DOI: 10.1111/jpn.12232
ORIGINAL ARTICLE
Effects of age and zinc supplementation on transport properties in the jejunum of piglets E. M. Gefeller1, H. Martens1, J. R. Aschenbach1, S. Klingspor1, S. Twardziok2, P. Wrede2, R. Pieper3 and U. Lodemann1 1 Faculty of Veterinary Medicine, Institute of Veterinary Physiology, Freie Universit€at Berlin, Berlin, Germany – Universit€atsmedizin Berlin, Berlin, Germany, and 2 Molecular Biology and Bioinformatic, Charite 3 Faculty of Veterinary Medicine, Institute of Animal Nutrition, Freie Universit€at Berlin, Berlin, Germany
Summary Zinc is effective in the prevention and treatment of post-weaning diarrhoea and in promoting piglet growth. Its effects on the absorption of nutrients and the secretory capacity of the intestinal epithelium are controversial. We investigated the effects of age, dietary pharmacological zinc supplementation and acute zinc exposure in vitro on small-intestinal transport properties of weaned piglets. We further examined whether the effect of zinc on secretory responses depended on the pathway by which chloride secretion is activated. A total of 96 piglets were weaned at 26 days of age and allocated to diets containing three different levels of zinc oxide (50, 150 and 2500 ppm). At the age of 32, 39, 46 and 53 days, piglets were killed, and isolated epithelia from the mid-jejunum were used for intestinal transport studies in conventional Ussing chambers, with 23 lM ZnSO4 being added to the serosal side for testing acute effects. Absorptive transport was stimulated by mucosal addition of D-glucose or L-glutamine. Secretion was activated by serosal addition of prostaglandin E2, carbachol or by mucosal application of Escherichia coli heat-stable enterotoxin (Stp). Jejunal transport properties showed significant age-dependent alterations (p < 0.03). Both absorptive and secretory responses were highest in the youngest piglets (32 d). The dietary zinc supplementation had no significant influence on jejunal absorptive and secretory responses. However, the pre-treatment of epithelia with ZnSO4 in vitro led to a small but significant decrease in both absorptive and secretory capacities (p < 0.05), with an exception for carbachol (p = 0.07). The results showed that, in piglets, chronic supplementation with zinc did not sustainably influence the jejunal transport properties in the post-weaning phase. Because transport properties are influenced by the addition of zinc in vitro, we suggest that possible epithelial effects of zinc depend on the acute presence of this ion. Keywords epithelium, ion transport, pig, small intestine, Ussing chamber, chloride secretion Correspondence Dr. Ulrike Lodemann, Institute of Veterinary Physiology, Freie Universit€at Berlin, Oertzenweg 19b, 14163 Berlin, Germany. Tel: +49 30 83862592; Fax: +49 30 83862610; E-mail: [email protected] Received: 25 April 2013; accepted: 30 June 2014
Introduction Around weaning, piglets are exposed to many stress factors that manifest themselves in increased intestinal permeability (Bruewer et al., 2003) and make piglets highly susceptible to pathogenic bacteria and their enterotoxins (Stevens et al., 1972; Lall es et al., 2004). Zinc supplementation at pharmacological levels has been shown to improve performance parameters in weaned piglets (Hahn and Baker, 1993; Hill et al., 2001; Zhang and Guo, 2009). The incidence of post-weaning diarrhoea can also be reduced by high doses of zinc (Owusu-Asiedu 542
et al., 2003), but the underlying mechanisms are poorly understood. Evidence has been provided for the importance of zinc in sustaining intestinal barrier function (Vallee and Falchuk, 1993). The influence of zinc on the intestinal absorption of nutrients is, however, controversial as it has been shown to both decrease (Lyall et al., 1979; Watkins et al., 1989; Yoldi et al., 1992; Rodriguez-Yoldi et al., 1994) and increase (Lee et al., 1989) glucose absorption. Similarly, contradictory results have been obtained regarding the intestinal transport of amino acids in the presence of zinc (Boldizsar and Simon, 1981; Monteilh-Zoller et al., 1999).
