Julia Dibner, Ph.D.
Novus International, Inc.
St. Louis, Missouri, USA

INTRODUCTION
A healthy gastrointestinal system is critical for the achievement of optimum genetic potential of young broilers. Recent literature has included data on patterns of early feed intake, ontogeny of digestive enzyme secretion, nutrient transporters and absorptive surface area. Much remains to be done. For example, the identification and description of factors that alter the normal development of intestinal tissue during early life are topics which have not frequently been reported. The objective of the research from the author's laboratory was to identify changes in the microscopic structure of the gastrointestinal system that accompany the growth and feeding of various dietary ingredients. Various nutritional regimes differing in texture and freshness were examined. This report will cover the early development of gut structure and effect of diet on that development. Results demonstrate that enormous changes occur normally in the microscopic structure of this organ system during the first week after hatching. Results also show freshness and texture affect gastrointestinal development, and this will be reflected in subsequent growth and performance.

GASTROINTESTINAL PHYSIOLOGY
The gastrointestinal (GI) system is the primary site of entry for any orally administered compound, including dietary ingredients. The functions of this organ system include digestion, absorption, and protection. The structure of the gut is well adapted to perform these functions. Several reviews have considered the relationship between structure and function in the avian gastrointestinal system (McLelland, 1979; Turk, 1982). The mucosa of the gut is the first tissue to encounter dietary ingredients and contaminants, and studies of its macroscopic and microscopic structure have been used to clarify the initial response of the animal to these materials. For example, it is well recognized that the presence of histamine and other biogenic amines in feeds can lead to macroscopic alterations in the gut, including ulceration and hemorrhage in the gizzard and intestine (Harry et al.,1975). Proventricular ulceration is associated with the feeding of high levels of copper (Poupoulis and Jensen, 1976). It is abundantly clear that such severe structural changes have important effects on performance. What is less clear is whether dietary variables such as texture and freshness cause microscopic effects and whether these could also influence performance, but to a lesser degree.

Other dietary constituents which cause changes in performance associated with changes in the gross structure of the GI system are antibiotic growth promotants. There are several of these, and most have been found to reduce the overall weight of the small intestine. This is due more to changes in the thickness of the intestinal wall rather than changes in intestinal length (Coates et al., 1954; Jukes et al., 1956; Franti et al., 1972; Henry et al., 1987; Izat et al., 1989; Izat et al., 1990). Microscopic examinations were rarely reported in these papers, but one publication suggests that the thinning is due to a significant reduction in the mucosal connective tissue (Jukes et al., 1956). Such structural changes have been proposed to effect improved performance through improved nutrient absorption, although other mechanisms involving reduced chronic low level infection and reduced competition for nutrients by endogenous microflora have also been suggested (Coates et al., 1954; Izat et al., 1989). Changes in microbial populations certainly have the potential to affect health in that the competitive exclusion of pathogens by the normal microflora could be disrupted, leading to opportunistic infections. An aspect of gastrointestinal growth, which has not been the subject of much research, is the effect of texture on growth and function. Effects of dietary fiber on gut microscopic growth and health in poultry have not been reported, although effects on mammals are well known.

The response of the gut itself to dietary ingredients has important implications for bird performance. Intestinal epithelial cells have a very high metabolic rate to support their secretory and absorptive functions and are constantly being renewed by stem cell proliferation in the crypts of Lieberkuhn. During the first weeks of life, the enormous growth of the GI system not only far exceeds that of other organ systems, it is essential if the bird is to achieve its genetic potential (Sell et al., 1991). For these reasons, damage to the gut mucosa can raise significantly the bird's maintenance requirement, leaving fewer nutrients for growth. The gut-associated lymphoid tissue (GALT) also demands nutrient support for metabolism and proliferation, and the unnecessary stimulation of this tissue by hypersensitivity reactions to dietary ingredients also diverts nutrients that could be used for growth. Thus, studies of the microscopic response of the GI system to dietary ingredients and additives may help the nutritionist to determine the optimum ingredients and additives required to achieve maximum nutrient efficiency.

