1. INTRODUCTION
2. OVERVIEW
3. ANATOMY AND GENERAL PHYSIOLOGY OF THE GUT
4. CHARACTERISTICS OF ENZYMES AND OTHER DIGESTIVE SECRETIONS
5. METHODS OF MEASUREMENT AND ANALYSIS FOR DIGESTION STUDIES
6. DIGESTIVE ANATOMY OF SELECTED FISHES
7. REFERENCES
L. S. Smith
University of Washington
Seattle, Washington
Literature about digestive functions in teleost fishes is extensive in some areas, almost nil in others. The material to be presented here is not a comprehensive review of that literature because there are two recent reviews (Harder, 1975; Kapoor et al., 1975), which have extensive bibliographies on both the anatomy and physiology of teleost digestive systems. Readers desiring additional information should consult both reviews because they tend to be complementary, with Harder emphasizing anatomy of European fishes while Kapoor and co-authors emphasize digestion in North American and Asian fishes.
The general organization of this chapter begins with the anatomy of the gut, proceeding from anterior to posterior. Then the route is retraced to discuss the organ physiology of the digestive tract. A third pass through the tract discusses details of the enzymes. Then the typical methods used for studying digestion are described as a basis for the reader to make detailed comparisons among different methodologies and specific results or to perform his own experiments. Finally, some anatomical and functional comparisons are made for four species of fish with distinctly different feeding habits: carp (plant-oriented omnivore), catfish (animal-oriented omnivore), milkfish (specialized microplanktivore), and rainbow trout (carnivore). These comparisons are best made when accompanied by dissections of specimens of each species, although drawings are provided for readers having no access to the fish.
2.1 Definition of the Gut and its Subdivisions
2.2 Evolution and Ontogeny of the Digestive Tract
2.3 Generalizations
The gut is a tubular structure beginning at the mouth and ending at the anus. It is commonly divided into four parts. The most anterior part, the head gut, is most often considered in terms of its two components, the oral (buccal) and gill (branchial, pharyngeal) cavities. The foregut begins at the posterior edge of the gills and includes the oesophagus, the stomach, and the pylorus. In fish, such as the cyprinus, which lack both a stomach and pylorus, the foregut consists of the oesophagus and an intestine anterior to the opening of the bile duct. This posterior demarcation is arbitrary and primarily for convenience during gross dissection and may have little relation to the functional aspects. The midgut includes the intestine posterior to the pylorus, often with no distinct demarcation posteriorly between it and the hindgut. The midgut often includes a variable number of pyloric caecae (pyloric appendages) near the pylorus, although pyloric caecae are always absent in fishes which lack stomachs. The midgut is always the longest portion of the gut and ma be coiled into complicated loops (often characteristic for each species) when longer than the visceral cavity. In some fish, the beginning of the hindgut is marked by an increase in diameter of the gut. The posterior end of the hindgut is the anus. Only rarely is there a hindgut caecum in fish comparable to that found in mammals. A cloaca (a chamber common to anal and urogenital openings and formed from infolded body wall) never occurs in teleost fish, except the Dipnoi, although it is universal in sharks and rays.
The gut of protochordates consists of a simple, straight tube through which food is propelled by ciliary action. An early elaboration of the gut is seen in lampreys where an infolding (typhlosole) of the gut wall presumably increases the absorptive area of the gut. A similar, but spiral, infolding of the hindgut occurs in sharks, rays, and the coelocanth (Latimeria) in the form of the spiral valve (spiral intestine). The gut wall in lampreys also contains diagonal muscle fibres, although true peristalsis (travelling wave of contraction) is thought not to occur. Teleost fish have a gut which is typical of the higher vertebrates in many respects, although the midgut villi (absorptive papilli) of mammals are absent in fish.
The gut forms very early during embryological development (ontogeny) and shows some of the same stages of development as in the evolution of the vertebrate gut, some larval fish having portions of their gut which are ciliated, for example. The general character and even the length of the gut may change during development. The gut appears to shorten, for example, in fish in which the larval stage is herbivorous and the adult stage is carnivorous. In other fish the gut length remains relatively constant in proportion to body size throughout life.
A number of generalizations about the gut of fishes have been attempted, many of them extrapolated from terrestrial vertebrates. The commonest of these, the observation that herbivores have longer guts than carnivores, appears only partially true in fish. While this may be true in limited groups of fish, it is not universal in teleosts as a whole. Gut lengths have been listed as 0.2-2.5, 0.6-8.0 and 0.8-15.0 times body length in carnivores, omnivores, and herbivores, respectively. Thus, the longest guts are found in herbivores, but not all herbivores have long guts; i.e., the gut lengths of some herbivores are shorter than those of some carnivores. Part of the explanation lies in the fact that many fish eat a variety of food, sometimes ingested with considerable indigestible material (e.g. mud) which often influences gut length. The size of the food particles - from submicroscopic plankton to whole fish - may also influence gut configuration.
One generalization so far appears to have no exception. In fishes having no stomachs, no acid phase of digestion occurs, even when the midgut develops stomach-like pouches anteriorly. Although gut tissues exhibit great versatility, the midgut appears unable (or does not need) to duplicate the stomach functions.
In general, most studies relating food habits to gut morphology show considerable relationship between the two. However, the gut also retains considerable reserve ability to respond to new foods, new environments, and new opportunities. This versatility has been demonstrated in a number of cases in which a single genus has adapted to new niches and evolved whole new modes of feeding and digestion to utilize otherwise unexploited food resources and done so over rather short evolutionary periods of time.
