Effects of Luminal Nutrient Absorption, Intraluminal Physical ...

METABOLISM AND NUTRITION Effects of Luminal Nutrient Absorption, Intraluminal Physical Stimulation, and Intravenous Parenteral Alimentation on the Recovery Responses of Duodenal Villus Morphology Following Feed Withdrawal in Chickens P. Tarachai and K. Yamauchi1 Laboratory of Animal Science, Faculty of Agriculture, Kagawa University, Miki-cho, Kagawa-ken, 761-0795 Japan ABSTRACT The aim of this study was to clarify which of the following three factors induces villus morphological recovery best: enteral nutrient absorption, intraluminal physical stimulation, or intravenous parenteral alimentation. At 142 d, male White Leghorn chickens (Gallus gallus domesticus) were divided into eight groups of five birds each as follows: 1) access given ad libitum to a commercial layer mash diet (CP, 17.5%; ME, 2,830 kcal/ kg) (control), 2) 5-d feed withdrawal (feed withdrawal), 3) 3-d feed withdrawal (3-FW), followed by refeeding the same diet as the control for 2 d (refeeding), 4) 3FW followed by force-feeding enteral hyperalimentation (enteral), 5) 3-FW followed by force-feeding an indigestible (nonabsorbable) substance (kaolin), 6) 3-FW followed by force-feeding water for 2 d (force-fed control), 7) 3FW followed by parenteral hyperalimentation (parenteral), and 8) 3-FW followed by no alimentation (sham control) for 2 d. In the refeeding and enteral groups, BW significantly recovered (P < 0.05), and in the parenteral group, BW tended to increase, suggesting that nutrients were enterally and parenterally absorbed, respectively. The BW in

the remaining three groups showed a significant decrease (P < 0.05), indicating that kaolin could not be absorbed enterally. Compared with the feed withdrawal group, villus height, cell mitosis, and villus tip surface morphology of refeeding and enteral groups exhibited rapid villus morphological recovery. Villus morphological recovery of the enteral group appears to have been caused by enteral nutrient absorption. However, villus morphology in the kaolin treatment was not different from that in the feed withdrawal group, which suggests that intraluminal physical stimulation had no effect on villus morphological recovery. On the other hand, the parenteral group showed no effect on villus morphological recovery, which suggests that the parenteral nutrient supplied to the villi via the blood could not induce villus morphological recovery; the intestinal mucosal atrophy might have been caused by the absence of enteral nutrients, and would only be stimulated by enteral nutrient absorption. In conclusion, the present findings suggest that villus morphology is governed neither by intraluminal physical stimulation nor by parenteral alimentation, but by enteral nutrient absorption.

(Key words: enteral alimentation, parenteral alimentation, intraluminal physical stimulation, villus morphology) 2000 Poultry Science 79:1578–1585

INTRODUCTION Compared with light strain chicks, heavy strain chicks have greater villus heights, greater DNA concentrations within the duodenum (Uni et al., 1995), larger villi (Yamauchi and Isshiki, 1991), and increased epithelial cell RNA (Yamauchi et al., 1992). In chicks, duodenal villus height and cellular proliferation within the crypt are reduced by fasting and rapidly recovered upon refeeding (Yamauchi et al., 1996; Shamoto et al., 1999), and this recovery is greatly influenced by the realimentation diet (Shamoto et al., 1999; Shamoto and Yamauchi, 2000).

The villus tip has a smooth appearance during feed withdrawal and returns to a normal morphology upon refeeding (Shamoto et al., 1999; Shamoto and Yamauchi, 2000), suggesting that luminally absorbed nutrients may influence villus morphology. However, intraluminal stimulation is also correlated with these villus structural changes, and stimuli arising from factors such as dietary fibers within the intestine might also influence the structural integrity of the intestine. It is of interest, therefore, to determine which of the following three factors is most responsible for inducing villus morphological changes: luminal nutrient absorption, intraluminal physical stimulation, or intravenous parenteral alimentation.

Received for publication November 5, 1999. Accepted for publication May 8, 2000. 1 To whom correspondence should be addressed: yamauchi@ag. kagawa-u.ac.jp.

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Abbreviation Key: 3-FW = 3-d feed withdrawal.

