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Articles

Small Intestine Epithelial Barrier Function Is Compromised in Pigs with Low Feed Intake at Weaning^{1 ,2}

M. A. M. Spreeuwenberg³, J. M. A. J. Verdonk^*, H. R. Gaskins and M. W. A. Verstegen^**

Swine Research Center, Nutreco, Boxmeer, The Netherlands; ^* ID TNO Animal Nutrition, Lelystad, The Netherlands; Departments of Animal Sciences and Veterinary Pathobiology, University of Illinois at Urbana-Champaign, IL; and ^** Division of Animal Nutrition, Department of Animal Sciences, University of Wageningen, The Netherlands

³To whom correspondence should be addressed. E-mail: Mirjam.Spreeuwenberg{at}nutreco.com.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Compromising alterations in gastrointestinal architecture are common during the weaning transition of pigs. The relation between villous atrophy and epithelial barrier function at weaning is not well understood. This study evaluated in vitro transepithelial transport by Ussing metabolic chambers, local alterations in T-cell subsets and villous architecture at low energy intake level and their relation with lactose/protein ratios in the diet. Pigs (n = 66, 26 d old) were sampled either at weaning (d 0), d 1, 2 or 4 postweaning. Piglets received one of three diets at a low energy intake level, which differed in lactose and protein ratio as follows: low lactose/high protein (LL/HP), control (C), or high lactose/low protein (HL/LP). Mean digestible energy intake was 648 kJ/pig on d 1, 1668 kJ/pig on d 2, 1995 kJ/pig on d 3 and 1990 kJ/pig on d 4 postweaning. The CD4⁺/CD8⁺ T-lymphocytes ratio decreased after weaning (P < 0.05). Decreased paracellular transport (P < 0.01), greater villous height (P < 0.01), shallower crypts and lower villus/crypt ratios (P < 0.01) were observed on d 2 compared with d 0. Piglets consuming the HL/LP diet tended to have less paracellular transport (P < 0.10) and greater villous height (P < 0.10) compared with piglets fed the other diets. During the first 4 d postweaning, the effect of diet composition on mucosal integrity was not as important as the sequential effects of low energy intake at weaning. Stress and diminished enteral stimulation seem to compromise mucosal integrity as indicated by increased paracellular transport and altered T-cell subsets.

KEY WORDS: • piglets • weaning • energy intake • epithelial barrier function • T lymphocytes

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Pigs are confronted by multiple stressors at weaning. Under commercial conditions, weaning may involve complex social changes, including separation from the sow, a new housing system, separation from littermates and exposure to unfamiliar pigs (1) . Diet composition also changes at weaning; the liquid milk from the sow is replaced by pelleted dry feed with carbohydrates instead of fat as the main energy source.

Abrupt weaning is typically accompanied by low feed intake, which seems to be the main reason for the growth stasis after weaning (2) . Weaning also causes morphologic and histologic changes of the small intestine of pigs (3 4 5 6 7 8 9 10 11 12) . These changes include reduction in villous height and an increased crypt depth. The magnitude of the intestinal responses seems to be related to feed intake of the piglets (7 , 12) , independent of diet composition (9 ,10) . Beers-Schreurs (13) found that the weaning transition itself explained part of the reduction in villous height and increased crypt depth. Villous height decreased and crypt depth increased in weaned piglets compared with unweaned piglets given sow’s milk at a high energy level after weaning. The reduction in villous height was even more pronounced when the piglets were fed a weanling diet or sow’s milk at a comparable low energy level (13) . Starvation itself decreased jejunal villous height and increased paracellular permeability in the ileum and jejunum of adult rats (14) . An inverse relationship was found between ATP concentrations in jejunal mucosa and permeability (15) , indicating that at a low energy level, permeability is increased.

The relationship between epithelial barrier function and villous atrophy at weaning is not understood. A compromise in epithelial barrier function possibly increases paracellular permeability. With increased paracellular permeability, toxins, allergenic compounds or bacteria may enter systemic tissues, resulting in inflammatory or immunologic responses (16 ,17) .

Providing piglets sow’s milk after weaning resulted in less villous atrophy compared with a weanling diet (13) ; thus milk components seem to be favorable. Sow’s milk is composed mainly of fat (40.6 g/100 g), protein (29.4 g/100 g) and lactose (28.3 g/100 g) (18) . Lactose is converted by lactase to galactose and glucose; glucose can be an energy source for epithelial cells (19) . Lactose seems, therefore, a key energy source for intestinal epithelial cells in young piglets. Some amino acids in the milk protein can be used as an energy source for epithelial cells (e.g., glutamine), as well as contribute to protein synthesis.