At the same time, there are usually severe constraints on adaptations to new food. As long as swimming continues to be important to a fish's lifestyle, any major change in body shape, such as a bulging visceral mass resulting from enlarging the stomach or lengthening the midgut, must extract a penalty in terms of increased effort needed for swimming. Feeding mechanisms must not interfere with the respiratory functions of the gills and vice versa. All in all, "packaged" so that any major change in the digestive system would call for major compromises in many other systems. Perhaps the best generalization is that teleost fish maintain an intimate relationship between the form and function of their gut and their food resource. In the final analysis, all of the other life processes continue to function only when sufficient materials and energy are obtained and assimilated via the gut.
3.1 Functional Anatomy of the Gut
3.2 Peristalsis and its Control
3.3 Gastric Evacuation Time and Related Studies
3.4 Digestion and Absorption
3.5 Specific Dynamic Action (SDA)
3.6 Interrelationship between Osmoregulation and Digestion
The mouth exhibits a variety of fascinating adaptations for capturing, holding and sorting food, ratcheting it into the oesophagus and otherwise manipulating it prior to entry into the stomach. Only two which have possible relevance to digestion Will be discussed.
In milkfish (Chanos), the gill cavity contains epibranchial (suprabranchia) organs dorsally on each side, consisting either of simple blind sacs or elaborate, spirally-coiled ducts. The organs occur in several relatively unrelated families of lower teleosts and apparently relate to the kind of food eaten. Those fish with simple ducts all eat macro-plankton and those with the larger ducts microplankton. Although their function is unknown, concentrating the plankton has been suggested as a possibility.
The common carp provides an excellent example of non-mandibular teeth being used as the primary chewing apparatus. Pharyngeal teeth occur in the most fully developed forms of the Cyprinidae and Cobitidae, although many other groups also show some degree of abrading or triturating ability with some part of the gill bars. In carp, the lower ends of the gill bars have a well developed musculature which operates two sets of interdigitating teeth so as to grind plants into small pieces before swallowing them. The grinding presumably increases the rather small proportion of plant cells which can otherwise be successfully attached by digestive enzymes.
Many fish which chew their food have some ability to secrete mucus at the same time and place. This would have some apparent benefit when ingesting abrasive food. Although one might be tempted to equate such secretions with saliva, enzyme activity in the mucus does not appear to have been demonstrated, so the mucus is only partly comparable to saliva.
The oesophagus, in most cases, is a short, broad, muscular passageway between the mouth and the stomach. Taste buds are usually present along with additional mucus cells. Freshwater fishes are reputed to have longer (stronger?) oesophageal muscles than marine fish, presumably because of the osmoregulatory advantage to be gained by squeezing out the greatest possible amount of water from their food (i.e., marine fish would be drinking seawater in addition to that ingested with their food and freshwater fish would have to excrete any excess water).
The oesophagus of eels (Anguilla) is an exception to this general pattern. It is relatively long, narrow, and serves during seawater residence to dilute ingested seawater before it reaches the stomach. A possible conflict between the osmoregulatory and digestive roles of the gut in marine fish in general will be discussed later (Section 3.5).
Fish stomachs may be classified into four general configurations. These include (a) a straight stomach with an enlarged lumen, as in Esox, (b) a U-shaped stomach with enlarged lumen as in Salmo, Coregonus, Clupea, (c) a stomach shaped like a Y on its side, i.e., the stem of the Y forms a caudally-directed caecum, as in Alosa, Anguilla, the true cods, and ocean perch, and (d) the absence of a stomach as in cyprinids, gobidids, cyprinodonts gobies, blennies, scarids and many others, some families of which only one genus lacks a stomach.
The particular advantage of any configuration seems to rest primarily with the stomach having a shape convenient for containing food in the shape in which it is ingested. Fish which eat mud or other small particles more or less continuously have need for only a small stomach, if any at all. The Y-shaped stomach, at the other extreme, seems particularly suited for holding large prey and can readily stretch posteriorly as needed with little disturbance to the attachments of mesenteries or other organs. Regardless of configuration, all stomachs probably function similarly by producing hydrochloric acid and the enzyme, pepsin.
The transport of food from the stomach into the midgut is controlled by a muscular sphincter, the pylorus. The control of the pylorus has not bean demonstrated in fish, but the best guess at this time is that it resembles that in higher vertebrates. The pylorus is developed to various degrees in different species for unknown reasons, in some species even being absent. In the latter case, the nearby muscles of the stomach wall take over this function, which may also include a grinding function by the roughened internal lining. In fish which lack a stomach, the pylorus is absent and the oesophageal sphincter serves to prevent regress of food from the intestine, i.e., in fish lacking a stomach and pylorus, the midgut attaches directly to the oesophagus.
The digestive processes of the midgut have not been studied extensively, except histo-chemically (see Section 4 for details on enzymes), but so far as known resemble the higher vertebrates. The midgut is mildly alkaline and contains enzymes from the pancreas and the intestinal wall, as well as bile from the liver. These enzymes attack all three classes of foods - proteins, lipids, and carbohydrates - although predators such as salmonids may be largely deficient in carbohydrases. The pyloric caecae attached to the anterior part of the midgut have attracted considerable attention because of their elaborate anatomy and their taxonomic significance. Histological examination has proved them to have the same structure and enzyme content as the upper midgut. Another suggestion was that pyloric caecae might contain bacteria which produce B-vitamins as in the rodent caecum. When tested, this hypothesis had no factual basis either. Pyloric caecae apparently represent a way to increase the surface area of the midgut and nothing more. This still leaves an interesting question of how food is moved into and out of the blind sacs which are often rather lone and slim: e.g., in salmonids.
The demarcation between midgut and hindgut is often minimal in terms of gross anatomy, but more readily differentiated histologically - most secretory cells are lacking in the hindgut except for mucus cells. The blood supply to the hindgut is usually comparable to that in the posterior midgut, so presumably absorption is continuing similarly as in the midgut. Formation of faeces and other hindgut functions appear to have been studied minimally, except histologically.