ALIMENTATION AND INTESTINAL VILLUS MORPHOLOGY TABLE 1. Composition of diet (air dry basis) Ingredients and nutrients

%

Ground corn Soybean meal Fish meal Rice bran Concentrate mixture1 Crude protein Crude fat Crude fiber Ash Calcium Phosphorus Metabolizable energy (kcal/kg) Lysine Methionine Methionine + cystine Tryptophan

63.0 19.0 7.0 1.0 10.0 17.5 2.5 5.0 13.0 2.6 0.55 2,830 0.79 0.33 0.65 0.18

1 Including 0.1% Premix; Premix provided the following per kilogram of diet: Iron, 189.33 mg; copper, 8.11 mg; manganese, 52.18 mg; zinc, 68.45 mg; vitamin A, 76,140.00 IU; vitamin D3, 3,500.00 IU; vitamin E, 25.35 mg; vitamin K3, 5.04 mg; vitamin B1, 4.69 mg; vitamin B2, 5.81 mg; pantothenic acid, 12.11 mg; vitamin B6, 7.98 mg; choline, 1,304.08 mg; vitamin B12, 0.01 mg; nicotinic acid, 45.29 mg.

MATERIALS AND METHODS Birds and Housing Male Single Comb White Leghorn chicks (Gallus gallus domesticus; Julia strain) were obtained from a commercial hatchery and maintained in individual cages in an environmentally controlled room with a 14-h photoperiod (06:00 h light to 20:00 h dark) at a mean environmental temperature of 20.0 C. Birds had access ad libitum to water and a commercial layer mash diet (Table 1).2

Experimental Design At 142 d, 40 birds of uniform BW (1.47 kg) were chosen and allotted into eight groups, each group including five birds, as follows: 1) access given ad libitum to a commercial layer mash diet (control); 2) 5-d feed withdrawal (feed withdrawal); 3) 3-d feed withdrawal (3FW), followed by refeeding the same diet as the control for 2 d (refeeding); 4) 3-FW followed by force-feeding enteral hyperalimentation (enteral); 5) 3-FW followed by force-feeding an indigestible (nonabsorbable) substance (kaolin); 6) 3-FW followed by force-feeding water for 2 d (force-fed control); 7) 3-FW followed by parenteral hyperalimentation (parenteral); and 8) 3-FW followed by no alimentation (sham control) for 2 d. In the enteral and kaolin groups, chicks were force-fed a 100-mL Har-

2 Japan Federation of Poultry Farmers Co-operative Association, Tokyo, 101-0041 Japan. 3 Yoshitomi Pharmaceutical Industries, Ltd., Osaka, 541-0046 Japan. 4 Maruishi Pharmaceutical Co., Ltd., Osaka, 541-0044 Japan. 5 Terumo Corporation, Tokyo, 151-0072 Japan. 6 Tanabe Seiyaku Co., Ltd., Osaka, 541-8505 Japan.

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monic威-M3 (Table 2) or a 100-mL kaolin solution of a mixture of 100 g powdered kaolin (Japanese pharmacopoeia 13 edi.4) (natural hydrated aluminum silicate) and 300 mL water, respectively, into their crops using an elastic tube at 09:00, 12:00, and 15:00 h (total 300 mL/ d). As a control for these groups, the water group was also force-fed a total of 300 mL/d water in the manner described above. In the parenteral group, birds were anesthetized with diethyl ether, and their legs and wings were fixed on the operation table, left side on top, during the operation. Feathers around the neck area 5 cm caudal to the mandibula were plucked. A 1-cm skin area was incised, and a 5-cm catheter of 2-mm diameter (type Fr. 55) was inserted into the left lateral jugular vein toward the heart and sutured to the closed skin part. The other end of the catheter was tunneled subcutaneously and exteriorized at the skin immediately caudal to the comb. Birds were fitted with a parabolic antenna type of collar to prevent removal of the catheter. Immediately after the surgical operation, chickens were returned to individual cages. The other end of the catheter was connected to a drip bag outside of the cage, and an amino acid solution including glucose and electrolytes (Amicaliq,威,5,6, Table 3) was intravenously dripped continuously (total 300 mL/d). As a control for this group, sham-operated chickens were employed using the same procedures described above, but without intravenous injection of Amicaliq.威 The protocol for these experiments was performed according to the humane care guidelines provided by the Kagawa Medical School. At the end of the experiments, BW was measured and expressed as relative BW (BW on each day during each treatment)/(initial BW) × 1,000 g.

