Ussing chamber

Intestinal barrier function and absorption in pigs after weaning have mainly been assessed in ex vivo studies with Ussing chambers. In Ussing chambers, a section of intestinal mucosa is mounted between two chambers. Marker probes are added to the solution in the chamber at the mucosal site. The appearance of these marker probes in the chamber at the serosal site represents the permeability for these probes. Table 1 gives an overview of probes that have been used in studies with pigs after weaning. In addition, Table 1 gives three electrophysical parameters that can be determined in Ussing chambers. First, the transepithelial electrical resistance (TEER) of the mounted intestinal mucosa can be determined. This is considered to reflect the opening of the tight junctions between epithelial cells, i.e. the paracellular permeability of the intestinal mucosa⁽Reference Boudry¹⁴⁾. An increased TEER reflects decreased paracellular permeability, and a decreased TEER reflects increased paracellular permeability. Second, the transepithelial electrical conductance can be determined. This is the inverse of the TEER⁽Reference Boudry¹⁴⁾. Finally, the short-circuit current (Isc) over the mucosa can be measured. This is a measurement of active electrogenic ion transport across the epithelium. Increased Isc reflects either increased electrogenic anion secretion (e.g. Cl⁻  and HCO₃⁻ ) or increased electrogenic cation absorption (Na⁺). In vivo, the water flow over the intestinal epithelium follows the osmotic gradient induced by actively transported electrolytes. Therefore, Isc can be used to indicate water movement over the epithelium⁽Reference Boudry¹⁴⁾. Moreover, the change in Isc after the addition of specific nutrients to the mucosal solution (e.g. glucose and glutamine) is an indirect measure of Na-dependent nutrient absorption.

Table 1 Marker probe characteristics and electrophysical parameters

Na-Flu, sodium-fluorescein isothiocyanate; TEER, transepithelial electrical resistance.

* Determined by measuring the change in short-circuit current after the addition of glucose or glutamine to the mucosal site of the chamber.

Orally administered marker probes

In vivo permeability tests have been used to assess intestinal permeability in human and medical research for many decades⁽Reference Menzies¹⁵⁾. The principle of the test is that orally administered test substances (probes) that are not metabolised in the body pass through the intestinal epithelium and are excreted in the urine within a short time after administering. Most frequently used probes are those monosaccharides and disaccharides that are not degraded by digestive enzymes, that are hardly metabolised in the body but that are fermented by ‘colonic’ bacteria⁽Reference Bjarnason, MacPherson and Hollander¹⁶⁾. Because of these three characteristics, the sugars are almost exclusively absorbed by the small intestine, and the influences of digestion and metabolism are minimised. Therefore, depending on their permeation route, they can be used as specific markers for small-intestinal permeability function, absorption function or both⁽Reference Bjarnason, MacPherson and Hollander¹⁶⁾.

In vivo permeability tests have been used in pigs around weaning. The most frequently used test in this respect is the d-xylose absorption test⁽Reference Pluske, Williams and Aherne^9, Reference Miller, Newby and Stokes^17–Reference Berkeveld, Langendijk and Verheijden²¹⁾. In studies with pigs, d-xylose is exclusively analysed in blood 1 h after an oral dose. In humans, both blood and urine are used for the d-xylose test. In animals, it is easier to sample blood than to perform a quantitative urine collection. However, a single blood measurement is affected by many factors, for instance, rate of gastric emptying, intestinal absorption rate and clearance rate from the blood⁽Reference Peled, Doron and Laufer²²⁾. Therefore, in general, a correct quantitative urine collection is better than a single blood sampling in order to get an accurate permeability or absorption estimate⁽Reference Peled, Doron and Laufer²²⁾. Even though, absorption and permeability tests based on quantitative urine collection can also be disturbed by several factors. Most important in this respect are a marked renal dysfunction, an incomplete urine collection and an increased luminal clearance of the marker probes by bacterial overgrowth⁽Reference Peled, Doron and Laufer^22, Reference Ehrenpreis, Salvino and Craig²³⁾.