This experiment investigated mucosal variables over time in response to low energy intake and compared the effectiveness of lactose vs. protein in preserving mucosal integrity during the weaning transition. We postulated that the energy supply is more limiting than the protein supply for epithelial cells in contributing to mucosal integrity, i.e., a diet with a high lactose/protein ratio would better preserve mucosal integrity. T-lymphocyte cellularity was measured as an indicator of inflammation. Transepithelial permeability was measured as a functional indicator of mucosal integrity.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Animals and weaning.

Barrows (n = 66) procured from a commercial maternal line herd [Great York x (Dutch Landrace x Finnish Landrace)] were used. The piglets were weaned at 25.9 ± 2.01 d of age. Creep feed was not provided during the suckling period to avoid adaptation to experimental diets and to make the piglets’ treatment uniform. At weaning, pigs were removed from the sow and transported 10 km to the TNO Nutrition research facility in Wageningen (The Netherlands). Upon arrival from the source farm, pigs were weighed and housed individually in 50 x 90 cm² floor pens. The walls of the pens were transparent plastic, enabling visual contact among the piglets. Each pen was equipped with a plastic trough. Water was supplied via the liquid milk replacer diets. Environmental temperature was maintained at 24°C. Lights were on continuously. The experimental protocol was approved by the Animal Care and Ethics Committee of the research institute TNO.

Feeds, feeding and experimental design.

The experiment was conducted in two consecutive batches. On the day of weaning, dissection was performed on 12 randomly chosen piglets to collect reference values. Additionally, the remaining 54 piglets were assigned to 3 x 3 experimental groups on the basis of body weight (BW)⁴ ; the groups differed in diet and day of dissection. The experimental groups were given one of three experimental diets that differed in the ratio of lactose to protein (Table 1 ). A control liquid milk replacer (C) was compared with a liquid milk replacer with a low lactose/high protein (LL/HP) ratio, and a high lactose/low protein (HL/LP) ratio. The percentage of fat was the same in each experimental diet.

(1)

where DE is the digestible energy intake (kJ/d) and BW is body weight (kg).

The amount of milk replacer offered to the piglets was calculated daily. Body weight was calculated on the basis of BW upon arrival and the expected growth of 60 g/d [based on Pluske et al. (12) ]. The milk replacer was fed at a concentration of 62 g/L of water. The pigs were fed 4 times per day at 0900, 1230, 1700 and 2130 h. Feed refusals were collected, weighed and subtracted from the amount of milk offered to calculate actual daily feed intake.

Growth and health.

Piglets were weighed upon arrival and on the day of dissection to determine individual growth curves. Feces consistency and shape were scored twice a day from 0 to 3 where 0 = normally shaped feces, 1 = shapeless feces, 2 = thick liquid (soft) feces, and 3 = thin liquid feces (watery diarrhea).

Sampling of gut for histology and permeability.

At d 0, 1, 2 and 4 postweaning, piglets to be killed were weighed and anesthetized by inhalation of a mixture of N₂O/O₂ (ratio 2:1) and isoflurane. The concentration of isofluorane was adjusted to the depth of the narcosis (Guedel, stadium III, phase 2). A midline laparotomy was performed. At three different segments of the small intestine, tissue samples were taken as follows: 0.5 m distal of the ligament of Treitz (proximal small intestine), 3.5 m distal of the ligament of Treitz (mid-small intestine) and 0.5 m proximal to the ileocecal ligament (distal small intestine). For the villous height, crypt depth and villus/crypt ratio, the mean value of the three sampled segments was calculated. After samples were taken, piglets were killed by an intracardiac injection (2 mL) of T61 (a watery solution containing a combination of embutramide, mebezoniumiodide and tetracainehydrochloride; Hoechst Holland, Amsterdam, The Netherlands).