Tissue Sampling Chickens under light anesthesia with diethyl ether were euthanized by decapitation. The entire small intesTABLE 2. Composition of Harmonic威,1 Nutrients

mg/mL

Protein Lipid Glucose Vitamin A, IU Vitamin B1 Vitamin B2 Vitamin B6 Ascorbic acid Vitamin E Nicotinic acid Folic acid, µg Pantothenic acid Vitamin B12, µg Biotin, µg Na K Ca Mg Fe Zn Cl P

48.0 30.0 135.0 1,600.0 0.0088 0.0024 0.004 0.2 0.017 0.04 0.9 0.032 0.0068 0.2 0.92 1.17 0.48 0.1 0.0072 0.007 1.12 0.49

1

Yoshitami Pharmaceutical Industries, Ltd., Osaka, 541-0046 Japan.

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TABLE 3. Composition of Amicaliq威 Nutrients

mg/mL

L-isoleucine L-leucine L-lysine HCl L-methionine L-phenylalanine L-tryptophane L-threonine L-valine L-thyrosin L-arginine L-arginine HCl L-histidine L-alanine L-aspatic acid Amino acetic acid (glycine) L-proline L-serine Glucose Electrolytes Potassium chloride Magnesium chloride Di-potassium hydrogen phosphate Sodium lactate (50%) Lactic acid Supplement Sodium hydrogen sulfite L-cystein Total free amino acids Electrolytes, MEq/ml Na+ K+ Mg2+ Cl+ HPO42 Lactate−

2.34 3.71 2.75 1.35 2.12 1.32 0.44 2.47 0.14 1.48 1.90 1.29 2.37 0.14 1.51 1.90 1.15 75.00 1.64 0.30 0.26 5.64 1.33 0.50 0.15 2.75 0.03 0.03 0.0003 0.05 0.0003 0.04

1 Teruno Corporation, Tokyo, 151-0072 Japan; and Tanabe Seiyaku Co., Ltd., Osaka, 541-8505 Japan.

tine was quickly excised and placed in a mixture of 3% glutaraldehyde and 4% paraformaldehyde fixative solution in 0.1 M cacodylate buffer (pH 7.4). The same fixative was also injected into the intestinal lumen. The segment from the ventriculus (gizzard) to the pancreatic and bile ducts was recognized as the duodenum. The tissue samples were taken at the middle part of the duodenum.

ing the intestinal crypt, was measured (Figure 1A). Sixteen villus height measurements per bird were made (a total of 80 measurements per treatment). Values of villus height were measured using an image analyzer (Nikon Cosmozone lS7). For measuring the number of cell mitoses in the crypt, mitotic cells having homogenous, intensely stained basophilic nuclei with hematoxylin were counted (Figure 1B). In the case of cells in late stages of division, cell mitosis number was counted as one mitotic event (Figure 1C). Cell mitosis was measured on four sections per bird (a total of 20 measurements per treatment). Scanning Electron Microscopy. A 2-cm duodenal section was taken immediately distal to the section collected for light microscopy. Each section was slit longitudinally, opened, and washed with 0.01 M phosphate buffered saline (pH 7.4). To prevent the slit intestine from curling, the edges were pinned to the paraffincovered bottom of a petri dish containing a mixture of 3% glutaraldehyde and 4% paraformaldehyde fixative solution in 0.1 M cacodylate buffer (pH 7.4). The slit intestinal section was fixed in this flattened position at room temperature for 1 h, cut into a 4 × 10-mm square, and fixed for 1 additional h. The pieces were rinsed with a 0.1 M sodium cacodylate buffer (pH 7.4) and postfixed with 1% osmium tetroxide in the same ice-cold buffer for 2 h. The specimens were dried in a critical-point drying apparatus (Hitachi HCP-18) using liquid carbon dioxide as the medium. The dried specimens were coated with platinum (RMC-Eiko RE vacuum coater9) and examined with a scanning electron microscope (Hitachi S-8008) at 8 kV.

