PHYSIOLOGICAL REVIEWS   Vol. 78 No. 2 April 1998, pp. 393-427
Copyright ©1998 by the American Physiological Society

Contributions of Microbes in Vertebrate Gastrointestinal Tract to Production and Conservation of Nutrients

C. EDWARD STEVENS AND IAN D. HUME

College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina; and School of Biological Sciences, Sydney, New South Wales, Australia

I. INTRODUCTION
II. VARIATIONS IN DIET, HABITAT, AND GENERAL CHARACTERISTICS OF THE DIGESTIVE SYSTEM
    A. Fish
    B. Amphibians
    C. Reptiles
    D. Birds
    E. Mammals
III. DIGESTA PASSAGE AND RETENTION TIME
IV. CHARACTERISTICS AND DISTRIBUTION OF INDIGENOUS GUT MICROBES
    A. Ruminant Forestomach
    B. Foregut of Other Species
    C. Midgut
    D. Hindgut
V. FERMENTATION OF CARBOHYDRATES
    A. Ruminant Forestomach
    B. Digestive Tract of Other Species
    C. Absorption of Short-Chain Fatty Acids
VI. PRODUCTION OF MICROBIAL PROTEIN AND RECYCLING OF NITROGEN
    A. Ruminant Forestomach
    B. Foregut of Other Species
    C. Hindgut
    D. Absorption of Ammonia and Amino Acids Produced by Microbes
VII. PRODUCTION AND ABSORPTION OF VITAMINS
VIII. MICROBIAL DETOXIFICATION
IX. DISEASES ASSOCIATED WITH MICROBIAL FERMENTATION
X. CONCLUSIONS
REFERENCES

	ABSTRACT

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Stevens, C. Edward, and Ian D. Hume. Contributions of Microbes in Vertebrate Gastrointestinal Tract to Production and Conservation of Nutrients. Physiol. Rev. 78: 393-427, 1998.  The vertebrate gastrointestinal tract is populated by bacteria and, in some species, protozoa and fungi that can convert dietary and endogenous substrates into absorbable nutrients. Because of a neutral pH and longer digesta retention time, the largest bacterial populations are found in the hindgut or large intestine of mammals, birds, reptiles, and adult amphibians and in the foregut of a few mammals and at least one species of bird. Bacteria ferment carbohydrates into short-chain fatty acids (SCFA), convert dietary and endogenous nitrogenous compounds into ammonia and microbial protein, and synthesize B vitamins. Absorption of SCFA provides energy for the gut epithelial cells and plays an important role in the absorption of Na and water. Ammonia absorption aids in the conservation of nitrogen and water. A larger gut capacity and longer digesta retention time provide herbivores with additional SCFA for maintenance energy and foregut-fermenting and copoprophagic hindgut-fermenting species with access to microbially synthesized protein and B vitamins. Protozoa and fungi also contribute nutrients to the host. This review discusses the contributions of gut microorganisms common to all vertebrates, the numerous digestive strategies that allow herbivores to maximize these contributions, and the effects of low-fiber diets and discontinuous feeding schedules on these microbial digestive processes.

	I. INTRODUCTION

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One of the most prevalent and interesting characteristics of the digestive system is the symbiotic relationship between an animal and the microorganisms that inhabit its digestive tract. The gastrointestinal tract of vertebrates becomes colonized with bacteria shortly after birth or hatching. This includes transient microorganisms and the autochthonous bacteria, which develop into relatively stable populations that are characteristic of the species (76). The number and species of microorganisms in a given gut segment are affected by the pH and retention time of digesta. The low pH of gastric contents and rapid transit of digesta through the midgut or small intestine of most species tend to inhibit the growth of many microbes. However, the relatively neutral pH and prolonged retention of digesta in the foregut of some and the hindgut or large intestine of all terrestrial vertebrates are accompanied by an increase in the number and variety of bacteria and, in some species, the additional presence of protozoa and even fungi.

Much of our understanding of the nutritional contributions of gut microbes comes from early studies of the ruminant forestomach. This led to the presumption and subsequent demonstration that gut microbes serve a similar purpose in the large intestine of simple-stomached herbivores, such as the horse and rabbit. However, we now know that bacteria in concentrations equivalent to those in the rumen perform similar functions in the hindgut of all terrestrial vertebrates. Microbial fermentation also occurs to a lesser degree in the stomach and midgut of all species, with the midgut serving as the principal site in a few herbivores.

Bacteria indigenous to the gastrointestinal tract ferment dietary and endogenous carbohydrates into short-chain fatty acids (SCFA) that provide energy for the gut epithelium and other tissues and facilitate the absorption of Na and water. They also convert dietary and endogenous nitrogenous compounds into ammonia and microbial protein and synthesize B vitamins. Absorption of ammonia serves to recycle and conserve endogenous nitrogen from the urea (or uric acid) and digestive enzymes that are released into the gut. Microbial protein and B vitamins produced in a forestomach or recycled through the gut after coprophagy provide many species with an additional source of essential amino acids and vitamins.

The advent of herbivores that can subsist largely on a diet of the fibrous portion of plants allowed a significant expansion in the numbers and ecological distribution of vertebrates. Subsistence on this type of diet requires the ingestion of large quantities of plant material and its reduction to small particle size. Some species pass this material rapidly through the gut for the extraction of readily available nutrients, but most herbivores subject it to prolonged microbial fermentation in some segment of their digestive tract.

This review emphasizes the ubiquitous role that gut microbes play in the provision and conservation of nutrients and their role in expanding the nutritional niches available to herbivores. The nutritional contributions of gut microbes are dependent on the diet, habitat, and anatomy of the digestive system and the pH and retention time of digesta. Therefore, we begin with a discussion of the variations in diet, habitat, and the structure and function of the digestive system that are related to the microbial production of nutrients. This is followed by a description of the gut microbes and their contributions to nutritional requirements. Section IX discusses some of the major dysfunctions in microbial digestive processes that can result from improper diets and feeding practices.

	II. VARIATIONS IN DIET, HABITAT, AND GENERAL CHARACTERISTICS OF THE DIGESTIVE SYSTEM

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The vertebrates are believed to have evolved ~500 million years ago and presently consist of ~45,000 species, which can be divided into 8 classes of fish, amphibians, reptiles, birds, and mammals. These animals show a wide range of variation in habitat, diet, and the structural characteristics of their digestive system. Because of species variations, comparisons of the digestive tract are best described under the general headings of headgut (mouthparts and pharynx), foregut (esophagus and stomach), and the midgut and hindgut, which are the small and large intestine in many species.

The fish include macrophagous and microphagous carnivores, omnivores, and herbivores, which may feed on living or dead material. Some can vary their diet from fish in the summer to plankton in the winter, even utilizing bacteria and algae as food (22). The anatomy of the fish digestive system has been reviewed by Harder (117, 118) and Kapoor and Khawna (148). The headgut shows a wide range of divergence with respect to diet. Jaws are absent in the cyclostomes (hagfish and lampreys), which are parasitic on other fish, but present in all other fish and advanced vertebrates. The teeth of fish vary in both their location (jaw, tongue, pharynx, or other surfaces of the orobranchial cavity) and function. In most species they are used for grasping, cutting, or tearing. The ability to reduce food to very small particles is limited to filter-feeders, grazers, or those that triturate their food with pharyngeal teeth. Potential loss of small food particles through the gills is prevented by gill rakers in many species and pharyngeal mastication in others.

The esophagus of fish is generally short, and the stomach is absent in cyclostomes and some advanced groups. When present, the stomach is either straight, U-shaped, or Y-shaped with a blind sac on its greater curvature. It is lined with regions of proper gastric mucosa, which secretes HCl, pepsinogen, and mucus, and pyloric mucosa, which secretes HCO₃ and mucus. The midgut can vary from one that is short and straight to a longer structure with spirals and loops. In some species with a small or absent stomach and short intestine, folds of the midgut extend varying distances into the lumen to form a spiral valve, which increases its surface area and delays digesta passage. In many species that have a stomach, the surface area and digesta retention time of the cranial midgut are increased by the presence of anywhere from 1 to 1,000 pyloric ceca. The hindgut of most fish is short and difficult to distinguish from the midgut. However, the intestine is divided into two distinct segments, separated by a valve or sphincter, in a few species.

The exocrine pancreatic tissue is present in primitive ceca along the midgut of cyclostomes and some species in the more advanced classes of fish. However, the pancreas is a compact organ in sharks, skates, rays, many teleosts, and all other vertebrates. The liver is a compact organ in all vertebrates, and biliary secretions are stored in a gallbladder in most vertebrates, but a gallbladder is absent in some species of fish and mammals.

Herbivores are relatively rare among fish and confined to teleosts. Most herbivores are of intermediate body size. Horn (130) classified the digestive tracts of herbivorous marine fish into four types, based on adaptations designed to degrade cell walls of algae and/or bacteria by acid lysis, mechanical trituration, or microbial fermentation (Fig. 1). The first type, which includes surgeon fish, has no mechanisms for the trituration of food and a thin-walled stomach, but a relatively long intestine. The second type, which includes the mullets, features a thick-walled, gizzardlike segment of stomach and an intestine of variable length. The third type, which includes the marine parrotfish and freshwater grass carp, has pharyngeal teeth that grind food to a small particle size but no stomach and a relatively short intestine. The fourth type, seen in the sea chubs, has a long intestine that includes a distinct hindgut.

View larger version (24K): [in this window] [in a new window]   FIG. 1.   Gastrointestinal tracts of 4 species of marine herbivorous fish. Surgeon fish is a browser with pyloric ceca and a relatively long intestine. Mullet is a grazer with a thick-walled gizzardlike stomach (shaded area) and pyloric ceca. Parrot fish is a grazer with pharyngeal teeth, no stomach or pyloric ceca, and an intestine that is relatively short but large in diameter. Sea chub is a browser with pyloric ceca and a distinct hindgut that contains sphincters or valves at its junction with midgut and a more distal location (shaded areas) and small paired ceca. [Modified from Horn (130).]

