchedchain amino acids is dependent on hydrogenase activity and methane formation. When carbon monoxide, a specific bacterial hydrogenase inhibitor, was added to mixed cultures containing peptides as an energy source, methane production decreased, the intracellular ratio of NADH to NAD increased, and levels of branched-chain volatile fatty acids and ammonia decreased (Hino and Russell 1985).

Death and Recycling The rumen environment is normally almost neutral, but pH can decline significantly if large amounts of starch are fed. Early work by Hungate et al. ( 1952) demonstrated that rumen acidosis was associated with an overgrowth of S. bovisand the inability of lactate-using bacteria to use all the lactate that was produced. Lactate is 10 times stronger an acid than the volatile fatty acids, and accumulation of it eventually exceeds the buffering capacity of rumen fluid. As pH declines, the growth rate of most rumen bacteria, including S. bovis, decreases, although S. bovis is more resistant to low pH than most other rumina! species (Russell and Dombrowski 1980). This greater resistance gives S. bovis a competitive advantage and causes even more lactate and lower pH. S. bovis, however, is eventually inhibited when pH reaches 4.5 and the lactobacilli, which are even more resistant to low pH, dominate the rumen (Hungate eta!. 1952). It is well documented that rumen protozoa ingest and digest a significant portion of the bacteria and smaller protozoa that are produced in the rumen (Eadie and Gill 1971; Coleman 1975, 1980; Cottle et al. 1978). Coleman (1964) reported that a single protozoan could ingest as many as 12,000 bacteria an hour, but later reports indicated that the predation rate in vivo is probably much slower (Kurihara et al. 1978). Whether the protozoa attack their prey selectively remains in doubt (Kurihara et al. 1968). One could speculate that free bacteria would be more easily ingested than bacteria attached to larger feed particles, but there are few data to support this hypothesis. Most of the rumen microorganisms (approximately 75%) are found associated with feed particles, and attachment may confer some advantages. Being adjacent to sites of feedstuff degradation, attached microorganisms can more readily capture the products of extracellular hydrolysis. If the microorganisms are firmly attached to large feed particles, they may be more resistant to

84

J. B. Russell

protozoal engulfment. The rumen is often used as an example of a natural chemos tat, but the contents do not tum over homogeneously. The solid fraction (large feed particles) turns over two to four times more slowly than the liquid small particle pool. Some of the rumen microorganisms (e.g., the protozoa) grow very slowly and could be washed out of the rumen by the liquid outflow. By associating with feed particles that remain in the rumen longer, they increase their chances for growth. Examination of rumen contents usually reveals an abundance of solid feed, but soluble energy sources can be present at low or negligible concentrations during much of the feeding cycle. When exogenous substrates run out, the rumen microorganisms must depend on endogenous substrates for their metabolism. Several studies indicate that rumen bacteria are able to survive for only a short period under starvation conditions (Respell 1984). Unfortunately, little is known about factors influencing the rate of bacterial death in the rumen.

Metabolic Regulation Versatility often contributes to survival, and metabolic regulation increases the options available to microorganisms. Many rumen bacteria are capable of using the same sugars and are, therefore, potentially able to compete for them. Comparison of the organisms, however, showed that they had catabolite regulatory mechanisms that enabled them to use some substrates to the exclusion of others (Russell and Baldwin 1978). Because they had different patterns of substrate preference, two bacteria capable of using the same sugar were not necessarily in direct competition. Studies by Bryant (1956) suggested that S. ruminantium contributes more to rumen fermentation in animals fed rations containing soluble carbohydrates. Because soluble carbohydrates are fermented rapidly by many rumen bacteria, the competition for these substrates is likely to be intense. S. ruminantium has both a high maximum growth rate and a high affinity for a variety of sugars, and these physiological characteristics make it well suited to compete for these substrates. S. ruminantium is also able to produce lactate as well as to ferment it, and this feature contributes to its versatility. S. ruminantium grows rapidly on the sugars that are present soon after feeding and converts them to lactate, a less desirable substrate. Later on in the feeding cycle, it is able to ferment the lactate and generate more ATP. S. bovisand S. ruminantium produce lactate when they grow rapidly, and the ATP yield of this fermentation is low (Scheifinger et al. 1975, Russell and Baldwin 1979b). Because readily fermented carbohydrate and, hence, energy are usually limiting in the rumen, one would expect the organisms to select fermentation pathways that yield more ATP per unit substrate fermented (e.g., acetate, propionate, or even butyrate). In the case of S. bovisand S. ruminan-