INGREDIENT TEXTURE
The gastrointestinal system of a hatchling must undergo tremendous change before it is capable of efficiently digesting many of the ingredients in a typical poultry diet. The first and most obvious limiting factor is surface area for absorption. During the first five to seven days post hatch, the growth of the gastrointestinal system may exceed that of the rest of the body by as much as five-fold (Nitsan et al., 1991a; Nitsan et al., 1991b; Sell, 1991). Interestingly, the microvilli of enterocytes also increase in length during the first week of life (Chambers and Gray, 1979), suggesting that the initial growth of the bird may be limited by the surface area of the gastrointestinal system. An important correlate is the relationship of gut organ development and the bird's growth rate. Lilja (Lilja, 1983) reported that avian species with high growth rate capacities were also characterized by a rapid early development of the digestive organs and liver. The converse was true for birds with low growth rate capacity, such as quail (Lilja, 1983). Similarly, birds selected for a high eight-week body weight were shown to have a greater relative weight of gastrointestinal tract at day 10 than did birds selected for low eight-week body weight (Dunnington and Siegel, 1995).

Effects of texture on gut structure in avians are well known. For example, the size of the crop and the gizzard is influenced both during life and evolutionarily by the texture and components of the bird's diet (McLelland, 1979). Little is known, however, about how these changes occur or whether the effect of low residue diets given during early gut development will persist into later stages and, if so, whether performance will be affected. Reduction in GI mass is seen when rats are fed an elemental diet (Evers et al., 1989). Such diets are very low in residue and similar changes in gut structure may occur in birds fed a low residue diet.

Experimental work
The focus of the work reported here is the neonatal period during which relative gut growth is greatest. Birds were hatched, brooded and housed in a battery cage system as previously described (Dibner et al., 1995). To evaluate effects of feeding various textures, isonitrogenous diets were formulated which contained natural ingredients in either normal or low residue form. The normal texture diet included ground corn and soybean meal (SBM), while isolated soy protein (ISP) and corn starch were used in the low texture treatment. These treatments were applied for the first three days only, after which the birds were fed a corn soy starter diet formulated to meet or exceed National Research Council recommendations. The short (days one to three) and long term (days six to 21) effects of these treatments were compared. Morphometry data were generated as previously described (Dibner et al., 1995) using four to five birds per treatment per day.
Figures 1 and 2 shows the effect of fasting or feeding normal or low residue diets during days zero, one, and two of the study. Starting on day three, all birds were fed a common corn soy starter feed ad libitum. Figure 1 shows effects on the weight of the small intestine during the treatment period and when measured four days after the feeding of a standard corn soy starter. Absolute rather than relative weights are presented because body weight loss in the fasted controls confounded the weight/100g body weight measure. Clearly, the small intestine was affected more by fasting than by either of the two residue treatments, with differences persisting at three days on the standard corn soy diet. Intestine weights from birds of either residue treatment were about the same after four days on the corn soy diet (day seven).

In Figure 2, effects on pancreas weight are presented. The results suggest that fasting is associated with minimal increases in relative pancreas weight, and that the birds fed the low residue diets had lower pancreas growth after two days on treatment and persisting through the first four days of being fed ad libitum a standard corn soy diet. The effects on pancreas growth may be the result of differences in the requirement for digestive enzymes to make the diets available to the bird. It might be expected that ISP and corn starch require less enzyme catalyzed breakdown than SBM and ground corn. Notice also that the pancreas does not achieve normal size as quickly as does the small intestine following replacement of the treatments with standard corn soy starter. This may result in lower ingredient digestibility, and in this way, affect growth during the first week of life.

INGREDIENT FRESHNESS
Several reports indicate that animals fed oxidized fats can exhibit poor performance, including decreases in gain and feed efficiency in rats (Raced et al., 1963) and broilers (Cabel et al., 1988; Balnave, 1970). It has been demonstrated on numerous occasions that animals require certain polyunsaturated fatty acids and that deficiencies can be associated with weight loss, fatty liver, kidney malfunction and poor reproduction (Balnave, 1971; Nakamura et al., 1973; Holman, 1986; Ashida et al., 1988). It has also been demonstrated that, once oxidized, dietary fatty acids still can be incorporated into cell membranes (Bunyan et al., 1968).
A study was conducted in the author's laboratory to study the effect of feed freshness on the gastrointestinal system (Dibner et al., 1995). Birds were fed diets containing control or oxidized fat with or without added ethoxyquin at the time of feed mixing. In addition to performance differences, a variety of effects of oxidized fat on the gastrointestinal system were examined, including changes in nutrient uptake, intestinal microflora, and the gut associated lymphoid tissue. Tissues from birds in this study were also evaluated for microscopic structural changes.