All ectothermic tetrapods (amphibians and reptiles) are found within 45° of the equator, and their greatest diversity is seen within 20° (90). Amphibians presently contain ~4,000 species belonging to three orders: the Gymnophionia (wormlike burrowing amphibians), Caudata (salamanders, newts, and congo eels), and Salientia (frogs and toads). Most species begin life as free-living aquatic larvae, which metamorphose into terrestrial adults. Larval amphibians may be carnivores, omnivores, or herbivores (218). Some have a large mouth and can engulf their entire prey of crustaceans, mosquito larva, or worms. Others have complex, microphagous, filtering mechanisms for ingestion of bacteria, zooplankton, or phytoplankton, which can be aided by the removal of encrusted material with horny teeth in anurans (frog and toad) tadpoles. Most larval anurans lack a stomach or gastric pouch. The gastric region usually forms a thickened sheath, which produces mucus, a proteolytic cathepsin, and a low pH. Pepsin has been rarely reported. The intestine is relatively long, with no distinct separation into a midgut and hindgut.

Adult amphibians are carnivores with weak dentition that serves for the grasping of food while it is being swallowed. Metamorphose of anurians is accompanied by profound changes in diet, feeding practices, and the structure and function of the gastrointestinal tract (133, 218). This includes the appearance of a stomach lined with areas of proper and pyloric glandular mucosa, joined by an additional area of cardiac glandular mucosa in some species. The intestine is shortened, with removal and regeneration of intestinal epithelium and the appearance of a distinct hindgut (Fig. 2).

View larger version (28K): [in this window] [in a new window]   FIG. 2.   Gastrointestinal tracts of an adult amphibian and a carnivorous caiman, an omnivorous turtle, and herbivorous tortoise and lizard. Note cecum, larger volume, and greater relative length of hindgut in reptilian herbivores as well as baffles provided by projecting tissue in cecum and proximal colon of iguana. [From Stevens and Hume (252). Reprinted with permission of Cambridge University Press.]

The anatomy of the reptilian digestive system has been reviewed by Parsons and Cameron (201), Luppa (171), and Bjorndal (26). The mouth parts of most reptiles are used for grasping, cutting, and tearing their food. This is accomplished with a beak in the chelonians and teeth in other reptiles. A few species have teeth that can crush their food, but the scissorlike action of the reptilian jaw prevents the grinding of food into small particles (196). The salivary glands of reptiles, birds, and mammals secrete mucus and fluid that aid in the deglutition of food, and digestive enzymes in some species. The stomach of reptiles tends to be tubular, but it is larger and more outpocketed in crocodilians. The pylorus of alligators is muscular and separated from the remainder of the stomach by a constriction. Gastroliths (stones, gravel, or sand) have been reported in crocodilians, chelonians, and lizards. These were found in 100% of the stomachs of Crocodylus nycloticus over 2 m in length (65), where they may provide ballast or serve the same triturative function as the gizzard in birds. Like the adult amphibians, the mucosal surface of the stomach is divided into regions of proper gastric, pyloric, and, sometimes, cardiac glandular mucosa.

The gastrointestinal tracts of a carnivorous, an omnivorous, and two herbivorous reptiles are illustrated in Figure 2. Most reptiles are carnivores or omnivores, and many subsist on insects during their early or entire life. Herbivores are limited to ~50 species of lizards (211), 40 species of tortoise (183), and a few turtles. The midgut tends to be longest in carnivores and shortest in herbivores. The opposite is true for the length of the hindgut, with the exception is the herbivorous Florida red-bellied turtle (Pseudemys nelsoni), which has an extremely long small intestine that contained 68% of the gut contents (27). The hindgut includes a cecum in most herbivores. The cecum and proximal colon of herbivorous lizards in the families Iguanidae, Agamidae, and Scincidae are compartmentalized by mucosal folds (Fig. 2), which slow digesta passage and increase absorptive surface area.

Reptilian herbivores include terrestrial, freshwater and marine species, and a few lizards and tortoises that have adapted to desert environments. Herbivores appear to be limited in both their minimum and maximum body size. Juveniles of herbivorous species of lizard are carnivores or omnivores, and the adults weighed more than 300 g (211). An increase in body mass was associated with an increase in the relative gut capacity of herbivorous iguanas (263) and fiber digestibility in the herbivorous green turtle (Chelonia mydas) (25). The largest herbivores are the arboreal common iguanas (Iguana iguana), which weigh up to 13 kg (157), and tortoises (Testudo elephantus and Testudo gigantea), which weigh up to 250 kg with a high proportion of their body weight comprised of carapace (183).

The relative paucity of reptilian herbivores has been attributed to a poor masticatory apparatus, ectothermy, and the limitations of a small body size on the gut capacity of most species. However, this is difficult to explain in light of the striking success of herbivorous dinosaurs, which were the dominant terrestrial vertebrates during the Jurassic and Cretaceous periods and included both the largest species and others with a wide range of body size (64). The presence of smooth stones with their fossil remains suggest that some species may have used a gizzard to reduce plant material to a small particle size (17). However, Norman and Weishampel (191) concluded that the ornithopods, a large and diverse group of late Mesozoic dinosaurs, had a masticatory apparatus that approached the efficiency of mammals.

Dinosaurs also may have differed from present-day reptiles in their ability to maintain a stable body temperature. Farlow (89) concluded that a low mass-specific metabolic rate would reduce the total energy requirements and rate of digesta passage in large dinosaurs, and fermentation could provide heat for thermoregulation. The possibility that dinosaurs were endotherms also has been raised, based on their success in number of species and distribution long after the first appearance of mammals and comparisons of the structure and oxygen isotope composition of dinosaur bones with those of present-day reptiles, birds, and mammals (17, 21, 44, 223).

Birds are endotherms that differ from other vertebrates in their cover of feathers and (with the exception of bats) modifications for flight, which include the absence of teeth, decreased weight of the jaw skeleton and muscles, acquisition of a gizzard as the organ for trituration, and limitations in gut capacity. Storer (256) listed the ~8,600 species of existing birds under 28 orders. They include carnivores, piscivores, insectivores, carrion eaters, and species that feed principally on fruit, seeds, pollen, nectar, leaves, or roots. Many of the common species are found in the orders Galliformes (cocklike) and Passiformes (sparrowlike).

Structural characteristics of the avian digestive system are described by Ziswiler and Farner (287) and Duke (77). Mouth parts show wide diversification with the diet. This is particularly true for the bill, which can serve for cutting, tearing, crushing, filter-feeding, or other purposes. However, as with other nonmammalian vertebrates, the jaws are not constructed for efficient trituration or grinding of food. Figure 3 shows the gastrointestinal tracts of a carnivorous, an omnivorous, and four herbivorous species. The functions attributed to the stomach of other vertebrates are carried out by a crop (storage), proventriculus (pepsinogen and HCl secretion), and gizzard (trituration). The proventriculus is lined with proper gastric and pyloric glandular mucosa. The relative size of these organs tends to vary with the diet. Granivores and herbivores generally have a larger crop and a larger, more muscular gizzard.

View larger version (25K): [in this window] [in a new window]   FIG. 3.   Gastrointestinal tracts of a carnivorous hawk, an omnivorous chicken, and 4 herbivorous birds. Note larger size of crop in omnivore and herbivores, and particularly in hoatzin. Ceca are small in hawks and relatively large in grouse. Although ceca are relatively small in hoatzin, emu, and ostrich, an expanded foregut (hoatzin), a much longer midgut (emu), or a much longer colon (ostrich) compensates for this. [From Stevens and Hume (252). Reprinted with permission of Cambridge University Press.]

Most birds have a relatively short midgut and a hindgut that consists of a short, straight colon and, often, paired ceca. Poppema (210) measured the relative lengths of intestinal segments in 644 specimens representing 24 orders, 51 families, 124 genera, and 166 species of birds. She found that ceca were absent or poorly developed in all species belonging to 13 of these orders. This included small passerine species and most of the larger species that feed on fruit, nectar, carrion, or small vertebrates. The most developed ceca and highest ratio of cecal length to total intestinal length were found in granivores and species whose diet contained high levels of plant fiber or chitin.

Most herbivores belong to the orders Galliformes and Rheiformes. Galliformes are predominantly ground birds that tend to fly only short distances. Suborder Opisthocomi contains only one species, the hoatzin (189), a 750-g South American folivore that is unique among birds in its use of an enlarged crop and distal esophagus (Fig. 3) for microbial fermentation (112). Suborder Galli contains a number of herbivorous grouse and two species of herbivorous partridge with a distensible crop, well-developed gizzard, and relatively large ceca (190).

Grouse consist of 11 genera and 18 species that weigh between 350 and 6,500 g. Prairie chicken inhabit North American steppes and semideserts, and hazel grouse, which include the ruffed grouse (Fig. 3), are inhabitants of mixed forests and semideserts of North America and Eurasia. Forest grouse include capercaillie, black grouse, spruce grouse, and ptarmigan. Capercaillie and spruce grouse can consume large amounts of conifer needles, and the ceca of spruce grouse doubled in length during the winter (202). Ptarmigans are distributed the furthest north of all land birds, in the tundra of North America, Greenland, and Eurasia, where they feed on twigs and buds during winter months. Most partridges are omnivores, but snow partridge and snow cocks are herbivores found in mountains up to 5,000 m above sea level (215).

Rheiformes (ratites) are flightless birds that include the herbivorous rheas, emus, ostriches, and recently extinct moas (234). Rheas, which inhabit the South American steppes and high plains, weigh up to 25 kg and use enormous ceca as the principal site for microbial fermentation. Emus, Australian birds that reach weights of 55 kg, have relatively short ceca and a short colon and long midgut (Fig. 3), which contained 37% of the gut contents and serves as the principal site of microbial fermentation (124). The largest living birds are the ostriches, which weigh up to 150 kg. Their principal site for microbial fermentation is their colon, which is much longer than that of other birds (Fig. 3). Although the moas of New Zealand are extinct, the presence of grass and twigs in the crop and feces of giant moas, which reached weights of 250 kg, indicate that they were herbivores as well.

Mammals, which also are endothermic, consist of ~4,150 species. Mammals can be subdivided into two subclasses, Prototheria (echidnas and platypus) and Theria (marsupial and placental mammals), and can be further subdivided into 20 orders. One of the major advances in the evolution of mammals was the development of an extremely efficient masticatory apparatus (67). Although a few species have either lost their teeth or adapted them for other purposes, the teeth of most mammals include incisors for cutting, canines or fang teeth for grasping and tearing, and premolars and molars with uneven occluding surfaces. The latter are used for crushing. The lateral movements of the lower jaw seen in most mammals or the anterior-posterior movements seen in rodents and elephants provide a grinding action as well. Eisenberg (82) divided mammals into 16 categories of dietary specialization, and Langer and Chivers (165) discussed other dietary classifications. However, Table 1 lists the mammalian orders divided more simply into three categories based on the principal diet(s) of inclusive species.