For histologic analysis, tissue samples of the proximal, mid-, and distal small intestine were cut open longitudinally at the antimesenteric attachment, prepared on dental wax with the villi on the upper side and fixed in 0.1 mol/L phosphate buffered formalin solution (40 mL/L). A 3-mm wide zone from the mesenteric site was cut at right angles to the surface of the mucosa and embedded in paraffin wax. Sections (5 µm) were cut and stained with either the periodic acid/Schiff procedure (PA/S) or a combination of the basophilic dyes, high iron diamine (HID) and alcian blue (AB). From the PA/S-stained sections, crypt depth (µm), villous height (µm) and the number of goblet cells (per 100 µm crypt) were determined. From the HID/AB-stained sections, goblet cells of 5 crypts were classified as either sialomucin-containing (blue) or sulfomucin-containing (brown) to investigate the chemical nature of the mucins in the goblet cells. The percentage of sulfomucin-containing cells was calculated. The percentage of sialomucin-containing cells was 100 minus the percentage of sulfomucin-containing cells (data not shown).

To measure the number of CD4⁺ and CD8⁺ cells, mid-small intestinal tissues (3 cm) were deep frozen in liquid nitrogen for 30 min, stored frozen at -80°C until cryosectioning at 5 µm thickness and fixed in acetone for 7 min at room temperature (CD or cell differentiation molecules are cell surface markers of various leukocyte subsets). Cell labeling was performed by incubating the preparations overnight with murine antibodies directed against either porcine CD4 (clone number MIL-17, # MCA 1749, Serotec, Oxford, UK) or CD8 surface antigens (clone number MIL-12, # MCA 1223, Serotec). Subsequently, the samples were incubated with horse anti-mouse antibodies for 30 min followed by Universal peroxidase AEC (3-amino-9-ethyl carbazole substrate solution) for 25 min. Isotonic PBS was used to repeatedly wash the preparations. The tissue sections were counterstained using hematoxylin, washed with tap water and mounted. The number of CD4⁺ and CD8⁺ cells was determined per µm² in the lamina propria of the crypts using light microscopy.

To measure transepithelial transport, mid-small intestinal tissue samples (5 cm) were taken. Transepithelial transport of two compounds was measured in TNO transport chambers, i.e., [¹⁴C] GlySar (Cambridge Research Biochemicals, Northwich, UK) and [2-³H] mannitol (ICN Biomedicals, Zoetermeer, NL). GlySar is a small hydrophilic compound with a molecular weight of 146 Da. It is transported mainly via a transcellular route with a H⁺-coupled di/tripeptide carrier (21) . Mannitol has a molecular weight of 182 Da and is transported mainly via a paracellular route (21) . Intestinal tissues were rinsed with an ice-cold buffer solution of HEPES-buffered phenol red–free Dulbecco’s modified Eagles medium (DMEM) and cut open longitudinally. The tissue was placed with the mucosa on the upper side on a flat underground; with a blunt razor blade, the mucosal layer was carefully stripped off the muscle layer to preserve mucosal integrity. Samples of the mucosal layer were taken using a 9-mm steel punch. Flat sheets, in which isolated intestinal segments (0.2 cm²) separate a 1.5 mL mucosal and a 1.5 mL serosal compartment, were placed in the Ussing chambers. The effective exposed area in the Ussing chamber was 0.196 cm². The radiolabeled GlySar and mannitol were mixed with unlabeled compounds to yield final concentrations of 10 µmol/L. The donor compartment (mucosal side) was filled with 1.25 mL HEPES DMEM medium containing radiolabeled GlySar (10 µmol/L) and mannitol (10 µmol/L). The receptor compartment (serosal side) was filled with 1.25 mL HEPES DMEM medium. Both compartments were aerated (O₂/CO₂, 95:5) at a temperature of 37°C and stirred by gas lift. At indicated time points (15, 30, 45, 75 and 105 min), 0.5-mL samples were taken from the serosal side and the volume was reconstituted with DMEM without phenol red. ³H and ¹⁴C radioactivity was determined in the samples and the tissue (at the end of the experiment) by liquid scintillation counting with the Digital Overlay Technique using the Spectrum Library and the External Standard Spectrum for quench correction. Permeability coefficients (P_ms) were determined on the basis of the appearance of the probe at the serosal side according to the following equation:

(2)

where P_ms is the permeability coefficient from mucosal to serosal side (cm/s); R is the permeability rate (mol/s); A is the exposed intestinal area (cm²); and C₀ is the initial mucosal concentration of the test substance (mol/mL).

Statistical analysis.