It is evident that knowledge about the permeation routes of the used marker probes is required in order to draw conclusions with physiological relevance. Of the marker probes that are most commonly used with this technique, we have gathered the most relevant information in Table 1. The test results with this technique can, however, be influenced by many premucosal factors (gastric emptying, intestinal transit time and bacterial degradation) and postmucosal factors (metabolism, endogenous production, completeness of urinary collection and renal function)⁽Reference Bjarnason, MacPherson and Hollander¹⁶⁾. To reduce the effects of those premucosal and postmucosal factors, the theory of differential urinary excretion of marker probes has been introduced⁽Reference Bjarnason, MacPherson and Hollander^16, Reference Menzies, Laker and Pounder²⁴⁾. In this approach, it is common practice to use both a disaccharide and a monosaccharide, which are both transported over the epithelium by unmediated diffusion. The transport of monosaccharides used for this test occurs either through tight junctions between the epithelial cells or through aqueous pores in the cell. For disaccharides, the transport occurs through the tight junctions of the crypts⁽Reference Uil, Van Elburg and Van Overbeek^2, Reference Bjarnason, MacPherson and Hollander¹⁶⁾. The ratio of the urinary recovery of the two sugars provides information about the intestinal barrier function. The assumption is that both probes are affected by the premucosal and postmucosal factors to a similar extent, and their ratio is not disturbed by those factors⁽Reference Uil, Van Elburg and Van Overbeek²⁾. In this so-called ‘dual sugar test’, most often, lactulose is used as a disaccharide to assess paracellular permeability and l-rhamnose or mannitol (a sugar alcohol) is used as a monosaccharide. As an example, an increase in the lactulose:l-rhamnose ratio indicates a decrease in the intestinal barrier function, whereas a decrease in the lactulose:l-rhamnose ratio indicates an improved intestinal barrier function. The use of the dual sugar test in pigs is to our knowledge limited to four studies. In two studies, the effect of parenteral v. enteral nutrition in neonatal piglets⁽Reference Kansagra, Stoll and Rognerud^25, Reference Bjornvad, Thymann and Deutz²⁶⁾ has been addressed with lactulose and mannitol as marker probes. In another study, the effect of an lipopolysaccharide challenge in 20 kg gilts has been evaluated with lactulose and l-rhamnose as marker probes⁽Reference Bruins, Hallemeesch and Deutz²⁷⁾. In the fourth study, enhanced dietary Zn concentrations in the weaner diet decreased the lactulose:mannitol ratio in pigs at 2 weeks after weaning⁽Reference Zhang and Guo²⁸⁾.

Thus, studies in pigs shortly after weaning with orally dosed marker probes so far have been limited to a few that have assessed the absorptive small-intestinal function with d-xylose. Up to now, the use of the dual sugar tests to address small-intestinal barrier function in pigs shortly after weaning is limited to one study.

Comparison of techniques

With orally administered marker probes, intestinal barrier function and absorption itself can be measured without killing the pig. This enables repeated measurements on the same pig over time, which allows correlating permeability and absorption parameters with performance and health parameters over time. This is a clear advantage over the Ussing chamber technique for which the pigs need to be killed. However, Ussing chamber measurements enable the assessment of the permeability at a specific intestinal site. Thus, both techniques are complementary.

Transcellular transport (endocytosis)

In two studies with pigs, it has been revealed that in the proximal jejunum, the HRP flux was decreased at 2, 5 and 15 d after weaning⁽Reference Boudry, Péron and Le Huërou-Luron³⁴⁾ and at 4 and 7 d after weaning⁽Reference Verdonk, Bruininx and van der Meulen³⁵⁾ compared with pre-weaning levels (Table 2). We suggest that after weaning, the natural maturation process, enhanced by weaning may reduce the permeability for macromolecules by a reduction in endocytosis rate. This is further supported by a 90 % lower HRP flux at 35 d after weaning compared with 15 d after weaning, as has been established by Boudry et al. ⁽Reference Boudry, Péron and Le Huërou-Luron³⁴⁾. This suggested that the maturation process may be a beneficial mechanism that prevents the animal suffering from an antigen overload. Such an antigen overload may result in an excessive activation of the immune system when the pigs are subjected to their new environment after weaning. In a study of van der Meulen et al. ⁽Reference van der Meulen, Koopmans and Dekker⁷⁾, in the mid-jejunum, the HRP flux increased at 4 and 7 d after weaning compared with 1 d after weaning. In that study, a pre-weaning measurement was not done (first measurement 1 d after weaning), and the pigs were transported and separated from the other pigs and fasted overnight before Ussing chamber measurements were performed. The handling stress in this experiment the day before the measurements can explain the contradiction with the results of the two studies that measured HRP flux in the proximal jejunum⁽Reference Boudry, Péron and Le Huërou-Luron^34, Reference Verdonk, Bruininx and van der Meulen³⁵⁾. Stress has been shown to increase small-intestinal HRP flux in rats⁽Reference Keita, Söderholm and Ericson³³⁾. The handling stress the day before the Ussing chamber measurements on top of the effect of weaning may have generated the increased HRP flux over time in the experiment of van der Meulen et al. ⁽Reference van der Meulen, Koopmans and Dekker⁷⁾. Unlike in the jejunum, the HRP flux in the ileum was not affected after weaning in the study of Boudry et al. ⁽Reference Boudry, Péron and Le Huërou-Luron³⁴⁾. These differences of the ileum compared with the jejunum may relate to the fact that the basal HRP flux at weaning in the ileum was only 35 % of that in the jejunum. This indicates that at weaning, antigen sampling is already at a lower level in the ileum, and a further reduction (maturation) in this respect may not be beneficial.