The earliest mammals are believed to have been small carnivores, and 13 of the 20 orders contain species that feed principally on invertebrates or other vertebrates. The gastrointestinal tract of most carnivores is relatively short and simple (Fig. 4). The stomach is generally a unilateral dilatation of the digestive tract. The only exceptions to this are seen in the cetaceans (dolphins, porpoises, and whales), which have a large multicompartmental stomach (Fig. 4) that is believed to be conserved from herbivorous ancestors (182), and vampire bats, which have a convoluted stomach that is approximately twice the length of their body. A distinct hindgut is absent in some Insectivora, Carnivora, cetaceans, bats, and marsupials, and it lacks a valvular separation from the midgut in some of these species. Where present, the hindgut is relatively short and seldom haustrated, but it includes a cecum in some species.

View larger version (33K): [in this window] [in a new window]   FIG. 4.   Gastrointestinal tracts of mammalian carnivores. Most carnivorous mammals have a simple stomach and a relatively short hindgut, which is indistinguishable from midgut in many species. However, complex stomach of cetaceans provides one exception, and some species, such as dog, have a cecum and a small but well-developed colon. [Dog from Stevens (247). Copyright 1977 by Cornell University. Used by permission of the publisher, Cornell University Press. Mole and bat from Stevens (248). Whale and quoll from Stevens and Hume (252). Reprinted with permission of Cambridge University Press.]

Ten orders include omnivores, which feed on both plants and animals or on plant concentrates such as seeds, fruit, nectar, pollen, or roots, usually in addition to invertebrates. Some examples of omnivorous mammals are illustrated in Figure 5. With the exception of some rodents and frugivorous and nectivorous bats, their stomach is simple. The intestine varies in both its total length as a function of body length and the relative length of the midgut and hindgut. Bears have an extremely long intestine, with a short and indistinct hindgut, but the opossum intestine is almost equally divided between a midgut (small intestine) and hindgut (large intestine). Digesta retention is aided by a cecum in the hindgut of many omnivores and haustrations of the cecum and varying lengths of the colon in some species. The colon of pigs, humans, a few monkeys, and the chimpanzee is haustrated throughout its entire length.

View larger version (29K): [in this window] [in a new window]   FIG. 5.   Gastrointestinal tracts of mammalian omnivores. With exception of some rodents, such as rat, omnivores have simple stomachs. Their intestine is generally longer than that of carnivores, but midgut-to-hindgut ratio varies greatly. Bears and pandas have a very long midgut and a short and indistinct hindgut, whereas opossum's hindgut constitutes one-half of intestinal length. A cecum may be absent or present; it is well-developed in rats, in common with other rodents. Cecum and varying length of colon are haustrated in domesticated pigs, many herbivorous monkeys, apes, and humans. Illustration of rat gastrointestinal tract demonstrates effects of feeding time on relative size of stomach and cecum. Specimen on left was collected 4 h after feeding, and that on right was collected immediately after feeding; animals were fed at 12-h intervals. [Pig from Argenzio and Southworth (12). Human from Wrong et al. (282). Rat from Stevens (247). Copyright 1977 by Cornell University. Used by permission of the publisher, Cornell University Press. Armadillo from Stevens (248). Opossum and bear from Stevens and Hume (252). Reprinted with permission of Cambridge University Press.]

Herbivorous species, defined as animals that can subsist largely on a diet containing the fibrous portion (leaves, petioles, stems) of plants, are found in 11 orders. The herbivores, which are the most abundant in numbers of animals and species, include the largest terrestrial species and show the widest ecological distribution. Björnhag (29) estimated that herbivores constitute >90% of the mammalian biomass. Pandas, which have a gastrointestinal tract similar to bears, appear to obtain their nutrients by selective feeding and rapid transit of digesta through a long but relatively simple gastrointestinal tract. However, most mammalian herbivores obtain a substantial part of their nutrients by the retention and microbial fermentation of plant material in a voluminous cecum, colon, or forestomach.

The predominant feature in small herbivores such as the lagomorphs, hyrax, herbivorous rodents, and most small herbivorous marsupials, is a large cecum that serves as the principal site of microbial fermentation (Fig. 6). This is accompanied by a partially compartmentalized stomach in some rodents and the hyrax and by an additional pair of colonic appendages in the hyrax. Rodents constitute over 50% of the mammalian species, including a large number of herbivores ranging from 6-g voles to large aquatic beavers and 40-kg capybara (101). They inhabit all types of environment, including the desert species of gerbils and jirds and Arctic lemmings. Landry (164) attributed much of their success to a flexible masticatory apparatus. Anterior movement of the mandible allows the occlusion of incisors outside of their mouth, enabling seizure of prey, clipping of stems and leaves, or removal of bark from shrubs and trees. Posterior movement occludes the molars for the grinding of food material.

View larger version (35K): [in this window] [in a new window]   FIG. 6.   Gastrointestinal tracts of small hindgut-fermenting mammalian herbivores. Note domination of hindgut by an extremely well-developed cecum that is characteristic of herbivorous rodents, lagomorphs, small marsupial herbivores, and hyrax. Unlike other vertebrates, hyrax have both a well-developed cecum and a pair of colonic appendages. Stomach is also expanded and partly compartmentalized in hyrax and some rodents. [Rabbit from Stevens (247). Copyright 1977 by Cornell University. Used by permission of the publisher, Cornell University Press. Rock hyrax from Clemens (50). Copyright J. Nutr., American Institute of Nutrition. Koala from Harrop and Hume (121). Guinea pig and hamster from Stevens and Hume (252). Reprinted with permission of Cambridge University Press.]

The lagomorphs consist of 11 genera of hares and rabbits and one genus of pika that weigh from 400 to 2,000 g (5). They are widely distributed throughout the world, including species that inhabit deserts, arctic tundra, and altitudes up to 6,000 m. The small marsupial cecum fermenters are members of three families of arboreal herbivores: Phascolarctidae (koala), Phalangeridae (cucuses and brushtail possums), and Pseudochieridae (greater gliders and ringtail possums) (137). Hyraxes presently consist of one family and three genera that range from 2,500 to 3,500 g in body weight (216). Tree hyrax are principally arboreal folivores, but rock hyrax inhabits forests, steppes, and mountainous plains up to 3,700 m, and the Sahara hyrax in the mountains of Nigeria can survive practically without drinking water. Nonhuman primates also have a cecum, which is quite well developed in some lemurs and monkeys, but only a few of these species are predominantly herbivores (41).

An enlarged colon is the principal site for microbial fermentation in many of the larger herbivorous mammals, such as the perissodactyls, elephants, wombats, sirenians (manatees and dugongs), orangutans, and gorillas (Fig. 7). Perissodactyls include the equids and white rhinos, which graze on grasses and forbs, and the tapirs and black rhinos, which are browsers (271). Some equids, such as the African and Asiatic asses, inhabit semiarid and arid environments. Elephants are found in the forests and steppes of Asia and Africa, where they graze on grasses and browse on shrubs and small trees and the bark of large trees (4).

View larger version (33K): [in this window] [in a new window]   FIG. 7.   Gastrointestinal tracts of large hindgut-fermenting mammalian herbivores. Note that although a cecum is present, hindgut of these animals is dominated by a long and capacious colon. It is haustrated throughout its length in most species and further compartmentalized in perissodactyls and elephants. Although sirenians also have an expanded stomach, their long colon serves as principal site for microbial digestion. [Rhinoceros and elephant from Clemens and Maloiy (53), with permission from Oxford University Press. Wombat, orangutan, pony, and dugong from Stevens and Hume (252). Reprinted with permission of Cambridge University Press.]

Gorillas and orangutans are chiefly folivores (91). Wombats consist of two genera and three species of marsupial grazers that weigh up to 50 kg and inhabit the forests and semiarid regions of Australia (113). Haustrations extend over the cecum and the entire length of the colon of these species, and the colon is additionally divided into permanent compartments in the perissodactyls and elephants. Manatees inhabit rivers, river mouths, and freshwater lakes, feeding on marine algae, seaweed, and freshwater plants, but dugongs inhabit coastal seas and feed principally on four species of large algae (163).

A large compartmentalized or haustrated stomach is the principal site for microbial fermentation in the remainder of the large herbivores (Fig. 8). This includes most artiodactyls, and the sloths, macropod marsupials (kangaroos, wallabies and rat-kangaroos), and colobus and langur monkeys. The artiodactyls can be divided into the suborders Ruminantia (bovids, sheep, goats, giraffe, antelope, and deer), Tylopoda (new and old world camels), and Suiformes (hippos, peccaries, Malayan pig deer, and pigs). All but a few species of pigs are herbivores with an enlarged, compartmentalized stomach. The stomach of Ruminantia is divided into a large multicompartmental forestomach (reticulum, rumen, and omasum) and a secretory compartment (abomasum) that is similar to the entire stomach of most other vertebrates.

View larger version (35K): [in this window] [in a new window]   FIG. 8.   Gastrointestinal tracts of foregut-fermenting mammalian herbivores. Note extremely large stomach of these animals, which is either haustrated as a result of longitudinal bands of muscle, as seen in kangaroo and colobus monkey, or compartmentalized, as seen in sloth, sheep, and llama. Hindgut is less developed in most of these species, but its continued importance for recovery of electrolytes and water is demonstrated by extremely long colon of camelids. [Kangaroo and sheep from Stevens (247). Copyright 1977 by Cornell University. Used by permission from the publisher, Cornell University Press. Sloth from Stevens (248). Colobus monkey from Stevens (249). Llama from Stevens and Hume (252). Reprinted with permission of Cambridge University Press.]

The rumen and reticulum serve as the major fermentation organ, and the omasum serves principally for the transfer of digesta from the reticulum into the abomasum. Hofmann (129) classified Ruminantia into bulk and roughage eaters that were relatively nonselective in their browsing or grazing, concentrate selectors that browsed on the more nutritious succulent portion of plants, and an intermediate group that varied their feeding habits according to the availability of food. Bulk and roughage eaters, such as cattle and sheep, had the most well-developed forestomach and smallest cecum. Concentrate selectors, such as the dik dik, had a comparatively small reticulorumen and omasum and larger cecum. Development of the forestomach and cecum of the intermediate group fell between these two.