The variables measured met the normality criterion. A General Linear Models procedure (SAS version 6.12, SAS Institute, Cary, NC) was used to estimate the least-square means of the three different treatments. The effect of day postweaning was evaluated across diets. Day postweaning, batch and the two-way interaction were the independent variables in the statistical model. The final model was as follows:

(3)

where y_ijkl represents the independent variables; µ is the overall mean; B_i is batch (i = 1, 2); S_j is the fixed effect of day postweaning (j = 1, 2, 3 and 4); (B x S)_ij is the interaction of batch (B) and day postweaning (S); and e_ijk is the error term.

The effect of diet composition was evaluated by including diet composition, day postweaning and batch as independent variables in the statistical model. All two-way interactions were examined, but because these dependent variables appeared not to be significant, these were excluded from the final model. The final model therefore was as follows:

(4)

where y_ijkl represents the independent variables; µ is the overall mean; B_i is batch (i = 1, 2); S_j is the fixed effect of day postweaning (j = 1, 2, 3); D_k is the fixed effect of diet composition (k = 1, 2, 3); and e_ijkl is the error term.

² analysis was used to analyze the diarrhea scores. Pearson correlation analysis was performed to evaluate functional correlation among mean energy intake, histologic parameters and epithelial transport. Significance was assigned at P < 0.05; tendencies were assigned at 0.05 < P < 0.10.

	RESULTS

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General.

BW at weaning was 7.8 ± 0.13 kg. Daily weight loss [g/(pig · d)] through the 4-d treatment period was 97.2 ± 128.59 for LL/HP, 65.3 ± 127.23 for C, and 69.4 ± 146.17 for HL/LP. None of the piglets developed watery feces during the experimental period (score 3). Two had thick liquid feces (score 2); of these, 1 piglet received the C treatment and 1 the HL/LP treatment. Eight piglets had shapeless feces (score 1). Of these, 2 piglets received the C treatment, 1 piglet received LL/HP and 5 received HL/LP. The diarrhea scores were not significantly different among groups (P > 0.10). Inclusion of an independent binomial variable in the statistical model indicating the occurrence/absence of diarrhea, or exclusion of the piglets with diarrhea from the data did not affect the results and conclusions; therefore, the piglets with a diarrhea score were left in the database. None of the piglets received medical treatment during the experimental period.

Energy intake.

Figure 1 shows the DE intake of pigs fed the three milk replacers for 4 d postweaning. The number of piglets for the calculation of the mean DE intake decreased from 54 piglets at d 1, to 36 at d 2 and to 18 at d 3 and 4, due to dissection. DE intake did not differ among diet groups on the different sampling days. DE intake was 648 ± 388.93 kJ/pig on d 1, 1668 ± 625.54 kJ/pig on d 2, 1995 ± 605.25 kJ/pig on d 3, and 1990 ± 670.80 kJ/pig on d 4 postweaning. Independent of diet, the DE intake was lower than the amount offered to the piglets. The percentage of actual energy intake compared with the total amount offered was 43% at d 1, 81% at d 2, 96% at d 3 and 94% at d 4. Over time, intake increased (P < 0.01) for pigs fed each of the three diets.

View larger version (26K): [in this window] [in a new window]   Figure 1. Digestible energy (DE) intake of piglets fed a low lactose/high protein (LL/HP), control (C) or high lactose/low protein (HL/LP) milk replacer for the first 4 d postweaning. Values are means ± SD.

Histologic parameters and weight of the small intestine per kg BW or per cm length of the small intestine at d 0, 1, 2 and d 4 postweaning are shown in Table 2 . Decreased villous height, shallower crypt depths and decreased villus/crypt ratios were most pronounced at the proximal and mid-small intestine. At the distal small intestine, no differences were observed. Villous height of the three sampled sites decreased significantly compared with d 0 (P < 0.01) with the shortest villi at d 2. Villous heights at the three sampled segments were 369 µm on d 0, 349 µm on d 1, 258 µm on d 2 and 317 µm on d 4 (SEM, 12.8). The same mean decrease in villous height over time postweaning could be seen at the proximal and mid-small intestine.

The villus/crypt ratio of the three sampled sites was significantly lower (P < 0.01) at d 2 (1.7) and d 4 (1.9) compared with the d 0 (2.2) and d 1 (2.3). The ratio between villous height and crypt depth also decreased significantly over time postweaning at the proximal and mid-small intestine (P < 0.05), with the lowest ratio on d 2.