Table 2 Small-intestinal barrier function in pigs after weaning as measured by horseradish peroxidase flux, mannitol flux and transepithelial electrical resistance in Ussing chambers

d, Day.

Values were significantly different from those at weaning or from the control treatment: *P < 0·05.

† Piglets were delivered by caesarian section and subsequently housed in isolators and fed ad libitum condensed cows' milk until the treatment started at 3 weeks of age.

The HRP flux in the proximal jejunum was not affected by feed intake level after weaning⁽Reference Verdonk, Bruininx and van der Meulen³⁵⁾, and in the mid-jejunum, it was not affected by feed intake level before weaning and by weaning age (4 v. 7 weeks)⁽Reference van der Meulen, Koopmans and Dekker^7, Reference Verdonk, Bruininx and van der Meulen³⁵⁾. Boudry et al. ⁽Reference Boudry, Lallès and Malbert³⁶⁾ changed the diet of pigs of 25 kg (4–6 weeks after weaning) from a milk replacer to a barley- or wheat-based diet. At 4 d after this dietary change, the HRP flux in the proximal jejunum of the barley- and wheat-fed pigs was not different from that of control pigs fed the milk replacer. Also, extra tryptophan in the diet after weaning (5 g/kg diet) did not affect the HRP flux in the mid-small intestine at 4, 5 or 6 d after weaning of pigs at 25 d of age⁽Reference Koopmans, Guzik and Van Der Meulen³⁷⁾. Egberts et al. ⁽Reference Egberts, de Groot and Van Dijk³⁸⁾ found no effect of enterotoxigenic E. coli infection on proximal jejunal permeability for HRP (measured in vivo) 48 h after the infection in 3-week-old pigs. In suckling piglets, Boudry et al. ⁽Reference Boudry, Douard and Mourot³⁹⁾ have reported that the effect of mast cell degranulation (this is a stressor that increases permeability) on ileal permeability to HRP decreased with age. This effect of age occurred earlier in piglets of sows fed n-3 fatty acids (minimum at 7 d of age) than in piglets of sows fed the control diet (minimum at 28 d of age). In line with this, Rådberg et al. ⁽Reference Rådberg, Biernat and Linderoth⁴⁰⁾ have shown that oral administration of red kidney bean lectins in 2-week-old suckling piglets reduced the small-intestinal permeability for large molecules (bovine serum albumin, 67 kDa; ovalbumin, 45 kDa). Thus, dietary treatments were not able to affect HRP fluxes after weaning, but specific dietary treatments before weaning could reduce small-intestinal permeability for macromolecules. This indicates that the level of antigen uptake is higher before weaning than after weaning, creating a window that enables dietary treatments to have an effect. This further supports the hypothesis that antigen uptake decreases over time after weaning as a result of a maturation process.