The forestomach of other herbivorous artiodactyls and sloths also is divided into permanent compartments, but the forestomach of macropod marsupials and colobid and langur monkeys is sacculated by haustra, like those seen in the cecum and colon of many mammals (Fig. 8). Sloths are small (4-9 kg) arboreal inhabitants of the forests of Central and South America that feed on leaves, young shoots, blossoms, and fruit. Macropod marsupial are represented by the small (1-3 kg) Potoridae (rat-kangaroos) and the Macropodidae (kangaroos and wallabies), which vary in size from 1-kg hare-wallabies to 70-kg kangaroos and are distributed in a wide range of habitats including arid environments (114). Most of the forestomach of rat-kangaroos consists of a cranial sac that serves for the immediate storage of ingesta (141), but the forestomach of kangaroos and wallabies consists principally of an enlarged, haustrated tube (Fig. 8) that resembles the colon of colon-fermenting herbivores. The small and many of the intermediate-sized macropod marsupials are browsers, but the larger species tend to be grazers.

The Colibidae consist of four genera of langur, snub-nosed, and proboscis monkeys in southeast Asia and one genera of African colobus monkeys with large sacculated forestomachs (91) (Fig. 8). Many occupy tropical rain forests or swamps, but the largest langur, Presbytis entellus schistceus, inhabits conifer and rhododendron forests of the Himalayas at elevations up to 4,000 m, which can be deeply covered with snow.

Digestion of food and passage of digesta require the secretion of large amounts of fluid and electrolytes by the salivary glands, stomach, pancreas, biliary system, and intestine. These are released intermittently into the digestive tract of carnivores and omnivores in association with feeding, but the more continuous feeding and greater microbial fermentation capacity of herbivores requires a more continuous secretion of larger volumes of fluid.

Gut contents represent a small percentage of the body water of most carnivores and only ~4% of the body water of humans, but the gastrointestinal tract of sheep contains 29% of its total body water, with much of this in the forestomach. Table 2 lists the daily secretions of fluid by various components of the digestive system of humans, sheep, and ponies. The digestive system (without the large intestine) of human subjects secreted 7 l/100 kg body mass, which is equivalent to ~35% of the extracellular fluid volume. However, daily secretions into the digestive tract of sheep and ponies were equivalent to >220% of their extracellular fluid volume. This was largely the result of salivary and gastric secretions in the sheep and salivary, pancreatic, and large intestinal secretions in the pony. Ninety-eight percent of this fluid was reabsorbed by the gastrointestinal tract of each of these species. Much of this is reabsorbed by the midgut or small intestine of most species, but the hindgut plays a critical role in terrestrial vertebrates and, particularly, hindgut-fermenting herbivores.

The reticulorumen also serves as a reservoir for water in some desert species. Black bedouin goats can survive 3 days without water, losing 40% of their body weight, and regain their weight rapidly by drinking (239). Slow release of digesta from the forestomach aids in the conservation of water during deprivation and prevents overhydration after drinking.

	III. DIGESTA PASSAGE AND RETENTION TIME

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The time that digesta are retained in a given segment of the gastrointestinal tract can also determine the number and type of indigenous bacteria and the degree of microbial fermentation. Techniques for the use of markers to measure the kinetics of passage for fluid and particulate digesta have been reviewed by Clemens (52), Faichney (86), Warner (275), and Van Soest (269). The gastrointestinal transit time of food or a digesta marker is defined as the interval between its administration as a pulse dose into the stomach and its first appearance in the feces. A much more useful measure is the mean retention time (MRT), which is the average time that a marker remains in the gut.

The rates of food intake and digesta passage in ectothermic vertebrates are highly dependent on the ambient temperature. The temperature sensitivity of a biological function can be expressed by its Q₁₀ value, which is the amount of change over a temperature span of 10°C. Information compiled by Fange and Grove (88) showed a Q₁₀ value of 2.6 for the effect of body temperature on digesta transit time in a group of fish, and Q₁₀ values varied between 2.7 and 3.8 in snakes and lizards (242, 272). Although reduction in body temperature reduced the rates of food intake and increased digesta retention time in desert iguanas, it had no effect on gross energy digestibility in animals that were force-fed in amounts based on the field metabolic rate calculated for each temperature (286). Therefore, the increase in digesta retention time at lower body temperatures appeared to compensate for reduction in the rates of microbial fermentation and absorption of end products.

Horn (130) summarized information on the rate of passage of food particles through the digestive tract of principally freshwater fish. The total gut-emptying time for carnivores ranged from 10 to 158 h, but the time for most herbivores was <10 h. Even an omnivorous hemiramphid, which feeds on seagrass during the day and crustaceans at night, passed seagrass through the gut at about twice the rate of crustaceans. Longer transit times have been recorded in a few marine herbivores (222). One of the most studied herbivorous fish is the grass carp, a freshwater species that can consume its weight in vegetation each day and reduce plant material to 3-mm³ particles with its pharyngeal teeth. However, it lacks a stomach and pyloric ceca and has an intestine only two to three times its body length. An increase in water temperature from 10 to 27°C increased its feeding activity (257) and reduced food transit time from 18 to 8 h (22, 125).

Hindgut retention of digesta appears to have evolved in response to the need of terrestrial vertebrates to conserve electrolytes and water secreted into the upper digestive tract and excreted by the kidneys. The hindgut of adult amphibians, reptiles, and birds exits, along with the urinary tract, into a cloaca (Fig. 9). Retention and resorption of electrolytes and water by some reptiles and most birds is aided by a pacemaker in the cloacal region that generates antiperistaltic waves of contraction that reflux digesta and urine the length of the hindgut. One exception is the ostrich, which does not reflux digesta and urine from its cloaca (78). The urinary and digestive tracts of most mammals develop separate exits before birth, and the colon is generally longer, with its pacemaker located in a more proximal segment. Retention of digesta in the hindgut also results in larger populations of indigenous bacteria.

View larger version (30K): [in this window] [in a new window]   FIG. 9.   Evolution of nephron and hindgut in relation to habitat. Nephrons of fish, reptile, and bird kidneys are limited in their ability to concentrate urine. However, urine is excreted into cloaca of adult amphibians, reptiles, and birds and refluxed along with digesta for recovery of electrolytes and water by hindgut of some reptiles and most birds. Microbial digestion of uric acid also aids in conservation of nitrogen. In most mammals, digestive and urine tracts exit body separately. Recovery of urinary electrolytes and water is aided by kidney's loop of Henle, and nitrogen conservation is aided by microbial digestion of urea. [From Stevens (247). Copyright 1977 by Cornell University. Used by permission from the publisher, Cornell University Press.]

The MRT values for digesta markers in a carnivorous, omnivorous, and two herbivorous reptiles are shown in Table 3. Particles were retained longer than fluid by the gastrointestinal tract of all of these species, and both were retained longest by the herbivores. Birds have a much shorter digesta retention time than reptiles (Table 4). Particulate markers are retained longer than fluid in most birds due principally to their retention in the gizzard, but fluid is selectively retained by the ceca of some species. Björnhag and Sperber (31) found that after ileal contents passed through the colon of geese, turkeys, and guinea fowl, they were mixed with urine in the cloaca and fluid, and fine digesta particles were refluxed along the colon wall and forced through the narrow openings into the ceca. Leopold (166) stated that the feces of gallinaceous birds contained two types of digesta, and one of these had the composition of cecal contents. Administration of digesta markers to the Alaskan rock ptarmigan resulted in prolonged excretion of the particles and a rapid periodic excretion of fluid markers at intervals of ~8.5 h, with an average discharge of ~56% of the cecal contents (106). This would account for the much longer retention of fluid than particles in their gastrointestinal tract (Table 4).

Particles were retained longer than fluid by the stomach of the dog, pig, and rabbit and the large intestine of the dog and pig (Fig. 10). However, fluid was retained by the cecum of the rabbit with a more rapid elimination of particles in the feces. Fluid and fine particles are selectively retained by the cecum of most small mammalian herbivores <10 kg in body weight. Parra (200) found that the relative volume of gut contents in small mammalian herbivores decreased with a decrease in the body mass of the species. This, plus a higher mass-specific metabolic rate, limits the extent to which small animals can delay digesta passage. Selective retention of fluid and small particles concentrates fermentative effort on the more readily digestible fraction and minimizes the gut-filling effect of larger particles. Because of this process, small herbivores are able to utilize plant material of lower protein and higher fiber content than would be predicted on the basis of body size alone.

View larger version (30K): [in this window] [in a new window]   FIG. 10.   Percentage of digesta fluid and particulate markers (±SE) recovered from gastrointestinal tract of dog, pig, and rabbit at various times after administration. Dogs were fed a meat diet; pigs were fed a pelleted, low-concentrate, high-fiber diet; and rabbits were fed a commercially prepared, pelleted rabbit diet. Animals were fed at 12-h intervals for at least 2 wk before study. Digesta markers were administered orally at time of feeding. Fluid markers consisted of polyethylene glycol or 51Cr-EDTA. Plastic markers consisted of polyethylene tubing with an outside diameter of 2 mm, cut into lengths of 2 mm. Animals were killed in groups of 3 at times designated after the meal, and sections of gut were immediately separated by ligatures for recovery of markers. Solid bars, fluid marker; open bars, particulate marker. S, stomach; SI, small intestine; Ce, cecum; C, colon; PC, proximal colon; TC, terminal colon; Fe, feces. [Dog from Banta et al. (19). Pig from Clemens et al. (57). Copyright J. Nutr., American Institute of Nutrition. Rabbit from Pickard and Stevens (207).]

Coprophagy, the ingestion of feces, offers another avenue for the recovery of nutrients produced by gut microbes. Ingestion of maternal feces serves as a source of nutrients and bacteria for neonates in a number of species, and coprophagy has been observed during periods of nutritional deficiency in domestic cats (179) and horses (236). However, coprophagy is a regular practice in many small herbivorous mammals. Diurnal coprophagy has been reported in rabbits, hares, pika, rats, beavers, ground squirrels, lemmings, guinea pigs, chinchillas, capybara, nutria, ringtail possums, and a folivorous lemur, Lepilemur mustelinus leucopus (252).