The weight of the small intestine per kg BW decreased significantly over time postweaning with the lowest weight at d 2 (23.6 g/kg body) (Table 2) . The weight (g) per cm of the small intestine did not change during time postweaning and was, on average, 7.7 ± 1.08 g/cm.

Figure 2 shows the villous height and crypt depth of the proximal small intestine, mid-small intestine, distal small intestine and the mean value of those three sites of piglets fed LL/HP, C or HL/LP milk replacers. In the proximal small intestine, the villi of the piglets receiving the LL/HP diet tended to be shorter (347 µm) than the villi of the piglets receiving the HL/LP diet (419 µm; P < 0.10). In the proximal small intestine, the villus/crypt ratio was significantly higher (P < 0.05) in piglets fed the HL/LP diet (2.6) compared with those fed the LL/HP (2.0) and the C (2.2) diets (SEM, 0.16; data not shown).

View larger version (52K): [in this window] [in a new window]   Figure 2. Villous height and crypt depth at the proximal small intestine, mid-small intestine, distal small intestine and the mean value of the three segments of piglets fed low lactose/high protein (LL/HP), control (C) or high lactose/low protein (HL/LP) milk replacer. Values are means ± SEM, n = 18.

Crypt goblet cells.

Overall, the number of goblet cell per 100 µm of crypt was not different over time postweaning or across dietary treatments (data not shown). The number of crypt goblet cells was 5.5 ± 1.39 at the proximal, 5.6 ± 1.46 at the mid-, and 7.8 ± 1.81 at the distal small intestine (data not shown). Furthermore, the percentage of sulfomucin-containing cells in intestinal crypts was not different over time postweaning or across dietary treatments (data not shown). The percentage of crypt sulfomucin-containing cells was 35.4 ± 24.73% at the proximal, 27.2 ± 25.56% at the mid-, and 32.8 ± 25.04% at the distal small intestine (data not shown).

T lymphocytes.

The numbers of CD4⁺ and CD8⁺ T cells (per 10⁶ µm² crypt) at the mid-small intestine d 0, 1, 2 or d 4 postweaning are shown in Table 3 . The number of CD4⁺ T cells tended to be lower at d 1 compared with d 0 and 4 (P < 0.10). The number of CD8⁺ T cells at d 0 or 1 postweaning was numerically lower than at d 2 and 4 postweaning, but this difference was not significant. The CD4⁺/CD8⁺ ratio was significantly lower on d 1 and 2 compared with d 0 (P < 0.05), with the lowest ratio on d 1. The ratio of CD4⁺/CD8⁺ T cell lymphocytes had increased significantly by d 4 compared with d 1 postweaning. Diet composition did not affect the number of CD4⁺ and CD8⁺ T cells or the CD4⁺/CD8⁺ ratio (data not shown). A positive correlation was found between the number of CD4⁺ and CD8⁺ T cells (Table 4 ; R = 0.49, P < 0.01). The number of CD4⁺ T cells tended to be negatively correlated with villous height (R = -0.23, P < 0.10) and the villus/crypt ratio (R = -0.22, P < 0.10) at the mid-small intestine. The number of CD8⁺ T cells was negatively correlated with villous height (R = -0.27, P < 0.05) and the villus/crypt ratio (R = -0.25, P < 0.05) at the mid-small intestine. The mean DE intake tended to be positively correlated with CD4⁺ T cells (R = 0.25, P < 0.10) and the CD4⁺/CD8⁺ ratio (R = 0.22, P < 0.10).

Table 3 presents transepithelial transport by GlySar (transcellular transport) and mannitol (paracellular transport) as affected by days postweaning. Figure 3 shows the effect of diet composition on the transepithelial transport. Transcellular transport did not differ among days postweaning or the different weaning diets. Paracellular transport, however, was significantly higher at d 2 and 4 compared with d 0 and 1 postweaning (P < 0.01). Paracellular transport tended to be reduced for piglets consuming the HL/LP milk replacer diet (9.2 x 10^-6 cm/s) compared with those fed the control diet (12.1; P < 0.10).