Statistical Analysis All data measured in light microscopic examination were statistically analyzed using one-way ANOVA, and significant differences among the treatments were determined with Duncan’s multiple range test using the Stat View program10 at the level of P < 0.05.

RESULTS

Microscopic Examinations

Relative Body Weight Change

Light Microscopy. A 2-cm segment of the duodenum was transversally cut, fixed in Bouin’s fixative solution, and embedded in paraplast. Five-micrometer transverse sections were cut, and every tenth section was collected and then stained with hematoxylin-eosin. For villus height measurement, one villus having the lamina propria was selected from one duodenal transverse section. The length from the villus tip to the bottom, not includ-

Figure 2 shows the relative growth (BW) of each treatment group. After 3-FW, relative BW declined 10.2% compared with the initial BW (1.47 kg). In the refeeding and enteral treatment groups, BW gain recovered significantly after 1 and 2 d of refeeding, respectively (P < 0.05). The BW in the parenteral group tended to increase. The BW in the remaining four groups showed a further decrease (P < 0.05).

Light Microscopic Observations 7

Nikon Co., Tokyo, 100-8331 Japan. Hitachi Ltd., Tokyo, 100-8220 Japan. 9 Eiko Engineering Co., Ltd., Tokyo, 151 Japan. 10 Abacus Concepts, Inc., HULINKS, Inc., Tokyo, 171-0022 Japan. 8

Figure 3 shows the measured changes in villus height and cell mitosis. Feed withdrawal for the entire experimental period (5 d) significantly reduced villus height

ALIMENTATION AND INTESTINAL VILLUS MORPHOLOGY

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FIGURE 1. Examples of villi (A, × 60.5) and cell mitosis in the crypt (B, × 173; C, × 355). Arrow in A indicates bottom of villus; small arrows in B and C show cell mitoses; large arrows in B and C indicate conventional epithelial cells and division stage of mitotic cell into two daughter cells, respectively. All scale bars = 5 µm.

(P < 0.05). There were no significant differences between the control, refeeding, and enteral treatments with respect to villus height, whereas in all other treatments, villus height was significantly reduced. With respect to mitosis, the refeeding treatment resulted in a significant increase compared with the control, and the increases in the refeeding, enteral, and control groups were significantly higher than in the other treatments (P < 0.05).

Scanning Electron Microscopic Observations

FIGURE 2. Relative BW change during each treatment for 2 d after 3-d feed withdrawal. The BW recovery can be observed in the enteral and parenteral groups, but not in the kaolin group. a−cMeans with different superscripts are significantly different from each other (P < 0.05) (means ± SE; n = 5).

Scanning electron microscopic pictures of the duodenal villus tip surface are shown in Figures 4 and 5. The appearance of the control villi revealed a clear cell outline between each cell, protuberating cells, and protuberances of cell mass, all of which resulted in a rough surface (Figure 4A). These conspicuous morphological features became faint after 5 d of feed withdrawal, when a smooth surface was observed (Figure 4B). However, in the refeeding group, clear cell outlines and protuberances of each cell were again apparent (Figure 4C). In addition

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(Takahashi et al., 1993). The recovered BW of the parenteral group may demonstrate that nutrients in this elemental diet were parenterally absorbed, corresponding to the results of rats administrated Amicaliq威 (Iwasawa et al., 1990). The present trend of the enteral group showing a more rapid BW recovery than the parenteral group may be related to observations in piglets, in which the total protein synthesis rate of midjejunal mucosa was significantly lower in parenterally-fed piglets than in enterally-fed piglets (Dudley et al., 1998). On the other hand, the BW of the kaolin group was almost the same as those of the feed-withdrawal, force-fed control, and sham control groups, suggesting that kaolin was not absorbed enterally and did not provide significant nutrients. The BW of rats fed an elemental diet plus 30% kaolin was also less than that of rats fed the elemental diet alone (Goodlad et al., 1989).