Peristalsis consists of a travelling wave of contraction of the circular and longitudinal layers of muscle in the gut wall such that material inside the gut is moved along. The pharmacology of this system has been investigated in isolated trout intestine demonstrating that an intrinsic nerve network exists to control peristalsis; i.e., cholinergic drugs stimulated and adrenergic drugs inhibited peristaltic movements. The oesophagus arid stomach are also innervated extrinsically by branches of the vagal (cranial X) nerve. No studies appear to have been made so far concerning details of food transport through the teleost gut except for measurements of gastric evacuation time and total food passage time, although gut stasis has been hypothesized to occur in the Pacific salmon, as in domestic animals.
Many studies have been performed relating to developing an optimum feeding schedule, mostly for salmonids, but also including a number of other cultured fish. Variables considered with feeding rate and gastric evacuation time included temperature, season, activity, body size, gut capacity, satiety, and metabolic rate. A relatively consistent finding has been that gastric emptying rate declines more or less exponentially (sometimes linearly) with time. Larger meals first are often, but not always, digested at a faster rate than small meals and the amount of pepsin and acid produced was somewhat proportional to the degree of distension of the stomach. Stomach mobility often increases with the degree of stomach distension also. The appetite, digestion rate, and amount of secretions produced all decreased with decreased temperature, but the secretions also decreased if tested at temperatures in excess of the acclimation temperature. Appetite, i.e., the amount of food eaten voluntarily at one time, appears to be the inverse of stomach fullness, although this does not explain the entire appetite phenomenon. Appetite continues to increase for a number of days after the stomach is empty, indicating that additional metabolic or neural mechanisms are operating. Data on gastric emptying time, digestion rate, and temperature for sockeye salmon have been shown to reflect the underlying phenomenon. Direct comparison of data on digestion among different workers is difficult, because of differences in species, food and methods used.
The total time for passage of food through the gut until the non-digestible portions of a meal are voided as faeces has not commonly been measured. Gastric emptying time and total passage time in skipjack tuna at 23-26 C was about 12 hours with the intestine being maximally filled about five hours after eating and empty after about 14 hours. Defaecation often occurred 2-3 hours after a meal, presumably being material from a previous meal. After a single meal, faeces were found 24, 48 and even 96 hours after the meal. Thus, there is considerable variation in food passage time, presumably relating to the digestibility of the food. Magnuson (1969) commented that the passage rates in skipjack tuna were at least twice as fast as known for any other fish.
The obvious importance of food passage time becomes apparent when one wishes to analyze faeces resulting from ingestion of a specific meal. If one waits to feed a test meal until the gut is completely empty, then the digestion processes observed will be typical only of starved fish. If one feeds the test meal as part of a regular feeding programme, then the problem is to mark the food for appropriate faecal analysis. Thus the problem is not as simple as it might appear at first.
Digestion is the process by which ingested materials are reduced to molecules of small enough size or other appropriate characteristics for absorption, i.e., passage through the gut wall into the blood stream. This generally means that proteins are hydrolyzed to amino acids or to polypeptide chains of a few amino acids, digestible carbohydrates to simple sugars, and lipids to fatty acids and glycerol. Materials not absorbed are by definition indigestible and are eventually voided as faeces. Digestibility ranges from 100 percent for glucose to as little as 5 percent for raw starch or 5-15 percent for plant material containing mostly cellulose (plant fibre). Digestibility of most natural proteins and lipids ranges from 80 to 90 percent.
Digestion is a progressive process, beginning in the stomach and possibly not ending until food leaves the rectum as faeces. Most studies of digestion simply compare the protein, lipid and carbohydrate content of the faeces with that of the feed. A study on digestion in channel catfish by Smith and Lovell (1973) showed continuing digestion (and absorption) of protein during passage through each part of the gut (Table 1). The methods employed in this study are discussed in Section 4 below. The comparison of faeces collected from the rectum and from the water also points out the hazard of incomplete recovery of faecal matter being likely when collection is done from outside the gut. Most of the protein digestion occurred in the stomach, but also continued in the intestine.
Table 1 - Apparent Digestibility of Protein by Channel Catfish 1/
|
Feed |
Stomach |
Upper intestine |
Lower intestine |
Rectum |
Trough |
|
20% protein |
61.6 |
65.4 |
75.0 |
80.9 |
96.7 |
|
40% protein |
61.4 |
72.2 |
86.5 |
96.6 |
98.3 |
1/ from Smith and Lovell, 1973
Temperature and pH play major roles in determining the effectiveness of digestive enzymes as a whole (details for specific enzymes are given in Section 4 below). Although most enzyme production decreases at temperatures above or below acclimation temperature, most enzyme activity (for a given amount of enzyme) increases in proportion to the temperature over a wide range of temperatures.
In general, enzyme reaction rates continue to increase at higher temperatures, even though the temperatures increase beyond the lethal temperatures for the species, until the enzymes begin to denature around 50-60°C. On the other hand, enzymes have limited ranges of pH over which they function, often as little as 2 pH units. Data for channel catfish are probably representative of many teleosts. Acid concentrations (pH) in the stomach ranged from 2 to 4, then became alkaline (pH = 7-9) immediately below the pylorus, decreased slightly to a maximum of 8.6 in the upper intestine, and finally neared neutrality in the hindgut (Page et al., 1976). Fish having no stomach have no acid phase in digestion.