Ingestion of feces can only benefit an animal if they contain more nutrients than the food they displace. This is overcome in many small mammalian herbivores by cecotrophy, the selective ingestion of highly nutritious feces that are derived from cecal contents (131). Domesticated rabbits excrete dry fecal pellets during the day and soft, moist pellets at night. Dry pellets are the result of a colonic separation mechanism (CSM), which allows the more rapid passage of larger particles into the feces. Interruption of this process results in the formation of cecal contents into soft pellets and their passage through the colon. The nutritional significance of cecotrophy was reviewed by Hörnicke and Björnhag (131) and Björnhag (30). The soft feces of rabbits contain high levels of SCFA, microbial protein, B vitamins, Na, K, and water. They provided up to 30% of the nitrogen intake, and much of this was microbial protein with a high content of essential amino acids. Their content of B vitamins can exceed the animal's needs. The nitrogen content of cecotrophs greatly exceeded that of the diet and normal feces of Scandinavian lemmings, nutria, guinea pigs and chinchillas, and Norwegian and kangaroo rats. Chilcott and Hume (43) estimated that cecotrophs provided the ringtail possum with 58% of its daily intake of digestible energy and 81% of its daily intake of nitrogen.

Björnhag (30) reviewed studies of the CSM in the rabbit and other species. The cecal contents of rabbits are passed periodically into the proximal colon, where fluid and small particles are collected along the walls of the haustra and returned to the cecum by antiperistaltic contractions (28). Larger particles, such as those derived from the thicker and more lignified cell walls of seed hulls, bark, and wood, are moved aborally with cecal digesta outflow. Finely ground diets result in no separation of fluid and particles, or differences in the composition of cecotrophs and other feces. However, the degree of separation increases with an increase in dietary fiber. The ringtail possum, greater glider, and koala appear to have CSMs similar to that of the rabbit. Lemmings and voles have a CSM that selectively returns bacteria to the cecum, compensating for a cecal digesta retention time too brief for their maintenance by replication alone. The CSMs of guinea pigs, chinchilla, and nutria are similar to, but less efficient than, that of lemmings and voles. Colonic separation mechanisms appear to be absent in the brushtail possum and rock hyrax. Although fluid is selectively retained by the equine cecum, this is followed by selective retention of particles and then fluid by the subsequent ventral and dorsal compartments of the colon.

Table 4 lists the MRTs for fluid and particulate digesta markers in the gastrointestinal tract of cecum-fermenting herbivores in relation to the body size of the species. Retention time of fluid was shortest in animals on finely ground, more readily fermentable, pelleted diets and longest in those fed eucalyptus foliage, with no consistent correlation between retention time and body size. Fluid was retained as long or longer than particles by most of these species. Cecotrophy exacerbates this difference in MRTs due to the higher water content of cecotrophs in comparison with other feces. Therefore, the total gastrointestinal tract MRTs in cecotrophic species is the net result of slower gastric release of particles, slower cecal release of fluid, and the recycling of digesta through the tract.

Conversion of guinea pigs from a pelleted to an alfalfa meal diet increased the MRT of both fluid and particles (Table 4). Choshniak and Yahav (45) measured the MRT for particles in voles and fat jirds fed alfalfa hay versus less digestible Panicum (rhodes grass). Voles fed rhodes grass showed a marked reduction in fiber digestibility with no change in the MRTs for particles from those observed on either the alfalfa hay or the pelleted diet shown in Table 4. However, conversion from alfalfa hay to rhodes grass almost doubled the particle MRT in jirds, with no reduction in fiber digestibility. Analysis of the excretion curves indicated that this was due solely to differences in cecal retention time, suggesting that cecal retention time of particles was dependent on the selectivity of the CSM and digestibility of the diet. Rapid fermentation can produce osmotically active end products faster than they are absorbed, with a net diffusion of water into the gut until osmotic equilibrium is reached (9, 47). Thus, except for periods of cecotroph passage, available evidence suggests that the rate at which fluid and small particles leave the cecum is governed by the rate of cecal fermentation and degree of fluid distension. This would explain the longer retention time in guinea pigs fed alfalfa meal, fat jirds fed rhodes grass, and the marsupials on eucalyptus foliage.

Unlike cecum fermenters, the colon-fermenting herbivores (wombats, pigs, equids, orangutans, and gorillas) retained particulate digesta as long or longer than fluid, and the retention time of both was reduced with the addition of more fiber to the diet of wombats and the conversion of equids from a pelleted to a hay diet (Table 4). Domesticated donkeys of desert tribesman in the Middle East showed a longer MRT for particles (37 h) and greater digestive efficiency than horses on alfalfa hay, and the donkeys increased their intake on less digestible wheat straw with no change in particle MRT (143). This suggests that colonic retention time is more dependent on the physical characteristics of the fiber than its rate of fermentation. Colon fermenters also showed no relationship between MRTs and body size (Table 4). Foose (99) found that although the MRT for dye-marked hay tended to increase with an increase in the body size of perissodactyls (tapirs < equids < rhinos), the MRT for elephants fell between that of equids and rhinos, and the MRT of rhinos was less than that of smaller, foregut-fermenting bovids and camels.

The retention time of fluid by forestomach fermenters also appears to be related more to gastric morphology than body size (Table 4). Mean retention times for fluid and particles were extremely and equally long in the sloth, with 65% of this time spent in their multicompartmental forestomach (94). Retention times for fluid and particles also were similar in the potoroo and rufus rat-kangaroos, whose forestomachs have large sacciform segments. However, particles were retained longer than fluid by the rufus hare-wallaby and larger kangaroos, which have longer segments of tubiform stomach, and by the llama, sheep, goat, and ox. The longest retention times for particles were seen in ruminants on a hay diet. Kangaroos maintained their rate of food intake with an increase in dietary fiber (102, 104), but an increase in the fiber content reduces intake of forage by ruminants, because of extended retention of large particles by the reticulorumen. Because of the ventral location of the reticulo-omasal orifice and a filter formed by projecting leafs of omasal tissue, particulate digesta are retained by the reticulorumen of cattle until they are reduced to a size and specific gravity that allows their release (158, 253).

Bedouin goats and donkeys digested wheat straw with equal efficiency (35, 143). However, the goats did so by reducing their intake of food and water on the wheat straw diet, and a reduction in the drinking frequency of goats resulted in a further decrease in food intake and an increase in the MRT of particles in the rumen (35, 36). Comparisons of grazing zebra and wildebeest of the Serengeti plains of Africa showed that the zebra could subsist on a lower quality forage than could the wildebeest if provided with access to sufficient water (197).

Ruminants can also differ in their relative dependence on forestomach versus hindgut fermentation. The principal large herbivores inhabiting arctic tundra are moose, caribou, reindeer, and muskoxen. Moose, which are concentrate selectors with a relatively small reticulorumen and omasum, are confined to the lower Arctic zones, where they adjust to winter conditions by an increase in rumination and digesta retention time (220). Caribou and reindeer are intermediate feeders, and the significantly larger cecum of high-Arctic subspecies of reindeer during winter months suggested that they relied more heavily on cecum fermentation (246). Muskoxen have both the large reticulorumen and omasum of nonselective grazers and large cecal size of concentrate selectors. Postruminal digestibilities of 53-64% indicated that their cecum played a more important role during summer months (245). However, an increase in both the MRT for particulate digesta and the digestibility of organic matter indicated a greater dependence on forestomach fermentation during winter months (1).

	IV. CHARACTERISTICS AND DISTRIBUTION OF INDIGENOUS GUT MICROBES

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Much of our understanding of gut microbiology derives from early studies of the ruminant forestomach. During the first weeks after birth, the forestomach becomes colonized with Escherichia coli aerogenes and streptococci, which are joined by lactobacilli in the suckling animal (79). Weaning is followed by development of the extremely complex microbiota that are characteristic of adult animals (3, 142, 277). Culture counts give estimates of 10¹⁰ to 10¹¹ of predominantly anaerobic bacteria per gram of fluid in rumen contents. Microscopic counts, which include organisms that are dead or require specific culture media, give higher numbers. Table 5 lists the principal bacterial species found in the rumen of sheep and cattle and their fermentative properties. Their interactions are discussed by Van Soest (269), but they collectively ferment carbohydrate into SCFA, utilize protein and other nitrogenous compounds for synthesis of microbial protein, synthesize B vitamins, hydrolyze lipids, and hydrogenate fatty acids.

Although protozoa are much less numerous (10⁴ to 10⁵/g), they can occupy an equal volume of forestomach contents. The predominant species are anaerobic ciliates, which are capable of fermenting carbohydrates, storing starch, digesting protein, hydrogenating fatty acids, and regulating the numbers of bacteria (100, 128, 192, 213). They contribute relatively little to carbohydrate fermentation but store starch and produce protein for subsequent digestion during passage through the abomasum and midgut. Uptake of starch and sugars by protozoa also has a stabilizing effect in ruminants fed high-grain diets (267), where rapid bacterial production of SCFA and lactic acid can result in ulceration of the forestomach, and systemic acidosis and dehydration (74). Defaunation of the rumen impaired the absorption of calcium, magnesium, and phosphorus and changed the peptide patterns of duodenal digesta. Thus the protozoa play an integral role in digestion in ruminants. In addition to the bacteria and protozoa, anaerobic fungi can be found at concentrations of 10³ to 10⁵ zoospores/g fluid digesta in animals on high-fiber diets (97). They contain relatively high concentrations of protein with an amino acid composition similar to that of alfalfa (98) and may be an important source of nutrients for ruminants on low-quality diets.

Bacterial counts similar to those of the ruminant forestomach have been demonstrated in the forestomach of other artiodactyls (camels, llamas, hippos, peccaries), cetaceans (gray, bowhead, and minke whales), macropod marsupials, sloths, colobus and langur monkeys, and the crop and distal esophagus of the hoatzin (252). The forestomach of minke whales included organisms that can digest the chitinous exoskeleton of krill (177, 178). Bacteria were accompanied by 10³ to 10⁶ protozoa/g digesta in the forestomach of camels, hippos, the hoatzin, and some macropod marsupials, but protozoa appeared to be absent from the forestomach of sloths and the colobus and langur monkeys. Colonies of fungal sporangia, like those of the rumen, were found in the forestomach of four species of kangaroos and wallabies (71). Where measured, bacterial counts in the secretory compartment of animals with a forestomach and the simple stomach of other vertebrates tend to be much lower than those of the forestomach. Mean concentrations of 10³ to 10⁴/ml of predominantly aerobic bacteria have been reported in the gastric juice of fasting humans (217).