Paracellular transport

Moeser et al. ⁽Reference Moeser, Ryan and Nighot⁴¹⁾ have shown that mannitol flux and TEER over the mid-jejunum were not different at 1 d after weaning compared with unweaned controls for pigs weaned at 28 d of age. However, for pigs weaned at 3 weeks of age, Moeser et al. ⁽Reference Moeser, Klok and Ryan^6, Reference Moeser, Ryan and Nighot⁴¹⁾ and Boudry et al. ⁽Reference Boudry, Péron and Le Huërou-Luron³⁴⁾ have reported that the mannitol flux over the proximal or mid-jejunum was significantly increased, and TEER was significantly decreased at 1 and 2 d after weaning compared with weaning or compared with unweaned controls (Table 2). This shows that weaning pigs at a higher age can prevent the loss of the paracellular barrier function after weaning. In addition, Moeser et al. ⁽Reference Moeser, Klok and Ryan^6, Reference Moeser, Ryan and Nighot⁴¹⁾ have shown that in 3-week-old pigs, TEER and mannitol flux were not affected by the weaning process when stress pathways were blocked with a corticotropin-releasing factor receptor antagonists or with a mast cell-stabilising drug. This shows that stress is a major factor with respect to the disturbed intestinal barrier function after weaning. Moreover, it shows that the immune system through mast cell activation has a critical role in the loss of the intestinal barrier function after weaning. Several other studies have shown that intestinal barrier function was compromised in pigs weaned at an age of 26 d⁽Reference Verdonk, Bruininx and van der Meulen^35, Reference Montagne, Boundry and Favier^42–Reference Verdonk⁴⁴⁾. Verdonk⁽Reference Verdonk⁴⁴⁾ measured the mannitol flux in the mid-jejunum of pigs with a low v. a high intake level of milk replacer after weaning (Table 2). The mannitol flux was not affected in pigs with a high feed intake level at 1, 2 and 4 d after weaning. However, mannitol flux was increased for pigs with low feed intake levels at 2 and 4 d after weaning compared with pre-weaning. As an average over days 1, 2 and 4, this resulted in a higher mannitol flux for pigs with low feed intake compared with pigs with high feed intake (at least 2·5 times energy maintenance). In line with this, a 2 d fast of 23-d-old pigs increased transepithelial electrical conductance (the opposite of TEER)⁽Reference Carey, Hayden and Tucker⁴⁵⁾. Thus, a sufficient feed intake after weaning prevents the loss of the barrier function of the tight junctions after weaning. This indicates the importance of a sufficient luminal nutrient supply to maintain the barrier function. Sufficient feed intake may be especially important for the proximal small intestine because this part depends more on luminal nutrient supply than the distal small intestine⁽Reference Stoll, Chang and Fan⁴⁶⁾. In the study of Verdonk⁽Reference Verdonk⁴⁴⁾, villus height in the proximal small intestine decreased after weaning for pigs at the low intake level and was hardly affected after weaning in pigs at the high intake level. This illustrates that luminal nutrient supply was adequate in the high-intake group and inadequate in the low-intake group. In line with the study of Verdonk⁽Reference Verdonk⁴⁴⁾ described earlier, Spreeuwenberg et al. ⁽Reference Spreeuwenberg, Verdonk and Gaskins⁴³⁾ have shown that the mannitol flux in the mid-jejunum increased at 2 and 4 d after weaning compared with pre-weaning. Moreover, Verdonk et al. ⁽Reference Verdonk, Bruininx and van der Meulen^35, Reference Verdonk⁴⁴⁾ published two other experiments in which the mannitol flux was measured over the epithelium of the proximal instead of the mid-jejunum in pigs after weaning (Table 2). In the first experiment, the mannitol flux was increased at 2 and 6 d after weaning compared with the pre-weaning flux. However, in the second experiment, the mannitol flux at 4 and 7 d after weaning was not different from pre-weaning fluxes and was not affected by feed intake level after weaning. Thus, the last study, in contrast to the first two studies of Verdonk⁽Reference Verdonk, Bruininx and van der Meulen^35, Reference Verdonk⁴⁴⁾ and the study of Spreeuwenberg et al. ⁽Reference Spreeuwenberg, Verdonk and Gaskins⁴³⁾, has shown no increase in mannitol flux after weaning, although feed intake levels were reasonably low. This discrepancy is probably related to handling of the pigs before the start of the experiment. In the first two studies of Verdonk and in the study of Spreeuwenberg et al. ⁽Reference Spreeuwenberg, Verdonk and Gaskins⁴³⁾, pigs were transported from another location before the start of the experiment. However, in the last experiment of Verdonk⁽Reference Verdonk⁴⁴⁾, piglets were weaned and kept at the same location at which the experiment was conducted. Thus, in this last experiment, the weaning process was probably less stressful because pigs were not transported in a trailer. Because stress is an important factor with respect to the intestinal barrier function (see above), this may explain the differences between the studies. Lodemann et al. ⁽Reference Lodemann, Hübener and Jansen^47, Reference Lodemann, Lorenz and Weyrauch⁴⁸⁾ have found that at 7 d after weaning, the mannitol flux in the mid-jejunum was either significantly or numerically lower from before weaning, and TEER was not different than before weaning. In these experiments, the timing of permeability measurements may have been too late to detect a disturbed barrier function or, again, the stress level around weaning may have been lower than in the other experiments. In the ileum, TEER was increased at 5 and 15 d after weaning compared with pre-weaning levels in a study of Boudry et al. ⁽Reference Boudry, Péron and Le Huërou-Luron³⁴⁾. Carlson et al. ⁽Reference Carlson, Poulsen and Sehested⁴⁹⁾ have shown similar results in the ileum with pigs weaned at 4 weeks of age. In their study, the transepithelial electrical conductance (the opposite of TEER) was not different from weaning at 1–2 d after weaning and was lower at 5–6 and 14–15 d after weaning compared with weaning. Thus, in the ileum, in contrast to the proximal and mid-jejunum, intestinal barrier function is not compromised due to the weaning process. Finally, Berkeveld et al. ⁽Reference Berkeveld, Langendijk and Verheijden²¹⁾ administered pigs an oral mannitol dose before and at 0·5, 2, 4 and 7 d after weaning and measured plasma mannitol concentrations 1 h after each dose. They observed that the plasma mannitol concentrations decreased gradually after weaning, being significantly different from pre-weaning concentration at 4 d after weaning. This contradiction between the results of Berkeveld et al. ⁽Reference Berkeveld, Langendijk and Verheijden²¹⁾ (orally administered mannitol) with the other studies (Ussing chambers) may relate to the different techniques used to determine intestinal mannitol transport. The study of Berkeveld et al. ⁽Reference Berkeveld, Langendijk and Verheijden²¹⁾ may reflect mannitol permeability in the whole small intestine, and in this study, mannitol was measured in the blood instead of in the urine, which is a complicating factor in evaluating the results (see above).