The site of secretion in teleost stomachs appears to be a single kind of cell which produces both HCl and enzyme(s). This contrasts with mammals where two types of cells occur, one for acid and one for enzymes. The production of acid in teleosts is presumably the same as in mammals - NaCl and H2CO3 react to produce NaHCO3 and HCl, with the blood being the source of both input materials, which are later mostly reabsorbed in the intestine. One possible explanation for the loss of stomachs in some species of fish is that they live in a chloride-poor environment and that providing large amounts of chloride ion for operating a stomach is bioenergetically disadvantageous. In addition to acid and enzymes, the stomach wall also secretes mucus to protect the stomach from being digested. As long as the rate of mucus production exceeds the rate at which it is washed and digested away, the gut wall is protected from being digested. When mucus production slows or fails, e.g. during gut stasis, during stressful conditions, or post mortem, the gut wall can be eroded or even perforated by the gut's own digestive enzymes.
Two sites produce enzymes in the midgut - the pancreas and the intestinal wall. The intestinal wall is folded or ridged in simple patterns which can be species specific. Secretory cells for both mucus and all three classes of enzymes develop in the depths of the folds, migrate to tops of the ridges (closest to the gut lumen), and then discharge their products. The pancreatic cells produce enzymes and an alkaline solution which are delivered to the upper midgut through the common bile duct. The control of pancreatic secretions (and the pyloric sphincter) in fish is probably the same as in mammals, but there is no information on teleosts yet.
The physical state of food passing through the gut varies with species and type of food. Fish, such as salmonids, which eat relatively large prey, reduce the prey in size layer by layer. Gastric digestion proceeds in a layer of mucus, acid, and enzyme wherever the stomach wall contacts the food. Food appears liquified only in the midgut and solidifies somewhat again during formation of faeces. Pellets of commercial feed seem to be treated similarly, i.e., pellets get smaller and smaller in size with time, although stomachs of some recently-fed salmonids have been found to contain moderate amounts of liquified pellets. Stomachs of juvenile Pacific salmon captured in the open sea contained a thick slurry of pieces of amphi-pods in various stages of solubilization. Fish whose food contains high levels of indigestible ballast, e.g., common carp feeding on a mixture of mud and plants, probably show minimal change in the appearance or volume of their food while it passes through the gut. Microphagous fish, such as the milkfish (Chanos) whose food starts out as a suspension of fine particles, probably also keep it in much the same form all the way through the gut. In general: there seems not to be the same degree of liquifaction of food in fish as is commonly described for mammals.
Absorption of soluble food could begin in the stomach - it occurs in mammals, but has not been investigated in fish - but takes places predominantly in the midgut and probably to some degree in the hindgut. The sites and mechanisms of absorption are largely unstudied, except histologically. Several histologists have identified fat droplets in intestinal epithelial cells following a lipid-rich meal. Increased numbers of leucocytes in general circulation following a meal by the sea bream and increased number of fat droplets in them have been described (Smirnova, 1966). It was hypothesized that leucocytes entered the gut lumen, absorbed lipid droplets, and then returned to the blood stream. It is clear that the mammalian type of villi with their lymph duct (lacteal) inside are absent in fish, although there is some folding and ridging of the gut wall to increase surface area. Lacteals serve as a primary uptake route in mammals for uptake of droplets of emulsified lipids (chylomicra). Teleost fish have a lymphatic system which includes extensions into the gut wall, but its role in lipid uptake is unknown. Absorption of amino acids, peptides, and simple carbohydrates have been little studied, but presumably they diffuse through or are transported across the gut epithelium into the blood stream. What light microscopists identified as a brush border on the surface of the epithelial cells facing the gut lumen, has now been clarified with electron-microscopy as microvilli; i.e., subcellular, finger like projections of the cell membrane whose greatly increased surface area is probably involved in absorption.
Digested food, particularly proteins, is not fully available to a fish even after it has been absorbed into the blood stream. Amino acids, if used for building new tissue, could be used as absorbed. If amino acids are to be oxidized for energy, however, deamination (removal of the amino group) must occur first - a reaction which requires input of energy. This process, known as specific dynamic action (SDA), can be measured externally in fish as an increase in oxygen consumption beginning soon after ingestion of food followed by an increase in ammonia excretion.
The proportion of amino acids which get deaminated varies with the food and the fish's circumstances. Fish which are not growing because of low temperature or have their ration at maintenance level or below, would deaminate most or all of their amino acids. Fish kept at high rearing temperatures or at high activity levels and therefore having very high metabolic rates would do likewise. On the other hand, fish having rapid growth and high protein intake would deaminate a relatively small proportion of their digested protein, although the absolute quantity of amino acids deaminated could still be large enough to produce a relatively large SDA. The energy for deamination need not necessarily come from amino acids, but will be preferentially taken from carbohydrate or lipid, if available. Thus, salmonid aquaculturists long ago discovered this "protein-sparing" action of limited amounts of inexpensive carbohydrate in the diet as a way of reducing the cost of feed and still achieving a desired level of growth. The protein-sparing action of lipids appears to have been minimally investigated. One can thus minimize SDA costs, but not avoid them completely.
Researchers studying osmoregulation and researchers studying digestion have rarely considered each other's data. Marine fishes drink significant amounts of seawater, a relatively-well buffered solution having a pH of about 8.5, while gastric digestion requires a pH of 4 or lower in most fish. The amount of HCl required just to acidify the seawater would be substantial, that is, if the entire stomach gets flooded with seawater. There are several likely alternatives, however. In fish with Y-shaped stomachs, the seawater could travel directly from the oesophagus to the pylorus, and traverse only a small fraction of the stomach surface. If, at the same time, digestion functioned primarily as contact digestion, then it could be largely separated from osmoregulation. On the other hand, marine salmon stomachs have been found to be filled with a liquid slurry which would prevent such separation. In such cases, alternation of digestion and seawater drinking might be possible, although fish whose stomachs seemed continuously filled, and therefore would have no time for drinking, have also been observed.