The numbers of bacteria in the mammalian midgut are generally much lower than those in the rumen. Savage (235) reported that the human small intestine contained 10⁴ to 10⁶ viable, predominantly anaerobic organisms per gram of digesta. Rambaud (217) also reported counts of 10⁵ bacteria/ml in the small intestinal contents of two-thirds of the human subjects. These were predominantly aerobes, but the number increased to 10⁸/ml, with the appearance of enterobacteria and strict anaerobes in the ileum of one-third of the subjects. Mackie and Wilkins (175) found that the counts of anaerobic bacteria in grass-fed horses ranged from 10⁶/g in the duodenum to 10⁸/g in the ileum. The high percentage of proteolytic organisms, particularly in the duodenum of these animals, raised the question of whether they digest or compete with endogenous enzymes.

Although there is little information available on bacterial counts in the midgut of birds, reptiles, or amphibians, aerobes and facultative anaerobes in concentrations of 10³ to 10⁸/g digesta were found in the midgut of a variety of teleosts (132, 167, 265). The number of anaerobes in the intestine of rainbow trout was insignificant, but Trust et al. (264) found 10⁷ to 10⁹ anaerobes/g in the intestine of the grass carp and 10⁸/g in the goldfish intestine. In a review of the bacteria of fishes, Cahill (39) concluded that large populations of facultative and obligate anaerobes were present in the gut of many species.

Chitinolytic bacteria in counts of 10⁷ to 10⁹/g reported in the intestine of a number of marine fish (68, 109) may aid in digestion of chitin in the exoskeleton of insects, crustaceans, and other invertebrates in their diet. Substantial numbers (10⁴ to 10⁵/ml contents) of extremely large (600 × 80 µm) bacteria were found consistently in the intestinal contents of herbivorous surgeonfish in the Red Sea (93) and on the Great Barrier Reef (62). They were identified as gram-positive bacteria related to the Clostridia (6). A study in brown surgeon fish showed that they reduced the amylase, protease, and lipase activity of host enzymes but may contribute to the digestion of lipid (209). Protozoa, in counts of 10³ to 10⁶/g digesta, were also reported in the midgut of the herbivorous herring cale (58).

Bacterial counts of 10⁷ to 10¹²/g have been reported in the hindgut of mammals (humans, vervet and samango monkeys, naked mole rats, capybara), birds (chickens), reptiles (green sea turtles, green iguanas), leopard frogs (252), and the sea chub Kyphosus cornelii (59). Substantial numbers of protozoa also were found in the hindgut of horses, rhinos, elephants, naked mole rats, capybara, green iguanas, and sea chubs. The bacterial species inhabiting the large intestine that have been described for a number of mammals are generally similar to those found in the rumen (3, 278). At least 400 species, representing 40 genera, were isolated from human feces (92, 235). Most were bacteroides (up to 30% of the anaerobes), bifidobacteria (up to 25%), eubacteria, clostridia, and a variety of gram-positive cocci belonging to the genera Ruminococcus, Peptococcus, Peptostreptococcus, and Streptococcus (173). However, bacterial populations associated with the epithelial surface and lumen contents of the hindgut can differ from one another and from those in the feces. They also can vary between segments of the hindgut. Methanogenic bacteria appear to preferentially colonize the distal colon of humans that are CH₄ excretors (208). The epithelial surface of the cecum and proximal colon of the koala is populated with large numbers of bacteria capable of degrading tannin-protein complexes in their normal diet of eucalyptus foliage (195).

Gossling et al. (111) examined the effects of hibernation on the hindgut bacteria of the leopard frog. They found counts of 10¹⁰/g wet wt of the intestinal contents and 10⁹/g wet wt of the mucosal scrapings of frogs before, during, and after hibernation at 4°C. Hibernation reduced the total bacterial count of the hindgut and, in some cases, the variety of bacteria present, but subcultures of isolates showed that the bacteria continued to grow at temperatures near freezing.

	V. FERMENTATION OF CARBOHYDRATES

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Although the metabolic characteristics of individual species of bacteria vary, the end result of their combined fermentation activities is shown in Figure 11. Rumen microorganisms ferment sugars, starches, cellulose, hemicelluloses, and pectins into SCFA, CO₂ , CH₄ , and H₂ . Their extracellular enzymes digest polysaccharides into monosaccharides, which are converted by intracellular enzymes to pyruvate, as in the body cells of vertebrates. However, rather than entering a Krebs cycle for aerobic metabolism to CO₂ and H₂O, pyruvate is reduced anaerobically to short-chain organic acids, principally acetate, propionate, and butyrate plus CO₂ , H₂ , CH₄ , and H₂O. These organic acids were called volatile fatty acids (VFA) in the earlier literature, due to their ready separation from other components of digesta by steam distillation, but they are now referred to as SCFA. Although fermentation of starch produces 20% less energy than its conversion to glucose by endogenous enzymes, microbial fermentation of structural carbohydrates is a tremendous advantage to animals on a high-fiber diet.

View larger version (28K): [in this window] [in a new window]   FIG. 11.   Pathways of carbohydrate metabolism in rumen. [From Van Soest (269).]

The total concentration of SCFA in the forestomach of sheep and cattle varies between 60 and 120 mM, depending on diet and time after feeding (206). The rate of SCFA production depends on the substrate: soluble carbohydrate (starches and sugars) > pectin > cellulose (Fig. 12A). In animals fed hay or other roughage, the SCFA consist of 60-70% acetate, 15-20% propionate, and 10-15% butyrate. The acetate-to-propionate ratio is reduced by either an increase in the concentration of soluble carbohydrates or a reduction in rumen pH, and both of these SCFA are replaced by lactate at a pH <5.0 (Fig. 12B).

View larger version (20K): [in this window] [in a new window]   FIG. 12.   A: rate of fermentation of alfalfa components in rumen. [From Baldwin et al. (18). Copyright 1977, with kind permission from Elsevier Science Ltd., The Boulevard, Langford Lane, Kidlington 0X5 1GB, UK.] B: relationship between ruminal pH and proportions of acetic, propionic, and lactic acid produced. [From Kaufmann et al. (152). Reproduced by kind permission of Kluwer Academic Publisher, Lancaster, UK.]

Rumen gases vary in both their rate of production and their composition with time after feeding (276). Carbon dioxide is derived from fermentation of carbohydrate and the neutralization of SCFA with HCO₃ . Methane production is directly proportional to acetate production and inversely proportional to the production of propionate, but it also depends on other factors that affect the growth and replication of methanogenic organisms. It appears to be almost totally derived from reduction of formate, H₂ , and CO₂ , which accounts for the low concentrations of H₂ in the rumen, except for the first few days of a fasting period. Nitrogen and O₂ are added from swallowed air, and N₂ can diffuse into the rumen from the blood as well. Oxygen is rapidly reduced by rumen microorganisms, and some of the CO₂ is directly absorbed into the blood, but much of the CO₂ and most of the CH₄ produced in the rumen is removed by eructation. Kleiber (159) found that an adult cow on a diet of 5 kg hay lost 191 liters of CH₄ through eructation and flatulence, which was equivalent to a 10% loss of their daily digestible energy intake.

View larger version (24K): [in this window] [in a new window]   FIG. 13.   Mean (±SE) values for concentrations of volatile fatty acids (VFA) along gastrointestinal tract of raccoon (Procyon lotor), dog (Canis familiaris), pig (Sus scrofa), bush baby (Galago crassicaudatus), vervet monkey (Cercopithecus pygerythrus), and pony (Equus caballus). All animals were fed at 12-h intervals for 3-4 wk before study. Raccoons, pigs, and ponies were given same pelleted diet. Each value represents mean from 12 animals, sampled at 2, 4, 8, and 12 h after feeding. Sections of tract were oral (S1) and aboral (S2) halves of stomach, 2 or 3 equal segments of small intestine (SI1 , SI2 , SI3), cecum (Ce), and 2 or 3 segments of colon (C1 , C2 , C3). [Pony from Argenzio et al. (14). Pig from Clemens et al. (57). Dog from Banta et al. (19). Raccoon from Clemens and Stevens (55). Copyright J. Nutr., American Institute of Nutrition. Bush baby and vervet monkey from Clemens (51). Reprinted with permission of Cambridge University Press.]

View larger version (36K): [in this window] [in a new window]   FIG. 14.   Volume, net transmucosal exchange of water, and net appearance and disappearance of volatile fatty acids (VFA), protein, and urea plus ammonia nitrogen in pony large intestine as a function of time. All values, other than volume, are corrected for exchange between segments resulting from digesta flow. [Adapted from Argenzio (7) and Wootton and Argenzio (280).]

View larger version (20K): [in this window] [in a new window]   FIG. 15.   Bacterial metabolism of nitrogen and fermentation of carbohydrates in hindgut of mammals. A: bacterial fermentation of dietary and endogenous carbohydrates to short-chain fatty acids (SCFA), which are largely absorbed before defecation. B: resident bacteria also convert protein and other nitrogen-containing compounds into ammonia and bacterial protein. [Modified from Stevens and Hume (252) and Wrong and Vince (283).]

View larger version (17K): [in this window] [in a new window]   FIG. 16.   Hypothesis for mechanisms of short-chain fatty acid transport across rumen and hindgut epithelium. [Modifications and combination of models from Stevens et al. (250) and Titus and Ahearn (262).]

	VI. PRODUCTION OF MICROBIAL PROTEIN AND RECYCLING OF NITROGEN

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The amount of microbial protein produced in the rumen is closely related to the rate of SCFA production. About 9-10 g microbial protein are synthesized per mole of ATP produced in the fermentation (69). Only 10% of the substrate energy is released as ATP, with most of the remainder appearing as SCFA. The SCFA are too highly reduced to be available to the microbes; they are instead absorbed and oxidized in the aerobic environment of the host animal's tissues. Anaerobiosis sets the limit to the amount of amino acids that can be supplied by the rumen microbes to the animal. This is sufficient for maintenance and slow growth, but for higher levels of production of meat or milk, the diet must include protein that escapes degradation in the rumen for digestion in the small intestine. Only 10-20% of the protein of fresh forage escapes ruminal degradation, but the proportion escaping can be increased by treating the forage with heat or with chemicals such as formic acid or formaldehyde.

The nitrogen required for microbial protein synthesis is also of endogenous origin. This includes urea, a waste product of protein metabolism in the mammalian liver. Much of the urea is normally excreted via the kidneys, but in ruminants, loss of urea nitrogen is reduced by recycling through the forestomach. This is achieved by release of urea into the saliva and by the diffusion of urea down a steep concentration gradient from blood across the rumen wall, which is maintained by the ureolytic activity of bacteria attached to the ruminal epithelium (80, 134). Therefore, much of the waste from nitrogen metabolism joins the rumen ammonia pool, and a portion is incorporated into microbial protein, then absorbed as amino acids after its digestion in the small intestine. Excess ammonia is absorbed and converted to nonessential amino acids and urea by the liver.