In general, dietary treatments after weaning showed only minor effects on paracellular intestinal permeability (Table 2). Supplementing different probiotics to the diets of the sow and piglets had no effect on mid-jejunal mannitol flux and TEER of piglets after weaning⁽Reference Lodemann, Hübener and Jansen^47, Reference Lodemann, Lorenz and Weyrauch⁴⁸⁾. In two studies of Carlson et al. ⁽Reference Carlson, Poulsen and Sehested^49, Reference Carlson, Sehested and Feng⁵⁰⁾, it was revealed that dietary Cu and Zn levels had no effect on the transepithelial electrical conductance of the jejunum and ileum at 5–7 d after weaning. However, in a study of Zhang & Guo⁽Reference Zhang and Guo²⁸⁾, urinary latulose:mannitol ratios were decreased after feeding enhanced dietary Zn concentrations for a period of 2 weeks after weaning. The discrepancy between the studies of Carlson et al. and the study of Zhang & Guo may again relate to the stress level during the study. In the study of Zhang & Guo, pigs were separated from the other pigs, individually housed and fasted overnight before the permeability test. Boudry et al. ⁽Reference Boudry, Lallès and Malbert³⁶⁾ changed 25 kg pigs from a milk replacer diet to a barley- or wheat-based diet on an equal intake level. The jejunal TEER was not different from the milk-fed pigs 4 d after this dietary change. Finally, the combination of an increased dietary lactose concentration (41 v. 24 %) and a decreased dietary protein concentration (15 v. 30 %) tended to decrease mannitol flux in the mid-small intestine⁽Reference Spreeuwenberg, Verdonk and Gaskins⁴³⁾. Thus, no distinction could be made between a possible (positive) effect of lactose and a possible (negative) effect of protein on mannitol flux. In the same study, the combination of a decreased dietary lactose concentration (8 v. 24 %) and an increased dietary protein concentration (45 v. 30 %) had no effect on mannitol flux. In contrast to the studies described earlier, Hamard et al. ⁽Reference Hamard, Mazurais and Boudry⁵¹⁾ weaned pigs at 7 d of age and showed that dietary threonine deficiency, for a period of 2 weeks after weaning, increased the ileal permeability for fluorescein isothiocyanate dextran (4 kDa) and also tended to decrease TEER. The aforementioned studies indicate that diet composition in general has not a major effect on paracellular permeability. However, when diets deficient in nutrients (e.g. threonine) are fed, paracellular permeability deteriorates. In line with this, intestinal barrier function is compromised at low feed intake levels, and, in this respect, a low feed intake level is similar to a diet being deficient in all nutrients. In addition, diet composition may affect paracellular permeability when permeability measurements are accompanied with additional stressors (i.e. fasting and individual housing).

Conclusions regarding barrier function

The present review clarifies that small-intestinal barrier function in pigs is affected by the process of weaning. In the literature, four factors (i.e. weaning age, weaning stress, feed intake and diet composition) have been identified that can have a major effect on the barrier function after weaning. In addition, barrier function is differently affected after weaning in the proximal and mid-jejunum than in the ileum. The relationships between these different aspects of the intestinal barrier function after weaning are illustrated in Fig. 1. The stress that is associated with weaning manipulates the immune system, resulting in mast cell activation that has a critical role in the loss of the barrier function of the tight junctions in the small intestine. In addition, the loss of the paracellular barrier function is prevented when feed intake after weaning is at an adequate level such that the loss of the villous height is prevented. This shows that low feed intake is another factor that seems to have a critical role in the compromised barrier function after weaning. In contrast to the proximal and distal jejunum, paracellular barrier function after weaning is not compromised in the ileum. Because luminal nutrient supply is most critical in the proximal small intestine, this indicates that the loss of the barrier function due to low feed intake is due to a shortage of luminal nutrient supply. In line with this, it was shown that barrier function was compromised in pigs after feeding a threonine-deficient diet for a period of 2 weeks. This effect of nutrient supply on the barrier function may be a secondary effect as a result of a compromised intestinal architecture. The loss of the paracellular barrier function is almost exclusively found in the first week after weaning and returns to the pre-weaning level at 2 weeks after weaning. Pigs are less susceptible to a compromised barrier function when weaned at an older age and also in the long term have a better barrier function⁽Reference Moeser, Ryan and Nighot^41, Reference Smith, Clark and Overman⁵²⁾. This is probably related to the earlier stage of maturation of the small intestine when pigs are weaned at a young age. Although paracellular barrier function is consistently compromised after weaning, one can argue whether this is a direct risk for the health of the pig. Moreover, transcellular barrier function for macromolecules through endocytosis improves after weaning. We suggest that this maturation process enhanced by weaning may prevent the animal suffering from an antigen overload. Such an antigen overload may result in an excessive activation of the immune system when pigs are subjected to their new environment after weaning. We hypothesise that the increased paracellular permeability also indicates that the intestine is extra susceptible for the disturbance of the transcellular barrier function when several stressors occur simultaneously.