The pH of seawater should cause little or no problem with intestinal digestion. Too high a salt content in the intestine might exceed the operational range of some enzymes and thus reduce the rate of digestion. However, one of the functions of the stomach (and in eels, the oesophagus) in osmoregulation is to dilute the incoming seawater until it is approximately equal to the osmolarity of blood, thus protecting the intestine.
The final osmoregulatory product of the gut is a rectal fluid composed of magnesium and other divalent ions having about the same total concentration as blood. Preliminary data from scale loss studies indicated that death occurred from toxic levels of magnesium in the blood. A possible cause of the high magnesium is that gut peristalsis stopped, leaving the rectal fluid to accumulate and the magnesium ions to be reabsorbed instead of being excreted.
Thus, digestion and osmoregulation are so inter-related that problems in one system could disrupt the functions of the other. Exactly how fish normally avoid such problems is largely unknown.
4.1 Digestion in the Mouth and Oesophagus
4.2 Digestion in the Stomach
4.3 Digestion in the Midgut and Pyloric Caecae
4.4 The Role of Bile, Gall Bladder and Liver in Digestion
The ability of any organism to digest a given substance rests predominantly on whether the appropriate enzyme is present or not and then whether the required conditions for operation of that enzyme exist or not. The following describes the enzymes and their requisite conditions according to their location.
The hard surfaces of the mouths of most teleost; fishes would not lead one to expect any kind of secretion. However, many fish which chew with pharyngeal teeth or similar structures also produce mucus while chewing. Tests of this mucus in a few species for enzyme activity have so far yielded negative results. Likewise, oesophageal mucus cells, when examined histologically, showed no sign of containing any enzymatic granules, although there are reports of gastric-like secretory cells in the posterior oesophagus of a few fish.
Pepsin is the predominant gastric enzyme of all vertebrates, including fish. Optimal pH for maximal proteolytic activity has been reported for several species, as follows:
(a) pH 2 - pike, plaice
(b) pH 3-4 - Ictalurus
(c) pH 1.3, pH 2.5-3.5 - salmon, probably similar for tuna (Kapoor et al., 1975)
Peptic activity has been shown in a number of cultures and commercial species including Anguilla japonica, Tilapia mossambica, Pleuronecthys, both Salmo and Oncorhynchus species, Ictalurus, Micropterus, Lepomis and Perca. The presence of pepsin is so universal in vertebrates having stomachs that its presence can be presumed in fish for which no data is available.
The histochemistry of gastric secretion has been little studied in fish, although there is agreement on the presence of only one type of secretory cell in fish which stains positively for indicators of pepsinogen (pepsin precursor) cells. There is some question whether there may be more than one pepsin present in some fish, but no chromatographic or other tests have been done to investigate this. Several attempts have been made to identify acid-secreting cells, but results were either negative or confusing.
Other gastric enzymes have been proposed, but not firmly identified. Chitinolytic activity with an optimum at pH 4.5 was claimed for the stomach of Salmo irideus, but in most cases is probably from exogenous sources. If fish are like higher vertebrates, then the stomach wall also produces the hormone gastrin which stimulates gastric secretion. A lipase may also be present.
There are two sources of enzymes for the midgut - the pancreas and the secretory cells in the gut wall - with the pancreas perhaps secreting the greater variety and quantities of enzymes in fish. Because of the variety of enzymes present in different species, there have been some attempts to correlate enzyme activities with diet. However, these enzyme studies are fragmentary and histochemical tests are too general. Much remains to be learned about intestinal digestion in fish.
Trypsin appears to be the predominant protease in the midgut. Since the enzyme appears not to have been isolated, most authors have just tested for proteolytic activity over the pH range of 7 to 11 and reported their results as tryptic activity. The diffuse nature of the pancreas in most cases has limited many researchers to making relatively crude extracts from mixed tissues, hampering localization of the enzyme. Tryptic activity has been found in four stomachless species in Japan: Seriola, two basses and a puffer. Since these fish lack pepsin, some such kind of protease in the intestine would be the primary means of protein digestion. Tryptic activity was found in extracts of both the pancreas of perch and Tilapia and in intestinal extracts of Tilapia, all having a pH optimum of 8.0-8.2. Proteolytic activity has been identified in the pyloric caecae and intestine of rainbow trout. In grass carp, tryptic activity was stronger in the intestine than in the pancreas. In a mixture of pancreatic and pyloric caecae tissue from chinook salmon, casein was digested maximally at pH 9. Tryptic activity has also been demonstrated in extracts of liver of Several species, probably because in fish having a diffuse pancreas, pancreatic tissue extends into the liver, around the portal veins, and around the gall bladder. In several of the cases above, when extracts of pancreas were mixed with extracts of intestine, the tryptic activity increased ten-fold or more, suggesting the presence in fish of the enzyme enterokinase in the intestinal wall which activates in mammals the pancreatic trypsin as it reaches the intestine.
Additional pancreatic enzymes are involved in midgut digestion, many of them yet to be discovered. For example, Japanese workers are studying the occurrence and characteristics of a pancreatic collagenase in several Japanese fishes (Yoshinaka et al., 1973). There have also been several reports of chitinolytic activity in some fish which eat crustaceans predominantly. This could also have resulted from bacterial activity.
The occurrence of at least one lipase may be assumed in all fishes and has been demonstrated for a number of species. In carp and killifish extracts of intestine showed lipolytic activity. In goldfish, lipase activity occurred in extracts of a mixture of liver and pancreas and in the intestinal contents. Esterase (another lipase) activity has been found in the liver, spleen, bile, intestine, pyloric caecae and stomach of rainbow trout. Use of radioisotope-labeled lipids in cod suggested that the cod's lipase acted in the same manner as mammalian pancreatic lipase, although it was not considered more than a suggestion that fish lipase is of pancreatic origin. Regardless of origin, some kind of lipase is essential to fish because fatty acids are essential dietary components for fish.