On low-protein diets, nitrogen recycling can contribute the greater part of the protein flowing out of the ruminant forestomach. Urea recycling also reduces the amount of water required for the renal excretion of urea. Conversely, a restriction of water intake reduces urea excretion and increases the rate of urea-nitrogen recycling through the forestomach (103, 170, 185, 203, 204, 268).

Urea-nitrogen recycling has been also reported in the forestomach of camelids, kangaroos, and wallabies. Camels fed low-protein dry desert grass recycled 95% of the urea synthesized by their liver (185). Wallabies on a low-protein diet recycled 84% of their endogenous urea (156). Dellow and Hume (70) followed the fate of several dietary components along the tubular stomach of kangaroos (Fig. 17). Soluble carbohydrate disappeared shortly after its entrance from the esophagus, indicating rapid fermentation to SCFA. In contrast, the disappearance of fiber indicated a continuous, but slower, fermentation of ~60% of the digestible fiber by the time it reached the distal end of the forestomach. However, the protein levels increased to 115% of intake in the proximal forestomach, because of the incorporation of endogenous nitrogen, mainly from urea, into microbial protein. The subsequent decline in protein concentration along the foregut was the net result of the continued recycling of urea nitrogen into microbial protein and the degradation of dietary and microbial protein by proteolytic bacteria.

View larger version (22K): [in this window] [in a new window]   FIG. 17.   Pattern of digestion and absorption of organic matter (a), acid-detergent fiber (b), crude protein (c), and total soluble sugars (d) along stomach of eastern gray kangaroos fed chopped alfalfa hay ad libitum. Sites of sampling were sacciform forestomach (SFS), tubiform forestomach (TFS), and hindstomach (HS). [From Dellow and Hume (70).]

Urea is released into all segments of the mammalian intestine, but nitrogen recycling is most effective in the hindgut, where a steep transepithelial concentration gradient is maintained by large numbers of ureolytic bacteria at the lumen surface. Figure 15B shows the major substrates degraded for the production of ammonia and synthesis of microbial protein in the hindgut of mammals. These include endogenous nitrogen contained in urea, creatinine, digestive enzymes, mucus, and sloughed cells as well as nitrogen contained in dietary residues (281). Most of the amino acids appear to be derived from mucin and amino acids that are least efficiently absorbed from the small intestine. High concentrations of urea found in the colon of germ-free rats (168), the experimentally cleansed colon of humans (282), and the perfused colon of sheep and goats (84) indicate that most of the urea entering the hindgut is hydrolyzed to ammonia by bacteria attached to its epithelial surface. Most of the ammonia is either incorporated into microbial protein or absorbed and recycled to the liver for synthesis of nonessential amino acids or urea.

Urea nitrogen was extensively recycled through the hindgut of the rabbit (219), pony (214), rock hyrax (140), greater glider and brushtail possum (95), and donkey (144). Nitrogen balance was maintained in rabbits on a low-protein diet by infusion of urea into the cecum (233), and utilization of urea by the hyrax hindgut increased with either a reduction in dietary protein or restriction of water (140), in a manner similar to that of the ruminant forestomach. The recycling of urea nitrogen improved the utilization of dietary energy by maintaining higher numbers of bacterial populations in the hindgut of the rock hyrax, greater gliders, and donkeys. Conversion of fat jirds, donkeys, and bedouin goats from alfalfa or lucerne hay to rhodes grass or wheat straw increased the recycling of urea from 53 to 89%, 16 to 75%, and 40 to 69%, respectively, with no loss in body weight (37, 144, 284). Black bears recycled 20% of their endogenous urea through their digestive tract during hibernation, when they do not drink, eat, or defecate (116). Recycling of urea also was demonstrated in the gut of sharks (160) and the gulf toadfish (274).

Uric acid, the principal waste product of protein metabolism in reptiles and birds, does not readily defuse across the gut epithelium. However, uric acid was converted to ammonia and microbial protein after the reflux of urine from the cloaca to the ceca of chickens (181) and ptarmigan (184) and increased with a reduction in dietary protein (30). Birds conserve water by urinary excretion of nitrogenous wastes and electrolytes in osmotically inactive uric acid-urate complexes into the cloaca and reabsorption of urinary and digesta electrolytes by the hindgut. Boykin and Braun (32) found that the ureteral urine of chickens contained 4.7 mmol uric acid and 4.2 g protein/l. Because uric acid has an aqueous solubility of only 0.4 mM, they suggested that the protein served as a coating to maintain uric acid in small spheres (0.5-15 µm in diameter) and prevent its precipitation as large, damaging crystals during passage through the urinary tract. Microbial degradation of uric acid to ammonia, with subsequent absorption of ammonia from the cecum, could play an important role in the nitrogen economy of birds (33).

Ammonia, like SCFA, is present in undissociated (NH₃) and ionized (NH₄) forms. However, with a pK of ~9, it is present principally as NH₄ ions at the normal pH of rumen and hindgut contents. As with SCFA, ammonia was assumed to be absorbed as the more lipid-soluble NH₃ . However, the pH and concentration of ammonia at the lumen surface, where much of the microbial hydrolysis of urea occurs, is unknown. Furthermore, there is some evidence for carrier-mediated exchange of NH₄ and Na ions by the rat ileum (161) and the rectal pad of locusts (205).

Microbial degradation of endogenous and microbial protein in the hindgut by proteolytic bacteria also produces amino acids. Carrier-mediated absorption of amino acids has been demonstrated in the ceca of birds and the colon of chickens on a high-Na diet (241). However, only passive diffusion of amino acids has been shown in the hindgut of adult tortoises (16), rats (24), prairie voles (138), and other mammals, and undigested microbial protein is voided in the feces and lost in the absence of coprophagy.

	VII. PRODUCTION AND ABSORPTION OF VITAMINS

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Gut microbial cells also synthesize B vitamins, a complex of 10 separate water-soluble compounds. Ruminants do not require a dietary source of B vitamins because they are synthesized by the microbes in their forestomach and subsequently absorbed from the small intestine. Microbial synthesis of vitamin B₁₂ requires Co, and the low efficiency of Co incorporation into microbial vitamin B₁₂ (3%) accounts for its relatively high requirement in ruminants compared with nonforegut fermenters, which require preformed vitamin B₁₂ (269). The same is assumed to be true for other foregut-fermenting herbivores. The B-complex vitamins appear to be absorbed mainly from the small intestine by carrier-mediated, Na-dependent transport mechanisms (149, 225).

B vitamins also are synthesized by hindgut bacteria, but the extent to which they are absorbed from the hindgut is unclear. Wrong et al. (282) concluded that there was good evidence that nicotinic acid, riboflavin, pantothenic acid, thiamin, biotin, pyridoxine, folic acid, and vitamin B₁₂ are synthesized by microbes in the human colon, and all but the first three were absorbed to some degree. Kasper (150) demonstrated that thiamin was absorbed from the rat cecum, and Sorrell et al. (244) found that pantothenic acid, pyridoxine, and B₁₂ were absorbed equally well after their oral versus large intestinal administration to humans. In contrast, limited uptake from the hindgut is suggested by several studies that showed that rats fed a diet lacking riboflavin, pantothenic acid, biotin, pyridoxine, folic acid, or vitamin B₁₂ showed severe deficiencies unless they were allowed to ingest their feces.

The data in Table 6, extracted from National Research Council reports on nutritional requirements (187, 189), also suggest limited B vitamin uptake from the hindgut of several small hindgut fermenters. The degree to which the mouse, guinea pig, and rabbit require a dietary source of these vitamins was inversely proportional to the degree that they recycle microbially synthesized B vitamins to their stomach and small intestine. Thus rabbits, which are cecotrophic, appeared to be independent of a dietary source of all but three B vitamins. However, guinea pigs, which are coprophagic but not cecotrophic, require 7 of the 10 B vitamins in their diet. Laboratory mice ingest little of their feces and require all of the B vitamins in their diet.

	VIII. MICROBIAL DETOXIFICATION

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Plants have evolved a wide range compounds (plant secondary metabolites, PSM) as protection against being eaten by animals, with the coevolution of detoxification mechanisms in either the tissues (principally the liver) of herbivores (46) or in the microbes inhabiting their gut. Foregut fermenters can often detoxify or reduce the toxicity of protected plants, although biotransformation in the gut can enhance the toxicity of some PSMs as well (180). Microbial adaptation to PSM often involves the induction of specific enzymes in a consortium of microbes and the growth of subpopulations. Microbial adaptation may also involve the selection of mutants with altered enzyme specificities or novel metabolic activities (180). Detoxification of PSM may have played an important role in defining the niche occupied by some foregut-fermenting herbivores.

	IX. DISEASES ASSOCIATED WITH MICROBIAL FERMENTATION

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Oral administration of broad-spectrum antibiotics can destroy indigenous microorganisms, and prolonged sequestration of digesta in any segment of the gastrointestinal tract, due to torsion, volvulus, herniation, or postsurgical adhesions, can result in their replacement with pathogenic organisms. However, a number of diseases can be attributed to inappropriate diets or feeding schedules.

Engorgement of grain, fruit, or other foods containing high levels of rapidly fermentable carbohydrate or rapid conversion to a high-grain diet can result in a fulminating production of SCFA and depression of the pH in the rumen of cattle, with an increase in lactobacilli and production of lactic acid (Fig. 12, A and B). High levels of SCFA and lactic acid result in hypertonic rumen digesta and systemic dehydration, and rapid absorption of these organic acids can produce rumen atony, ulceration of the forestomach epithelium, and systemic acidosis (74). High-concentrate diets also produce bloat or tympani, because of the rapid production and entrapment of fermentative gasses (75), with intraruminal pressures that can result in cardiovascular collapse and death. They can also produce distension and displacement of the abomasum (258). Increased concentrations of SCFA may also account for the abomasal ulcers found in calves and adult cattle fed high-concentrate diets. The high incidence of ulcers in the stratified squamous epithelium of the stomach of pigs subjected to intensive husbandry practices may be similarly attributed to the high levels of SCFA and low pH in this region (8).