Fig. 1 Scheme representing the relationship between small-intestinal barrier function, small-intestinal location and factors (age, stress, feed intake or diet composition) that affect the barrier function. The thickness of the arrows indicates the significance of the relationship. Barrier function is less affected at high than at low weaning age, which relates probably to the intestinal maturation rate. Weaning stress compromises the paracellular barrier function indirectly through mast cell activation (immunity). Adequate feed intake levels after weaning prevent the loss of the barrier function probably indirectly through the preservation of intestinal architecture. The direct effect of diet composition on the intestinal barrier function seems to be limited unless diets are deficient in specific nutrients. Barrier function is most affected in the proximal and mid-small intestine and hardly in the distal small intestine.

Based on the present review, three different approaches can be followed to improve the intestinal barrier function after weaning by ways of dietary composition: first, the old-fashioned approach to improve the palatability of the diet to increase feed intake after weaning. This has only been partially successful up to now; second, to identify crucial nutrients (e.g. protein or specific amino acids) that may be supplied to pigs with low feed intake in a concentrated form or through the drinking-water in order to prevent the loss of the intestinal barrier function; third, to add specific biologically active components to the diet to modulate the stress response or the subsequent immune response, to prevent the loss of the barrier function. With this last approach, it is essential that the diet should be eaten, otherwise the active component needs to be supplied through the drinking-water.

Active absorption

In a study of Boudry et al. ⁽Reference Boudry, Péron and Le Huërou-Luron³⁴⁾, pigs were weaned at 21 d of age. In that study, the Na⁺-dependent glucose absorption increased in the proximal jejunum at 2 d after weaning and decreased at 15 d after weaning compared with pre-weaning absorption (Table 3). Moreover, in the same study, ileal glucose absorption decreased at 2, 5 and 15 d after weaning. In line with this, Smith⁽Reference Smith⁵⁶⁾ has found that the Na⁺-dependent alanine uptake by enterocytes of the mid-small intestine (measured with a rapid uptake apparatus, using radiolabelled tracer amino acids) decreased considerably at 5 d after weaning at 2 or 3 weeks of age. Furthermore, in 4-week-old pigs, Na⁺-dependent alanine uptake by enterocytes of the mid-small intestine was lower for weaned pigs (5 d after weaning, thus weaning at 23 d) than for unweaned pigs⁽Reference Miller, James and Smith⁵⁷⁾. However, the alanine uptake of 6-week-old pigs (both weaned and unweaned) was similar to the alanine uptake of 4-week-old weaned pigs⁽Reference Miller, James and Smith⁵⁷⁾. This study suggests that weaning (before 4 weeks of age) and ageing appear to decrease the number of enterocytes that are involved in active alanine uptake⁽Reference Miller, James and Smith⁵⁷⁾. The aforementioned studies have shown that active small-intestinal absorption decreases after weaning when pigs are weaned between 14 and 23 d of age. However, when weaned after 4 weeks of age, active absorption is not affected by the weaning process. Miller et al. ⁽Reference Miller, James and Smith⁵⁷⁾ have observed that the decreased absorption rates after weaning also occur in unweaned pigs but over a much longer time course. This suggests that this decrease in active absorption is part of a maturation process that is enhanced by the process of weaning. This may relate to a decrease in the relative demand for nutrients when pigs get older because weight gain expressed relative to body weight decreases when pigs get older. Several studies have investigated the active absorption of glucose, glutamine or GlySar in the proximal, mid and distal small intestine for pigs that were weaned at 26 or 28 d of age⁽Reference Verdonk, Bruininx and van der Meulen^35, Reference Spreeuwenberg, Verdonk and Gaskins^43, Reference Verdonk^44, Reference Lodemann, Hübener and Jansen^47–Reference Carlson, Poulsen and Sehested⁴⁹⁾. In all these studies, active absorption between 1 and 15 d after weaning was either similar to or higher than absorption before weaning. This confirms that active small-intestinal absorption after weaning is only suppressed when pigs are weaned before 4 weeks of age. The only contradiction to this is the study of Buddington et al. ⁽Reference Buddington, Elnif and Puchal-Gardiner⁵⁸⁾. They measured carrier-mediated asparagine, leucine, lysine, methionine and proline absorption (per unit of wet mass) in the mid-small intestine for pigs weaned between 32 and 35 d of age. They showed that for all amino acids, active absorption was lower after weaning (42 d of age) than before weaning (at 28 d of age). The discrepancies of the study of Buddington et al. ⁽Reference Buddington, Elnif and Puchal-Gardiner⁵⁸⁾ with the other studies may relate to the fact that they did not use Ussing chambers or a rapid uptake apparatus.