Carbohydrases have perhaps excited the most interest of all the enzymes, particularly because salmonids do not handle the large carbohydrate molecules very well, and many workers wanted to determine the reason. Further, because there are several carbohydrases, the possibility that different enzyme combinations might show adaptations to different diets also intrigued some investigators. Also, herbivorous fish might be expected to have more carbohydrase activity and less tryptic activity than carnivores or omnivores.
Amylase is a widespread starch-digesting enzyme which occurs in human saliva and in pancreatic secretions into the small intestine. Amylase activity has been found in goldfish and bluegill sunfish in extracts of mixed liver and pancreas, oesophagus (contamination from regurgitated food suggested) and intestine, but not in large-mouth bass. Similar activity has been seen as well in rainbow trout, perch, Tilapia, Pacific salmon, cod, common carp, eel, and flounder. In fish with a diffuse pancreas there may be no pancreatic duct and so amylase activity appears in the bile. In mackerel. Scomber spp., which have a compact pancreas, the bile had no amylase activity.
Other carbohydrases identified included glucosidases (rainbow trout, chum salmon, common carp), maltase (common carp, red sea bream, Archosargus, marine ayu, Plecoglossidae), and sucrase, lactase, melibiase, and cellobiase, all of the latter in common carp. The hypothesis that carnivores might be deficient in one or more carbohydrases is largely disproved by the widespread presence of amylase in salmonids and other predators and by the presence of maltase in sea bream and ayu. The apparently larger diversity of carbohydrases in common carp than in other fish seems mostly a lack of information about fish other than carp. The question of whether dietary differences influence the kind of enzymes present must remain open but the evidence so far remains largely negative. However, there seems to be some evidence to show that the amounts of various enzymes may relate to the diet. Data in Table 2 suggest that herbivores have de-emphasized the production of proteases compared to the carnivores and the reverse for carbohydrases.
Table 2 - Relative Activity Levels of Amylase and Trypsin in Selected Cyprinids (Kapoor et al., 1975)
|
Fish |
Feeding habit |
Amylase activity |
Trypsin activity |
Amylase Trypsin |
|
Scardinius |
herbivorous |
1.0 |
0.4 |
2.5 |
|
Blicca |
omnivorous |
1.1 |
0.9 |
1.2 |
|
Alburus |
omnivorous |
1.0 |
0.9 |
1.1 |
|
Aspius |
carnivorous |
0.15 |
1.2 |
0.125 |
|
Cyprinus |
omnivorous |
5.8 |
1.7 |
3.4 |
Similarly, in studies of Trachurus, Scomber, Mullus, Mugil, and Pleuronectes, the predatory species, Trachurus and Scomber had the highest proteolytic and lipolytic activities, while the planktivore, Mugil, had the lowest proteolytic and the highest amylolytic activities. Also, stomachless fish (which lack pepsin) are usually herbivores or omnivores, while carnivorous fish have true stomachs with peptic digestion. On the other hand, differences in proteolytic activity between Tilapia and Perca were small, and some other investigations of a variety of species failed to find any species differences. Apparently, where fish are somewhat specialized in their diets, differences in their enzyme activities are apparent. Many fish, however, remain non-specialized and have diversified diets and enzymes.
The functions of bile have scarcely been studied in fish, but presumably resemble those in higher vertebrates. In mammals bile is composed mainly of bilirubin and biliverdin, which are breakdown products of haemoglobin, and is produced continuously. These salts act like detergents and serve to emulsify lipids, thus making lipids more accessible to enzymes because of the increased surface area, allowing some lipids to be absorbed undigested as micro-droplets. In mammals, about 80 percent of the bile is recycled through the liver and gall bladder.
There are a few studies in fish which suggest that bile serves similar functions in fish. Several histologists have histochemically identified micro-droplets of lipid in midgut epithelium of fishes. That the gall bladder in fish reabsorbs water as in mammals has been confirmed. That bile is produced continuously in fish is suggested by the presence of green mucus in the lumen of the atrophied gut of spawning salmon. There appear to be no studies in fish of gall bladder contraction or other mechanisms controlling the release of bile during digestion. An observation of salmon having impacted gall bladders seemed related to diet because the gall bladders returned to normal when their dry pellet diet was changed to a moist pellet. Fish having impacted (and presumably non-contractile) gall bladders were normal otherwise and were indistinguishable in appearance and growth rates from fish in the same population with normal gall bladders.
Anatomists have tried for many years to correlate the shape of the liver and the position of the gall bladder in the liver with some of its functions. The basic functions of the liver in processing the foods which have been digested and absorbed are entirely cellular and molecular in scope. Thus, there is no functional requirement for shape at any level above the cellular level; i.e., livers basically could be of any shape. On the other hand, some restrictions are created by its position in the circulatory system between the gut and the heart, and the necessary interdigitation of the portal and hepatic veins, hepatic arteries, and bile ducts, all of which must serve essentially every cell of the liver. In common carp, the liver seems to have no shape of its own and simply fills every available space between the loops of the intestine. On the other hand, many fish (e.g., salmonids) have distinctive shape and colour to their livers. Changes in normal size and shape can indicate dietary or other problems. For example, a large, yellowish liver, often with white blotches suggests fatty degeneration of the liver caused by too much starch or by using saturated (mammalian) fats in the diet.