Birds were fed a corn soy broiler starter diet formulated to meet or exceed National Research Council (1984) nutrient requirements. Fat was provided as a combination of control poultry fat with an initial peroxide value (IPV) of 1.04 milliequivalent/kg fat (mEq/kg), oxidized poultry fat with an IPV of 212.5 mEq/kg, or lard with an IPV of 3.2 mEq/kg. Oxidation of the poultry fat was achieved by bubbling air through poultry fat heated to 900C.

There were four treatments in the broiler study: For treatment 1, fat was provided as non-oxidized poultry fat and the diet contained no ethoxyquin. For treatment 2, fat was provided as non-oxidized poultry fat and the diet contained ethoxyquin (Santoquin, Monsanto Company, St. Louis, MO) added at 125 ppm (4 oz/ton). For treatment 3, half of the fat was provided as oxidized poultry fat and half the fat as lard, and the diet contained no ethoxyquin. For treatment 4, half the fat was provided as oxidized poultry fat and half the fat as lard, and the diet contained ethoxyquin (125 ppm). The peroxide level for treatments 3 and 4 was 4.2 mEq/kg diet. Birds were randomly selected for histopathology studies, as well as nutrient uptake, cell proliferation and intestinal microflora studies previously described (Dibner et al.,1995; Shermer et al., 1995; Dibner et al., 1996).

Figure 3 shows effects of the dietary treatments on cross sectional area of ileum and both cecal tonsils. The most obvious difference is that the birds fed oxidized fat had smaller ileal cross sectional area, while simultaneously having much larger cecal tonsils. This is interesting in light of the observation, in the same study, that villus epithelium half life is reduced in animals fed oxidized fat in the absence of ethoxyquin (Dibner et al., 1996). The observation that cecal tonsil cross sectional area is increased in the same treatment is another example of an effect of oxidized fats on immune tissues, although no obvious effect was seen in this study on the proliferative activity of lymphocytes in the gut associated immune tissue. This would suggest increased recruitment of lymphocytes to the area, perhaps in response to the reduction in tissue IgA reported earlier (Dibner et al., 1996).

Figure 4 shows morphometry results of the same study. As this figure illustrates, crypt villus ratios in the small intestine of birds fed either oxidized fat diet were greater than those of birds fed control fat, both with and without ethoxyquin. Increased crypt villus ratio indicates high proliferative activity, which would be expected in light of the reduction in enterocyte half-life reported earlier. Villus length at day six indicated that the birds fed control fat, with or without ethoxyquin, and birds fed the oxidized fat with ethoxyquin, all had significantly longer villi than the birds fed oxidized fat without ethoxyquin. Interestingly, villus length on day 11 (Figure 4) indicates that although the birds fed oxidized fats had higher proliferative activity on day six, the resulting villi on day 11 were still shorter than were those of birds fed the control fat. Since this was observed in both oxidized fat treatments, it appears that even the presence of an antioxidant is not sufficient to completely protect the gut from the toxins present in oxidized fats.

Among the toxins in a sample of oxidized fat are carbon centered radicals and the product of secondary autoxidation such as ketones and aldehydes (Nakamura et al., 1973). Such compounds are not substrates for ethoxyquin or any other antioxidant and remain in the diet as toxic byproducts. A decrease in villus length may increase the proportion of enterocytes which are not yet fully functional and in this way, reduce the surface area available for secretion of digestive enzymes and absorption of nutrients. This may be a causative factor in the poor performance seen with oxidized fat. The fact that the longest villi were seen in birds fed the control fat with the antioxidant in the diet suggests that even with fresh feed ingredients, effects of oxidation which occur during feed mixing and storage may reduce availability of feed nutrients.

SUMMARY
The gastrointestinal system of the young bird grows at three to five times as fast as the rest of its body. Factors which influence gut growth include dietary form and ingredient freshness. Poor growth and health of this essential supply organ will limit performance and may cause the death of the animal if not corrected. Some of these factors, particularly ingredient freshness are determined, in part, by the supplier of the ingredient. Proper antioxidant stabilization, storage conditions and microbial control are essential for the maintenance of ingredient quality.

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