View larger version (49K): [in this window] [in a new window]   Figure 3. Transcellular (GlySar) and paracellular (mannitol) transport (10-6 cm/s) of the mid-small intestine of piglets fed low lactose/high protein (LL/HP), control (C) or high lactose/low protein (HL/LP) milk replacers. Values are means ± SEM, n = 12.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
These data demonstrate an acute and sequential decline of mucosal barrier function in the pig small intestine during the first 4 d postweaning. The piglets were weaned abruptly at 26 d of age and fed one of three liquid milk replacers. For each of the three diets, the piglets consumed only 648 kJ/pig on d 1 postweaning; this corresponded to 43% of the amount offered. Voluntary milk consumption before weaning was not measured, but averages 5 MJ ME/(pig·d) according to Harrell and colleagues (22) . Thus, the small intestine was subject to a brief but substantial decrease in enteral stimulation at weaning. The importance of enteral stimulation for mucosal homeostasis is well documented (7 ,9 ,10 ,23 24 25) , although the functional consequences of diminished enteral stimulation for the gut wall during the weaning transition in pigs are not clear. These data demonstrate a temporal relationship between low feed intake, increased paracellular transport, decreased ratio of CD4 and CD8 T-cell subsets and compromised epithelial architecture.

Stress and starvation both precede an acute temporal increase in paracellular transport and thereby affect mucosal integrity (14 ,15 ,26 ,27) . Weaning may be regarded as a stressor as indicated by an increase of plasma cortisol concentration and certain behavioral modifications (28) . Plasma cortisol concentrations were 258% greater in weanling pigs on d 2 postweaning compared with unweaned pigs (29) . Kiliaan and coworkers (27) demonstrated that macromolecular protein uptake (horseradish peroxidase) increased in rats after exposure to restraint stress at 8°C, via both the transcellular and paracellular pathways. They found that acetylcholine release during the stress response was critical in the enhanced uptake of the macromolecules across the epithelium. Starvation also increases paracellular transport across intestinal epithelium (14 ,15) . Moreover, Spitz and others (26) demonstrated that the combination of starvation and stress (by glucocorticosteroid injection) resulted in a larger decrease in transepithelial resistance, indicating decreased tight junction resistance, compared with animals either starved or stressed. An increase in intestinal permeability can occur quickly. For example, within 12 h after administration of nonsteroidal anti-inflammatory drugs (NSAID), intestinal permeability to ⁵¹Cr-EDTA was increased (30) .

By increased paracellular permeability, luminal antigens rather than bacteria may enter the lamina propria, resulting in inflammation. This is suggested by the fact that starvation alone does not appear sufficient for bacterial translocation, but after endotoxin challenge, starvation predisposes to bacterial translocation (31 32 33) . Locally increased intestinal permeability leads to an imbalance in normal interactions between luminal aggressive factors (in the small intestine, mainly bile, pancreas secretion, bacteria and their degradation products) and intestinal mucosa, resulting in low grade inflammation perhaps similar to that observed with NSAID-induced enteropathy (30) . Although a significant difference in paracellular transport was not observed between d 0 and 1 in this experiment, a numeric increase was noted (P = x.xx). The positive correlation, however, between either para- and transcellular transport and the CD8⁺ T cell subset predicts the direct involvement of acute inflammation in small intestinal permeability. We postulate that initial translocation of luminal antigens due to increased paracellular transport might have contributed to the alteration in CD4⁺ and CD8⁺ T-cell populations, which might have led to a further increase in paracellular transport during the following days.

These data demonstrate a brief decline in the number of CD4⁺ T cells at d 1, followed by an expansion of CD8⁺ T cells at d 2 and 4 postweaning. The changes in T-cell subsets resulted in a significant decrease in the ratio of CD4⁺ to CD8⁺ T cells at d 1 and 2 compared with d 0. The ratio of the number of CD4⁺ to CD8⁺ T cells seems critical. The number of crypt goblets in cells was not affected by time postweaning or diet composition in this trial and was similar to that observed in an earlier piglet study (34) . Dunsford and co-workers (35) showed incidentally a decrease in the number of goblet cells in the crypts after weaning. The results, however, were inconsistent across the small intestinal sites or across diets. In piglets administered total parenteral nutrition (TPN), the number of goblet cells increased in the villi but did not change in the crypts compared with baseline and orally fed piglets. The chemical composition of mucins was also altered in piglets administered TPN compared with baseline and orally fed piglets (25) . A possibly adaptive response of goblet cells in the crypts to compromised integrity of the mucosal barrier at low feed intake level was not observed in the present study, although villous goblet cells were not evaluated.