5.1 Measurement of Stomach Contents
5.2 Measurement of Digestibility and Related Factors
As great diversity of gut anatomy and function occurs in response to the wide variety of foods found in nature, so also is there a great variety of methods to study digestion. This multiple diversity often makes comparisons between species impossible and comparisons within species using different methods difficult. Although the impact of methods on interpretation of results is always important in scientific experiments, the problems of methodology in studying fish digestion seem more severe than in most experimentation. Further, there are significant gaps in information about fish digestion, particularly in the areas of mechanisms controlling the gut, which have probably come about because of the lack of appropriate methods.
The most common method of determining gastric evacuation time and the digestive action of the stomach has been serial slaughter. This involves feeding a population of fish to a specified level (usually a percentage of body weight), then killing portions of the population at various times afterwards and analyzing the remaining stomach contents (analytic methods discussed below). Problems involved in this method include the variability of food intake by different individuals and the stress imposed on the population by capturing sub-samples: chasing and fright would be expected to inhibit digestion.
There are a number of variations on this basic technique. In one of these, sockeye salmon were frozen immediately after sampling, and the stomach contents removed as a single frozen mass as the fish thawed, thus enabling a complete and reproducible removal of the contents of a specifically delimited part of the gut. Proximate analysis was then performed on the stomach contents. Several investigators have devised stomach pumps for several species of fish. Most commonly it consists of a plastic syringe of a diameter to fit the oesophagus with the lower (needle) end cut off to give an opening the same size as the bore. The syringe is inserted under anaesthesia and a sample of food drawn from the stomach into the syringe. This provides a sample of food for qualitative, rather than quantitative, analysis for determining enzyme action, acidification rates, etc. For measuring gastric evacuation in predatory fish, several investigators have fed whole, smaller fish (pre-weighed), then put the predators into narrow holding chambers (to reduce the water thickness), and followed the digestion of the prey by watching the gradual disappearance of the prey's skeleton by making X-ray plates. The advantages are: not having to handle the fish to make measurements, and obtaining repeated measurements from the same fish. Several investigators have added inert (non-digestible, non-absorbable) substances to commercial diets and measured periodically the amount remaining in the stomach. Substances used include chromium oxide (Cr2O3) and radio-isotopes such as cerium-141.
A variety of inert materials have been inserted into fish stomachs to obtain samples of gastric juices. Spongey foam plastic has been inserted under anaesthesia and removed later. The contents of the sponge were then squeezed out and analyzed for acid and enzyme content. Glass beads were inserted into the fish stomachs to test the effects of stomach distension. A disadvantage of both methods was that the inserted materials were much easier to acidify (not as well buffered) than food, and that the stomach may not have responded to them in typical fashion.
Chromium oxide (Cr2O3) mixed with prepared diets and measured in the faeces provides a general comparison of the overall digestibility of a feed expressed as:
More specific measurements of digestibility now seem to be replacing the use of Cr2O3 as an indicator. Measurements of the caloric values of ingested food and faeces produced provides some part of the information for estimating the energy balance for a fish (oxygen consumption and growth rate are also needed). Alternatively, protein (measured as nitrogen content) or lipid content of food and faeces could be measured. In both cases the equation would be:
In measuring protein nitrogen, one really should take the nitrogen from the deaminated amino acids into account, which requires measuring the ammonia excreted by the gills (metabolic nitrogen). That equation should be:
Some of the most extensive studies on the digestibility of food components have been performed and reported by Phillips (1969) and readers should see these for detailed methods.
Several authors have combined the use of an indicator or other general measurement with measurement of one food component, generally protein. As an example. Smith and Lovell (1973) combined measurement of protein nitrogen with Cr2O3. Their equation was:
An earlier author (cited by Kapoor et al., 1975) used the same idea and substituted caloric values in feed and faeces for the Cr2O3 in the equation above. By comparing the digestibility of pure protein and of protein in practical diets. Smith and Lovell (1973) concluded that their combined indicator method gave results for catfish similar to the digestive coefficients in livestock feeding tables for concentrated feedstuffs.
Many of the methods described so far require the collection of faeces. A great variety of devices have been designed to do this, most of them producing a place with a low velocity water flow so that finely particulate faecal matter is not swept away. That this can be a problem is illustrated by Table 1 of this paper which shows significant differences between faecal composition in the trough and in the rectum. Post (1965) designed a holding chamber to reduce this problem by having static water around the posterior half of the fish and collecting the faeces from this water. Even collecting faeces from the rectum of fish does not solve all problems of faecal analysis because it is difficult to determine by their location in the hindgut when water reabsorption is complete. Thus faecal collection always includes some degree of compromise which affects the subsequent results and the comparability with other experiments.
The use of Mette's rods (tubes) has been made primarily by Russian investigators. The rods consist of short sections of glass tubing filled with solidified substrates; such as gelatin, coagulated egg white, starch paste, etc. The tubes are placed in the gut and recovered at some later time when the length of the tube emptied of substrate is measured as both a qualitative and quantitative indicator of digestion. Placing and recovering such tubes in the stomach is usually no problem, but getting them into the midgut is more difficult. In studying intestinal functions in carp and European bream (another cyprinid), one solution to placing the tubes in position was to cut one of the long loops of intestine and bring the ends out through the body wall, i.e., surgically make an intestinal fistula. The gut openings normally kept plugged, were opened to insert and remove the Mette's rods.
6.1 Rainbow Trout (Salmo gairdneri)
6.2 Channel Catfish (Ictalurus punctatus)
6.3 Common Carp (Cyprinus carpio)
6.4 Milkfish (Chanos chanos)
The gross anatomy of four cultured fishes, having diverse feeding habits are described in this section, emphasizing noteworthy structures which show relationships to feeding and digestion. The line drawings are intended to serve as a general guide for the examination and dissection of actual specimens and are not intended to show definitive details of visceral organs. The four species illustrated were selected primarily for variety of feeding habits and life styles among a dozen or more cultured species with no intent to indicate their importance or representativeness compared to other culture species.