Energy Use by Rumen Microorganisms

85

tium the type of fermentation product qepends on the growth rate. They produce lactate when they grow rapidly, but they switch to acetate, formate, and ethanol or acetate and propionate when carbohydrate concentration and growth rate are low. This pattern of metabolic regulation maximizes the amount of ATP produced per unit time (Hungate 1979). When carbohydrate is limiting, the efficiency of ATP synthesis controls the A TP produced per unit time, but when carbohydrate is in excess, fast-growing organisms can still produce a large amount of A TP per unit time even if A TP per unit carbohydrate is low. One can then ask: Why do S. bovis and S. ruminantium not produce either acetate and ethanol or acetate and propionate, respectively, at rapid growth rates to obtain even more ATP? An obvious, but teleological, answer is that they would have been unable to grow as fast (Russell 1984). Westerhoff (1983) recently proposed that microbial growth efficiency must be sacrificed to make the process run faster. The switch between lactate and volatile fatty acid production inS. ruminantium is regulated by the intracellular pyruvate, which serves as a homeotropic activator of its lactate dehydrogenase (Wallace 1978). When carbohydrate concentrations increase, growth rate, intracellular pyruvate, and lactate dehydrogenase activity increase. With S. bovis, the pattern of regulation is more complicated. Increasing carbohydrate and growth rate is associated with an increase in intracellular fructose 1 ,6-diphosphate; the lactate dehydrogenase has an obligate requirement for this activator (Wolin 1964). At slow growth rates, intracellular fructose 1 ,6-diphosphate is low, the lactate dehydrogenase is not activated, and acetate and ethanol are the principal products. At low pH, however, intracellular pH declines significantly. The decline in intracellular pH in tum decreases the requirement of lactate dehydrogenase for fructose 1 ,6diphosphate, increases the maximum velocity of the lactate dehydrogenase, and inhibits the pyruvate-formate lyase. Once the extracellular pH is low, the organism will thus continue to make more lactate, even if carbohydrate availability and growth rate are low. This pattern of regulation leads to a spiraling action of even more lactate and lower pH and explains why rumen acidosis is a difficult condition to reverse (Russell and Hino 1985).

Quantification Microbial growth occurs exponentially, and the rate is usually expressed as a specific rate constant (f.L) or doubling time (g) f.L =In 2/g

Growth rates are most often measured in batch cultures with an excess of required nutrients and these values represent the maximum growth potential of

86

J. B. Russell

the organism. In the rumen, soluble nutrients can be plentiful soon after feeding, and microorganisms may attain their maximum growth rate. Later, the situation is quite different. The rumen may still contain an abundance of solid feed, but nearly all of the food is composed of large and sometimes complex polymers that must be hydrolyzed by extracellular enzymes. Microbial growth rates are limited by the concentration of products that are released by the enzymes. In the 1940s, Monod (1949) studied the effect of energy source concentration on bacterial growth rate and found that the two were related according to saturation kinetics typical of enzyme systems

where k is the growth rate obtainable under these conditions, kmax is the maximum growth rate, S is the substrate concentration, and ks is the affinity constant for the substrate. Microorganisms are very efficient scavengers of nutrients and the affinity constants are generally low. In enzymatic studies, initial velocities are usually determined at specific substrate concentrations below as well as above the affinity constant. The estimation of bacterial growth rates at low substrate is confounded by the propensity of many bacteria, especially anaerobic bacteria, to "lag" in the period soon after inoculation. Substrate is often depleted before a precise estimate of growth rate is possible. In chemostats, the kinetics of bacterial growth can be measured under steady-state conditions. Bacterial growth rate is controlled by the dilution rate, and the amount of substrate remaining at a dilution rate is inversely proportional to the affinity for substrate. In enzyme assays, the substrate concentration is set, and the researcher measures velocity. In continuous culture, a reverse approach is used. The velocity (growth rate) is set and the concentration of substrate needed to obtain this rate is measured. Because the measurements can be made under steady-state conditions, problems inherent to batch cultures do not apply. The biggest obstacle to the continuous culture approach is growth on the wall of the containing vessel (Munson 1970). Bacteria attached to the wall are not subject to the same degree of dilution as the bacteria in suspension. Growth on the wall leads to an overestimation of kmax and k 5 • In the 1950s Herbert et al. (1956) noted that bacterial growth yields were generally lower at slow growth rates than they were at faster growth rates. On the basis of these continuous culture experiments, they introduced the idea that energy sources were used for maintenance functions as well as for growth. At slow growth rates maintenance makes up a larger percentage of total energy use, and the yield of bacteria decreases proportionally. Marr et al. (1962) and Pirt (1965) introduced maintenance energy derivations that used double re-