Cytokine profiles were not measured here. In a study of De Winter and colleagues (36) , however, downregulation of CD4⁺ T cells altered interleukin 10 and transforming growth factor ß. Regulatory CD4⁺ T cells normally antagonize the expansion, localization, differentiation or effector function of T cells involved in inflammatory responses (36) . Expansion of CD8⁺ cells likely results in the secretion of proinflammatory cytokines (e.g., tumor necrosis factor- and interferon-), which further compromises barrier function (37 ,38) . A systemic increase of proinflammatory cytokines decreases feed intake, resulting in starvation (39) . The T-cell alterations affected the villi more than the crypts, indicated by the negative correlation between the number of CD8⁺ T cells and villous height. The relationship between DE intake and the ratio of CD4⁺ to CD8⁺ T cell numbers tended to be positive, indicating that after weaning, DE intake might be important. The CD4⁺ and CD8⁺ T-cell subsets did not differ among dietary treatments. This is in agreement with the results of McCracken (10) , who also showed that a low feed intake rather than diet composition contributes to local inflammation and affects the mucosal architecture after weaning.

The data demonstrate the onset of repair at d 4 postweaning for villous height, crypt depth, CD4⁺ T cells and the ratio of CD4⁺ to CD8⁺ T-cell subsets. McCracken and co-workers (9) reported the lowest villus/crypt ratio at d 5 instead of d 2, in comparing the sequential effect of the villus/crypt ratio of a liquid milk replacer on d 0, 1, 2, 5 and 7 postweaning. The resolution of inflammation is dependent on full restoration of epithelial barrier function, and the data indicate that paracellular transport remains elevated at d 4 postweaning. Plasma cortisol returned to preweaning levels on d 8 postweaning, comparing preweaned piglets and piglets at d 2 and 8 postweaning (29) . Cessation of the stress likely corresponds with the observation that repair has begun at d 4.

Interestingly, despite the wide range of protein and lactose, diet effects were generally less pronounced than the sequentials of low feed intake at weaning. A high lactose/protein ratio in the diet tended to result in greater villous length and less paracellular transport compared with the other diets. This observation is consistent with the hypothesis that energy from lactose is more limiting than protein for epithelial cells in contributing to mucosal integrity during the first days after weaning. However, diminished feed intake seems to override the effect of diet composition. Nutrient composition and availability may be more important in a reparative phase.

In summary, the effect of diet composition on mucosal integrity is not as important as the sequential effects of low feed intake during the first 4 d postweaning. Low feed intake and stress seem to predispose to decreases in mucosal integrity. The data demonstrated an increase in paracellular transport, an alteration in T-cell subsets and a decrease in villous height. Diet composition did not have a pronounced effect on the variables measured. In a reparative stage, diet effects might be more pronounced, which will be investigated further.

	ACKNOWLEDGMENTS

 
We are grateful to C. Smits, M. Hessing, and J. Meijer (Nutreco BV, The Netherlands), G. Bakker and J. Huisman (ID TNO Animal Nutrition, The Netherlands) for their helpful discussion and useful suggestions. We thank P. van Leeuwen (ID TNO Animal Nutrition, The Netherlands), R. Onderwater (TNO Nutrition, The Netherlands) and the laboratory of J. van Dijk (University of Utrecht, The Netherlands) for technical assistance.

	FOOTNOTES

 
¹ Presented in part in abstract form [Spreeuwenberg, M.A.M., Verdonk, J.M.A.J. & Verstegen, M.W.A.J. (2000) The effect of composition of liquid milk replacer at a low energy level on the small intestinal permeability of piglets after weaning. J. Anim. Sci. 78 (suppl. 1): 137 (abs.)].

² Supported by the Ministry of Economic Affairs and the Ministry of Agriculture of the Dutch government.

⁴ Abbreviations used: AB, alcain blue; BW, body weight; C, control diet; DE, digestible energy; DMEM, Dulbecco’s modified Eagle’s medium; HID, high iron diamine; HL/LP, high lactose/low protein diet; LL/HP, low lactose/high protein diet; NSAID, nonsteroidal anti-inflammatory drugs; PA/S, periodic acid/Schiff procedure; P_ms, permeability coefficient; TPN, total parenteral nutrition.

Manuscript received October 20, 2000. Initial review completed November 21, 2000. Revision accepted January 17, 2001.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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