Rainbow trout are largely carnivorous, but show few anatomical specializations for capturing and digesting prey. Teeth are simple and small with no other elaboration of structures to capture, hold, or swallow prey. Salmonids swallow their food whole via a wide oesophagus into a Y-shaped stomach. Many pyloric caecae branch near the pyloric end of the midgut, their numbers often being of taxonomic importance among the various salmonid species. The pancreas is diffusely scattered in the fat and connective tissue around the pyloric caecae and is not readily visible. The gall bladder extends from the middle lobe of the liver and the bile duct can usually be traced from there to the upper midgut in larger specimens. The midgut merges into the hindgut without any particular demarcation.
Other visceral organs include a thin-walled, nearly transparent swim bladder, the kidney just dorsal to that and running the full length of the visceral cavity. The kidney covers the dorsal aorta on the ventral surface of the vertebral column and encloses the posterior vena cava. The urinary ducts can usually be seen on the ventral surface of the kidney. They meet somewhat anterior to the posterior end of the kidney and descend as a single duct around one side of the swim bladder. An expansion of this descending portion of the urinary duct serves as a urinary bladder. The bladder is connected to the urogenital papilla as are testes in mature males. The gonads develop dorso-laterally in the anterior visceral cavity in both sexes, but the ovaries have no ducts connecting the urogenital papilla, eggs are simply shed into the visceral cavity. The only major organ remaining unmentioned is the spleen. In salmonids this lays ventrally, just above the pelvic fins, attached to the posterior side of the major visceral mass.
In general.) the rainbow trout is representative of most salmonids. It is a relatively primitive (unspecialized) fish, a typical carnivore with good swimming ability for capturing prey, a stomach which can easily extend posteriorly for ingesting relatively large prey, and a short intestine for handling food containing minimal amounts of indigestible material. The total length of the gut (oesophagus to anus) is 0.6 to 0.8 times the body length, about as short as any teleost (Figure 1a).
The channel Catfish is characteristic of most Ictaluridae. The mouth is wide and large, is fringed with gustatory barbels, and is generally adapted for foraging and sorting in mud to obtain the organic material - predominantly insects, but also snails, worms, plants, and general organic debris. The buccal cavity can be closed completely for squeezing mouthfuls of mud through the gill rakers and gill bars. The oesophagus is longer than in salmonids and leads to a round stomach which is located ventrally. The intestine originates at the anterior, ventral edge of the stomach, then turns dorsally to form several convoluted, half-circles around the stomach before proceeding posteriorly.
Arrangement of several non-digestive organs in the visceral cavity is noteworthy. The swim bladder has bulges on each side which raise the centre of bouyancy above the centre of gravity so that the fish do not turn belly-up when sick or unconscious, as in salmonids. These bulges also come very near to the body surface and probably enhance hearing. The relatively high position of the swim bladder is displaced anteriorly and dorsally so that it partly overlays the stomach. The gall bladder is in the mesenteries just posterior to the liver.
In general, ictalurids are versatile omnivores. They are relatively inactive, although marine catfish (family Arridae) appear to be more active than freshwater species and spend more time off the bottom. None of the species are particularly streamlined for efficient or rapid swimming, except that some species (channel cats, marine cats) have forked tails, which suggests some degree of swimming specialization. The maxillary and other barbels maximize their ability to find food at night or in turbid water where sight is largely useless. Other than the barbels, the digestive tract is of moderate size and length, showing little specialization (Figure 1b).
Common carp are representative of many cyprinids, including goldfish, squawfish, minnows, dace, chubs, and tench in North America. Most of these fish, including common carp, are omnivores, similar in several respects to catfish, but also differing significantly in several respects. Carp have maxillary barbels (most cyprinids do not) and forage in mud like catfish. However, carp ingest a considerably greater amount of plants than catfish and then chew the plants using a set of interdigitating pharyngeal teeth placed just anterior to the oesophagus. Carp lack a stomach, but have a long intestine which winds extensively throughout the visceral cavity. The gall bladder rests on the dorsal surface of the anterior midgut and the bile duet opens into the intestine just anterior to the gall bladder. In addition, the liver has no specific shape, but seems to serve as packing material around the intestine. Food seems to be ingested in small particles in a relatively steady stream instead of intermittently in large units, so the storage function of a stomach probably is not missed. With the liver filling all the available visceral space, there would be no room for accommodating the stomach expansion of a large meal anyway. The remainder of the visceral organs are relatively unremarkable (Figure 1c).
a. Rainbow trout (carnivore);
b. Catfish (omnivore emphasizing animal sources food);
c. Carp (omnivore, emphasizing plant sources of food);
d. Milkfish (microphagous planktovore).
Milkfish are specialized in several respects. The body shape, the streamlined cover over the eyes, and the widely forked tail all go with a fast-swimming life style. Fine (almost membranous) gill rakers suggest filter feeding, making a designation as carnivore, omnivore, or herbivore impossible since plankton is a mixture of many life forms, including some which are too simple to be clearly plant or animal. Milkfish are most frequently designated as microphagus planktovores. A specialized epibranchial organ above and behind the gills may help to concentrate microplankton, although no one has really demonstrated how it might do so. The stomach is a simple tube, somewhat convoluted, and of moderate size. The pyloric end of the stomach has thick, muscular walls and is usually described as a gizzard. The pyloric region of the long, narrow intestine has numerous pyloric caecae, also of small diameter. The swim bladder and the lining of the visceral cavity are membranous, similar to those in salmonids except for being jet black (Figure 1d).
In general, milkfish are cultured in enriched, saltwater lagoons in which they swim rapidly, straining their food from the typically turbid water.
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