87

Energy Use by Rumen Microorganisms

ciprocal plots of growth rate and cell mass or yield. Transformation of the data as a reciprocal, however, introduced a statistical bias (Neijssel and Tempest 1976, Stouthamer 1979, Tempest and Neijssel 1984). Plots employing the specific rate of substrate use (q) and growth rate (k) tended to provide a more accurate estimate of maintenance (m) and the theoretical maximum growth yield (Y0 ) k

q=-+m

Yo

where Y 0 is defined as the growth yield that would be obtained in the absence of maintenance. The maintenance derivation gives a reasonable estimate of yield, provided that energy is the factor limiting growth. When growth is limited by other nutrients (e.g., N, S, or P), the yield, as expressed in terms of energy source, decreases (Neijssel and Tempest 1976). Decreases in yield are more dramatic at lower dilution rates, but there is little information concerning the regulation ofthis "energy spilling." The death and turnover of rumen microorganisms is not fully understood. Mink and Hespell (1981a, 1981b) studied the starvation of several species of rumen bacteria and described the death rate with a half-time function that they termed the "ST50 ." Since the ST50 differed greatly among the species tested, the death rate could be an important variable determining the distribution of rumen microorganisms. A recent study showed that cells grown quickly were less resistant than cells grown slowly and nitrogen-limited cells died faster than cellobiose-limited cells (Wachenheim and Hespell 1985). Equations describing death may need to specify growth conditions as well as the types of individual microorganisms. 15 N studies indicated that as many as half of the rumen bacteria are eaten by protozoa (Nolan and Stachiw 1979), but mathematical assessments of predation are difficult for three reasons: ( 1) it is still not known if protozoal predation of bacteria is selective, (2) the effect of bacterial concentration on the rate of protozoal predation has not been adequately described, and (3) there is little information about the growth rates of protozoa. All of the rumen microorganisms are subjected to the passage of rumen contents to the lower gut, but there are at least two major dilution rates: that of the large particles and that of the liquid small particle pool. Some of the rumen bacteria, including S. bovis and Megasphaera elsdenii, were found almost exclusively in the fluid phase (Latham et al. 1980). For these organisms, the liquid dilution rate might provide a reliable estimate of the washout rate. Most of the selenomonads, ruminococci, and Butyrivibrio fibrisolvens and Bacteroides amylophilus cells were found with feed particles (Minato and Suto 1976, Latham et al. 1980). Not all sizes offeed particles pass from the rumen at the same rate, but an average solid dilution rate could provide a reasonable estimate for the organisms more closely associated with the feed.

88

J. B. Russell

Simulation Even though our understanding of rumen microbial ecology has increased, researchers still find it difficult to judge intuitively yet accurately the effect of a particular perturbation (e.g., change in diet or feed additives) on the microbial population. The dominant effect may be obvious, but secondary or synergistic responses are sometimes difficult to predict. The development of computer technology over the past three decades has made possible the simultaneous integration of a large number of variables. Baldwin and his colleagues constructed several models to test an understanding of rumen digestion and fermentation. In one of these models, the rumen microflora was subdivided into eight metabolic groups, but during solutions "considerable simplification ... occurred" (Reichl and Baldwin 1976). The authors concluded that "current data and concepts ... do not adequately accommodate competition among the several rumen microbial species and thus additional data and concepts regarding microbial interactions are required.'' More recently, we published a less ambitious but more mechanistic model of rumen microbial competition (Russell and Allen 1984). This model, lactate production by S. bovis and its use by M. elsdenii, was based on physiological characteristics that were easily quantified from simple chemostat kinetics. Early work by Hungate et al. (1952) demonstrated that rumen acidosis was invariably associated with an initial overgrowth of S. bovis and an inability of lactate-using bacteria to ferment all the lactic acid that was produced. Recent experiments by Counotte et al. (1981) indicated that M. elsdenii was the primary lactic acid-fermenting bacterium in starch-fed cattle. When S. bovis and M. elsdenii were co-cultured in chemostats with starch as the limiting energy source, relative numbers were dependent on both pH and dilution rate (Fig. 2A). The interaction between S. bovis and M. elsdenii involves two types of cross-feeding, a competition and an antagonism. M. elsdenii is unable to grow on starch, but it is able to use the maltose that is released by S. bovis amylases. Because both organisms are able to use maltose, they compete for it. For many years it was assumed that S. bovis carried out a homolactic fermentation, but lactate is produced only if growth rates are high or pH is low (Russell et al. 1981). When growth rates are low and pH is near neutral, S. bovis produces acetate, formate, and ethanol rather than lactate, and there is less energy source forM. elsdenii. If lactate accumulates, the buffering capacity of rumen fluid can be exceeded and rumen pH declines. S. bovis is more resistant to low pH than M. elsdenii (Russell and Dombrowski 1980, Therion et al. 1982). The relative numbers of S. bovisand M. elsdenii appeared to be influenced by five physiological factors: (1) the relative capacity (affinity and maximum velocity) of each organism to use maltose, (2) the production of lactate by S.

89

Energy Use by Rumen Microorganisms

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