Any condition that leads to malabsorption of carbohydrates in the midgut can result in diarrhea due to their hindgut fermentation to SCFA and lactic acid at rates too rapid for efficient absorption. This includes the ingestion of lactose-containing milk or milk products by neonates or adults with an inherent lactase deficiency, or infectious diseases that interrupt the normal processes of carbohydrate digestion and absorption. Neonates are particularly susceptible. Transmissible gastroenteritis (TGE) is characterized by atrophy of the villi in the small intestine, profuse diarrhea, and 100% mortality in pigs infected the first week after birth, but only a 2% mortality rate in pigs that are infected at three or more weeks in age. Argenzio et al. (11) found that TGE produced a marked increase in the volume of fluid secreted into the small intestine and a twofold increase in the amount of fluid presented to the hindgut. Three-day-old pigs were unable to ferment carbohydrate and absorb the excess fluid, but the more highly developed hindgut of older pigs converted most of the unabsorbed carbohydrate to SCFA, resulting in a sixfold increase in the absorption of water.

High levels of dietary starch also resulted in high concentrations of SCFA and lactic acid, a reduction in pH, and atony in the cecum of cattle (258) and horses (47). The cecal contents of horses showed a 25-fold increase in lactate concentration and a one-unit reduction in pH, which remained low for 5-6 h after feeding even after prolonged adaptation to the diet. At a pH of 5.5 or less, colonic epithelium can be severely damaged or destroyed by either SCFA or lactic acid (10). Therefore, rapid production of these organic acids and gas could account for the high incidence of large intestinal torsion, impaction, and colic in horses fed high-concentrate diets.

Secretion and absorption by the equine hindgut is also influenced by the feeding schedule. Horses that would normally spend 12-16 h of the day grazing are often given one or two meals of a pelleted ration for convenience in feeding. The large intestine of ponies fed a pelleted hay-grain diet at 12-h intervals showed marked cyclic changes in volume (Fig. 14). This was most marked in the ventral colon, which underwent a fivefold increase in volume during the first 8 h after feeding. Clarke et al. (48) found that the plasma volume of ponies fed at 12-h intervals dropped 15% within 1 h after feeding, followed by a return to normal values within 2 h and a subsequent, smaller reduction 6 h after feeding. They attributed the initial reduction to salivary and pancreatic secretions and the second reduction to the hindgut response to increased microbial fermentation. The reduction in plasma volume was associated with increased plasma levels of renin and aldosterone. Release of renin from the kidney promotes the release of angiotensin. Angiotensin reduces urinary excretion and stimulates both thirst and the release of aldosterone, which stimulates Na and water absorption by the kidney and hindgut. Renin and aldosterone concentrations returned to prefeeding levels by 12 h after the meal. Ponies fed at 2-h intervals showed neither the cyclic changes in large intestinal and plasma volume nor the increase in renin or aldosterone levels.

Early studies of Burkitt (38) showing a relationship between low-fiber, high-protein, high-fat diets and the higher incidence of colorectal cancer and other diseases of the human large intestine in affluent Western civilizations have been confirmed by numerous epidemiological and case-control studies (147). However, the reasons are unclear. Insoluble fiber (cellulose, lignin, and some hemicellulose) can reduce digesta retention time and increase the volume of digesta in the large intestine. Soluble forms of dietary fiber (pectins, gums, and hemicellulose) can form gels that may sequester potential carcinogens, and all forms but lignin can be fermented to SCFA (269). Although wheat bran and cellulose had a protective effect against colonic cancer, more rapidly fermentable carbohydrates (corn bran, pectin, carrageenan, agar, and metamucil) enhanced the development of chemically induced tumors in laboratory animals (172). Lack of enhancement in germ-free animals suggested that the end products of fermentation were the causative agents, but SCFA, particularly butyrate, also play an important role in the maintenance and repair of large intestinal epithelium (285).

Colonic cancer also has been attributed to the toxic effect of high levels of ammonia produced by microbial catabolism of protein. Administration of ammonium acetate increased the incidence of chemically induced colonic cancer in rats (63), and perfusion with ammonium acetate or ammonium chloride damaged and decreased the life span of colonic epithelial cells (135). Ammonia concentrations increased with an increase in dietary protein, and an increase in dietary fat increased the pH of colonic digesta, suggesting that the damage was due to absorption of the more lipid-soluble NH₃ (136). There also is evidence that dietary fat may promote colonic carcinogenesis by influencing the amount and type of microbial metabolism of bile acids and cholesterol. Therefore, the advantages of a high-fiber, low-protein, low-fat diet may be due to either an increase in the digesta passage, digesta volume, or population of indigenous microbes available for destruction of toxic substances, or to an decrease in the rate of microbial production of SCFA, ammonia, or other agents toxic to the epithelial cells.

	X. CONCLUSIONS

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Bacteria colonize the gastrointestinal tract of all vertebrates. The longer retention time and relatively neutral pH of digesta in the hindgut of most terrestrial vertebrates and the foregut of a few of these species result in large numbers and greater diversification. These bacteria are accompanied by colonies of protozoa in the foregut or hindgut of some herbivores and by colonies of fungi in the forestomach of a few of these species. The bacteria produce SCFA by fermentation of carbohydrates, convert nitrogenous compounds into ammonia and microbial protein, and synthesize B vitamins. Protozoa can store starch and synthesize protein and appear to aid in the absorption of magnesium and phosphorus. Fungi can also synthesize protein useful to their host.

Hindgut bacteria produce substantial levels of SCFA by microbial fermentation of dietary starches that escape digestion in the upper digestive tract and the endogenous carbohydrates contained in mucus and sloughed epithelial cells. They also produce ammonia and microbial protein from digestion of amino acids that escape absorption in the midgut, and from urea (or uric acid), digestive enzymes, mucus, and sloughed cells released into the gut. Absorption of SCFA provides energy for the hindgut epithelium and makes a minor contribution to the maintenance energy of carnivores and most omnivores. It also plays a critical role in hindgut absorption of Na and water. Absorption of ammonia serves to recycle urea- or uric acid-nitrogen, conserving both nitrogen and water. Some of the microbial protein is digested by proteolytic bacteria, but, with the exception of birds, it appears that the hindgut lacks the ability to actively absorb amino acids. Hindgut absorption of B vitamins also appears to be limited. Therefore, the nutritional contributions of microbial protein and B vitamins synthesized by hindgut microbes are restricted in animals that do not practice coprophagy.

The greater gut capacity and digesta retention time of most herbivores allow the additional fermentation of structural carbohydrates of plant cell walls and increases the amount of nitrogen that can be recycled for hepatic synthesis of nonessential amino acids. Use of the midgut as the principal site of microbial fermentation in most herbivorous fish, larval amphibians, and one herbivorous species of turtle and bird would seem to provide a conflict between microbes and endogenous digestive enzymes. However, the cecum or colon serves as the principal site for microbial fermentation in most herbivorous reptiles, birds, and mammals, and the foregut is the principal site in one species of bird and a number of mammals. Increased production and absorption of SCFA provides a substantial amount of the maintenance energy required by these animals. Hindgut fermenters can recover nutrients from food digested by endogenous enzymes before its less efficient fermentation by bacteria. Coprophagic hindgut fermenters and foregut fermenters can utilize their midgut for digestion of microbial protein and absorption of amino acids and absorption of the B vitamins synthesized by bacteria.

Most small herbivorous birds and mammals compensate for a limited gut capacity and higher rate of metabolism by selective retention of fluid and small particles in their cecum and more rapid excretion of larger digesta particles. Some species can adapt to a reduction in the digestibility of dietary fiber by increasing the cecal retention time of fluid, bacteria, and, to varying degrees, the smaller digesta particles. Selective retention of fluid, and the ingestion of water-rich feces by cecotrophic species, also aids in the conservation of water. The ability of cecum fermenters to recover soluble nutrients in their upper digestive tract gives them an advantage over small forestomach fermenters. Their ability to increase cecal retention time on high-fiber diets and the recovery of microbial protein and B vitamins by cecotrophic species also give them an advantage over small colon fermenters. This could account for the absence of colon fermenters and rarity of forestomach fermenters among small avian and mammalian herbivores. However, increased cecal retention time limits their ability to increase food intake and recover soluble nutrients on poorly digestible high-fiber diets.

Greater gut capacities and lower mass-specific energy requirements allow larger herbivores to adapt to less digestible diets by either increasing digesta passage, food intake, and soluble nutrient recovery, as seen in colon fermenters, or increasing digesta retention and fermentation time in a forestomach. Some colon fermenters can adapt as well or better than forestomach fermenters to low-quality forage. However, prolonged retention of plant particles in the forestomach increases the extent of fiber fermentation and favors the destruction of some plant toxins. Foregut-fermenting herbivores also have the advantage of utilizing both their midgut and hindgut for recovery of the large secretions of fluid and electrolytes required for microbial fermentation. This ability to adapt to conditions where forage, water, or both are sparse accounts for the fact that ruminants are the only large herbivores found at high altitudes and in arctic or extremely arid climates.

The absence of herbivores among adult amphibians and low percentage of herbivorous fish and reptiles have been attributed to an inefficient masticatory apparatus, limited gut capacity, and the effect of low ambient temperatures on food intake, digesta transit, and microbial fermentation in ectotherms. However, limitations in gut capacity would not explain the rarity of large herbivorous fish and reptiles, and reductions in body temperature result in compensatory reductions in the rate of digesta passage and requirements for nutrients. Therefore, the rarity of herbivorous fish and reptiles may be due to additional factors related to ectothermy. The restrictions of flight on the capacity and distribution of gut contents has also limited the number of herbivorous birds to a few species that are either flightless or limited in their flight.

The extreme success of mammalian herbivores can be attributed to endothermy, an efficient masticatory apparatus and an expansion in gut capacity. The parallel success of herbivorous dinosaurs suggests that they were endotherms with an efficient masticatory apparatus and similar suite of digestive strategies, according to their body size, habitat, and the quality and availability of forage and water.

Many of the gastrointestinal diseases of domesticated and captive herbivores can be attributed to inappropriate diets and/or feeding schedules. The digestive tract of most herbivores is constructed for almost continuous feeding on a diet high in plant fiber and relatively low in protein, starches, and sugars. Therefore, major problems can arise from the intermittent feeding or diets that contains low levels of fiber and high levels of rapidly fermentable starch or sugar. The hindgut of omnivores with a well-developed large intestine also appears to require a minimal amount of plant fiber for normal function, as evidenced by the higher incidence of cancer and other diseases in the colon of humans on low-fiber diets. The protective effects of dietary fiber may be attributed to a more rapid rate of digesta passage, to a greater volume dilution of hindgut digesta, or to differences in the quantity or composition of the bacterial populations.

  

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