Table 3 Small-intestinal molecular absorption in pigs after weaning as measured for Na⁺-dependent glucose, Na⁺-dependent glutamine, glycylsarcosine and sodium-fluorescein isothiocyanate absorption

d, Day.

Values were significantly different from those at weaning or from the control treatment: *P < 0·05.

In a study of Verdonk⁽Reference Verdonk⁴⁴⁾, energy intake during the first 2 d after weaning was either low (close to 0 for pigs on a dry diet) or moderate (0·5 times maintenance for pigs fed a wet diet). In this study, the average GlySar absorption at days 2 and 6 was higher for pigs with low feed intake than for pigs with moderate feed intake. In rats, starvation increased the expression of mRNA of peptide transporter 1, and along with the up-regulation of this transporter protein, the activity of GlySar uptake was enhanced⁽Reference Shimakura, Terada and Saito⁵⁹⁾. This may explain why GlySar absorption increased in pigs with low feed intake levels after weaning. In another study of Verdonk⁽Reference Verdonk⁴⁴⁾, pigs were fed milk replacers, and the energy intake during the first 2 d after weaning was either moderate (0·5 times maintenance) or high (3·0 times maintenance). GlySar absorption in the mid-jejunum at 1, 2 and 4 d after weaning was not different from pre-weaning for treatments with moderate energy intake. However, GlySar absorption at day 1 was lower compared with pre-weaning in pigs with high energy intake. Thus, low feed intake stimulates active absorption. In agreement with this, active glucose absorption in the proximal jejunum was stimulated after pigs were fasted for a period of 2 d⁽Reference Carey, Hayden and Tucker⁴⁵⁾. Moreover, Boudry et al. ⁽Reference Boudry, Péron and Le Huërou-Luron³⁴⁾ have shown that active glucose absorption in the proximal jejunum increased when pigs were fasted for a period of 2 d after weaning. In contrast, in the same study, ileal glucose absorption decreased after weaning. This difference in glucose absorption between the proximal and distal small intestine is probably because the proximal region depends more on luminal nutrient supply than the distal region⁽Reference Stoll, Chang and Fan⁴⁶⁾. Boudry et al. ⁽Reference Boudry, Lallès and Malbert³⁶⁾ changed pigs of 25 kg (4–6 weeks after weaning) from a milk replacer to a barley- or wheat-based diet. It was observed that 4 d after this dietary change, the Na⁺-dependent glucose absorption in the proximal jejunum of the barley- or wheat-fed pigs was higher than that of the control pigs fed the milk replacer. This indicates that after weaning, the shift from a milk- to a cereal-based diet increases active small-intestinal absorption. In conclusion, the short-term increase in active absorption in the proximal small intestine after weaning is due to the low feed intake after weaning. The long-term increase is related to the shift from a milk- to a cereal-based diet. The addition of fish oil or DHA to gestation diets of the sow increased Na⁺-dependent glucose and glutamine absorption in the proximal jejunum at 24 h after weaning in 15- to 20-d-old pigs⁽Reference Gabler, Radcliffe and Spencer^60, Reference Gabler, Spencer and Webel⁶¹⁾. Dietary threonine deficiency tended to increase ileal Na⁺-dependent glucose absorption at 14 d after weaning⁽Reference Hamard, Mazurais and Boudry⁵¹⁾. Studies with differences in dietary minerals (Cu and Zn), lactose and protein concentration and dietary probiotic addition have found no effect on active small-intestinal glucose, glutamine or GlySar absorption after weaning⁽Reference Spreeuwenberg, Verdonk and Gaskins^43, Reference Lodemann, Hübener and Jansen^47–Reference Carlson, Sehested and Feng⁵⁰⁾. Thus, the shift from a milk- to a cereal-based diet and dietary fatty acid composition have a significant effect on active small-intestinal absorption, whereas some other dietary changes have no effect on absorption.