Comparison of Villus Morphological Recovery Among Enteral Alimentation Treatments

FIGURE 3. Alterations of the duodenal villus height and cell mitosis number in the crypt in each treatment for 2 d after 3-d feed withdrawal. Note that both parameters show a rapid recovery in the enteral group but not in the kaolin and parenteral groups. a−cMeans with different superscripts are significantly different from each other (P < 0.05) (means ± SE; n = 5).

to these conspicuous morphologies, protuberances of large cells with no microvilli were frequently observed in the enteral group (Figure 4D). The villus surface of the kaolin, force-fed control, parenteral, and sham control groups showed a smooth surface (Figure 5).

DISCUSSION The aim of this study was to clarify which factor is most responsible for inducing villus morphological changes: luminal nutrient absorption, intraluminal physical stimulation, or intravenous parenteral alimentation. The effects of each experimental treatment were evaluated by BW recovery after 3-FW, and then villus morphological changes in these birds were compared.

Recovery of Body Weight After 3-FW followed by 2-d realimentation, the BW of the refeeding, enteral, and parenteral groups recovered. The rapid recovery of the refeeding group is thought to have been induced by adequate nutrient enteral absorption. The recovered BW of the enteral group may demonstrate that nutrients in this hyperalimentation (elemental diet) solution were enterally absorbed, corresponding to the increased BW of rats administered Harmonic威-M

It is reasonable to conclude that the rapid recovery of villus height and cell mitosis, and the activated villus surface morphology in the refeeding group were induced by the adequate enteral nutrient absorption and the intraluminal physical stimulation due to the diet. In the case of the soluble hyperalimentative enteral group, a similar rapid recovery was obtained as well; especially, morphological recovery was observed in the villus surface. Not considering villus recovery in the force-fed control group, enteral nutrient absorption is important for villus morphological recovery. On the other hand, the kaolin substance that filled up the intestine might physically stimulate the intestinal lumen. However, villus morphological recovery was not obtained in this group, indicating that intraluminal physical stimulation seems to have no effect on villus morphology. Goodlad et al. (1989) reported no stimulated intestinal epithelial cell proliferation in rats fed an elemental diet plus 30% kaolin. These observations indicate that the villus morphological alterations are not affected by intraluminal physical stimulation, but by enteral nutritient absorption.

Comparison of Villus Morphological Recovery Between Enteral and Parenteral Alimentation Groups Compared with the rapid morphological recovery of villi in the enteral alimentation (refeeding and enteral) groups, the parenteral intravenous alimentation of the parenteral group showed no villus recovery. The feed withdrawal, force-fed control, and sham control groups showed the same results. On the contrary, in the parenteral group, cell mitosis tended to decrease more than in the other groups. This result corresponds with reductions in the villus height, cell mitotic number, and DNA


content of the ileum (Heitman et al., 1980), the mucosal and protein contents of the jejunum (Kotler et al., 1981), and the mucosal weights and villus heights of the jejunum and ileum (Czernichow et al., 1992) after parenteral feeding in rats, as well as decreased mucosal height and villus surface area of the duodenum in parenterally

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treated miniature pigs (Shulman, 1988). Recently, Dudley et al. (1998) described shorter villi and lower weight of the jejunum in parenterally fed piglets than in those fed enterally. The present observations and findings of similar studies in the literature lead to the general conclusion that the parenteral nutrients supplied to the villi

FIGURE 4. Duodenal villus surface in control (A), 5-d feed withdrawal (B), 2-d refeeding a commercial diet after 3-d feed withdrawal (C) and 2-d force-feeding an enteral hyperalimentative solution after 3-d feed withdrawal (D) groups. Luminal nutrient absorption induces the active morphological recovery from atrophied villi. Scale bar = 20 µm (× 830).

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FIGURE 5. Duodenal villus surface in groups 2-d force-fed an indigestible substance solution (A) and a force-fed control (B) after 3-d feed withdrawal, and in 2-d intravenous parenteral hyperalimentative dripping after 3-d feed withdrawal (C) and its sham control (D) groups. Intraluminal physical stimulation and parenteral alimentation do not recover the atrophied villus morphology. Scale bar = 20 µm (× 830).

via the blood could not induce villus morphological recovery, although they elevated BW; intestinal mucosal atrophy may be induced by the absence of enteral nutrients and stimulated only by enteral nutrient absorption.

In conclusion, the present findings suggest that villus morphology is governed neither by intraluminal physical stimulation nor by parenteral alimentation, but by enteral nutrient absorption.


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