Passive absorption

Results of three studies⁽Reference Miller, Newby and Stokes^17, Reference Hampson and Smith^19, Reference Berkeveld, Langendijk and Verheijden²¹⁾ revealed that d-xylose absorption in piglets before weaning is hardly affected over time after 3 weeks of age. Results of five studies with pigs after weaning showed that absorption of d-xylose⁽Reference Pluske, Williams and Aherne^9, Reference Miller, Newby and Stokes^17–Reference Kelly, Smyth and McCracken²⁰⁾ decreased gradually to about 50 % of the pre-weaning level at 7 d after weaning. In line with this, the absorption of Na-Flu in the proximal jejunum decreased at 4 and 7 d after weaning compared with pre-weaning levels in pigs weaned at 26 d of age⁽Reference Verdonk⁴⁴⁾. Berkeveld et al. ⁽Reference Berkeveld, Langendijk and Verheijden²¹⁾, however, have observed that plasma d-xylose concentrations were significantly higher at 2 and 7 d after weaning than pre-weaning and were not different between pre-weaning and 0·5 and 4 d after weaning. Apart from the results of the study of Berkeveld et al. ⁽Reference Berkeveld, Langendijk and Verheijden²¹⁾, passive absorption seems to decrease consistently after weaning. This seems to be a permanent effect because even at 14 d after weaning d-xylose absorption was only at 65 % of the absorption level measured before weaning⁽Reference Miller, Newby and Stokes¹⁷⁾. We hypothesise that the reduced passive transcellular absorption after weaning is a defence mechanism that prevents uncontrolled transport of potential harmful agents to enter the body. This is more or less in line with what was described before with respect to the transcellular transport of macromolecules after weaning. In all these studies, weaning age, varying from 14 to 29 d of age, does not seem to have a major effect on the response after weaning. Creep feed intake of piglets before weaning had no effect on d-xylose absorption from 1 to 14 d after weaning⁽Reference Miller, Newby and Stokes^17, Reference Hampson and Smith^19, Reference Kelly, Smyth and McCracken²⁰⁾. Moreover, Na-Flu absorption was not affected by creep feed intake before weaning or by weaning age (4 or 7 weeks of age) at 1, 4 and 7 d after weaning⁽Reference van der Meulen, Koopmans and Dekker⁷⁾. Kelly et al. ⁽Reference Kelly, Smyth and McCracken⁶²⁾ have found no difference in d-xylose absorption at 5 d after weaning for tube-fed pigs fed at a low (0, 0·25, 0·5, 0·75, 1·0 times energy maintenance on days 1–5, respectively) or at a high (1·5, 1·75, 2·0, 2·25, 2·5 times energy maintenance on days 1–5, respectively) intake level of the diet. In addition, Pluske et al. ⁽Reference Pluske, Williams and Aherne⁹⁾ have shown that d-xylose absorption after weaning was not affected by the intake level of cows' milk after weaning (1·0, 2·5 or 4·0 times energy maintenance) and was not different for pigs fed cows' milk v. pigs fed a dry weaner diet. Thus, passive absorption is not affected by feed intake level before or after weaning. Finally, the addition of extra tryptophan (5 g/kg diet) after weaning had no effect on Na-Flu flux in the mid-small intestines at 4, 5 or 6 d after weaning of pigs weaned at 25 d of age⁽Reference Koopmans, Guzik and Van Der Meulen³⁷⁾.

Conclusions regarding absorption

The present review shows that active and passive absorption are differently affected after weaning. Active absorption after weaning is influenced by three important factors (weaning age, feed intake level and feed composition). However, passive absorption decreases after weaning irrespective of these three factors. This reduced passive transcellular absorption after weaning may be a defence mechanism that prevents uncontrolled transport of potential harmful agents to enter the body. This is more or less in line with what was previously described with respect to the transcellular transport of macromolecules after weaning. The decreased passive absorption may reflect a natural maturation process of the intestine that occurs rapidly after weaning, as hypothesised earlier by other authors⁽Reference Smith^56, Reference Miller, James and Smith⁵⁷⁾. In general, active small-intestinal absorption decreases after weaning when pigs are weaned at 3 weeks of age or at a lower age. In line with the decrease in passive absorption, this decrease in active absorption may be part of a maturation process that is enhanced by the process of weaning. This may relate to a decrease in the relative demand for nutrients when pigs get older because of a decrease in weight gain relative to body weight. However, when weaned at an age of 4 weeks or later in life, active absorption is not affected by weaning or stimulated by the weaning process. This may indicate that with respect to active absorption, the small intestine is mature at 4 weeks of age. The shift from a milk- to a cereal-based diet and addition of fish oil or DHA to the diet increase active absorption. Thus, diet composition after weaning can have a significant effect on active small-intestinal absorption. Moreover, this indicates that the shift from a milk- to a cereal-based diet may be responsible for the long-term increase in active absorption after weaning. It seems that passive and active absorption after weaning adapt accurately to the changed environment after weaning with respect to feeding status when weaned after 3 weeks of age. Only when weaned at 3 weeks of age or earlier, the decrease in active absorption indicates an insufficient adaptation to the new environment that may result in an insufficient absorptive capacity. A diet that stimulates active absorption, for instance DHA, may help to overcome or prevent this sudden decrease in absorption.