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As a possible explanation for the antidiarrhoeal effect of zinc, decreased secretory responses to several secretagogues have been observed when zinc has been applied at the basolateral side of isolated intestinal epithelia (Feng et al., 2006; Carlson et al., 2008). However, even this information is contradictory (Canani et al., 2005; Hoque et al., 2005; Berni Canani et al., 2010). Given the inconsistent and sometimes inconclusive results of previous studies, this study aimed at a very systematic and broad-based approach to assess the effect of dietary zinc supplementation at different dosages on both absorptive and secretory transport properties in the jejunum of piglets of various age groups after weaning. To dissociate long-term effects of zinc feeding from short-term effects attributable to the actual presence of zinc, intestinal epithelia from all zinc supplementation levels and age groups were additionally tested for the effects of an acute serosal application of zinc in vitro. Finally, the secretory response and the mode of activation of chloride secretion were also studied. Material and methods The experiments were conducted in accordance to German law, and legal permission was granted by the Landesamt f€ ur Gesundheit und Soziales (G 0347/09). Animals and diets
The experiment included 96 purebred Landrace piglets that were weaned at 26 1 days (d) of age with a mean bodyweight of 7.2 1.2 kg. Animals were housed in pens (n = 2 animals per pen) with straw bedding. From 12 d of age, piglets had free access to the same non-medicated pre-starter diet formulated to meet the requirements of piglets with an average bodyweight of 5–10 kg (GfE, 2006). After being weaned, the piglets were assigned randomly to one of three feeding groups balanced for gender, litter and bodyweight. The piglets received a mash starter diet (Table 1) until the 53th d of life; this diet provided zinc at final concentrations of 50 (Zn50), 150 (Zn150) or 2500 (Zn2500) mg/kg (ppm) by replacing corn starch with analytical grade zinc oxide (ZnO) (Sigma Aldrich, Taufkirchen, Germany). All diets were mixed in the feed mill at the Institute of Animal Nutrition of the Freie Universit€at Berlin. Dietary zinc levels were determined by atomic absorption spectrometry in an AAS vario 6 spectrometer (Analytik Jena, Jena, Germany) to be 57, 164 and 2425 ppm in the Zn50, Zn150 and Zn2500 feeding groups respectively. The piglets Journal of Animal Physiology and Animal Nutrition © 2014 Blackwell Verlag GmbH
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Table 1 Ingredients and chemical composition of the starter diet g/kg Ingredients Wheat Barley Soybean meal Corn starch/ zinc oxide* Limestone Monocalcium phosphate Mineral and Vitamin Premix† Soy oil Salt Lysine HCl Methionine Chemical composition Dry matter ME (MJ/kg) Crude ash Crude protein Crude fibre Ether extract Starch Lysine Methionine Threonine Tryptophan Calcium Phosphorus Sodium Magnesium Zinc mg/kg‡ Iron mg/kg Manganese mg/kg Copper mg/kg
380 300 232 10 20 20 15 17.5 2.0 2.5 1.0 879 13.0 81 194 36 34 376 11.7 4.0 7.2 2.4 11.0 8.0 3.1 2.2 34 309 81 18
*Corn starch in the basal diet was partially replaced in the diets containing 50, 150 and 2500 mg/kg zinc with analytical grade zinc oxide (Sigma Aldrich, Taufkirchen, Germany) to adjust for the desired zinc level. †Mineral and Vitamin Premix (Spezialfutter Neuruppin, Neuruppin, Germany) providing per kg feed: 1.95 g Na (as sodium chloride), 0.83 g Mg (as magnesium oxide), 10 500 IU vitamin A, 1800 IU vitamin D3, 120 mg vitamin E, 4.5 mg vitamin K3, 3.75 mg thiamine, 3.75 mg riboflavin, 6.0 mg pyridoxine, 30 lg cobalamine, 37.5 nicotinic acid, 1.5 mg folic acid, 375 lg biotin, 15 mg pantothenic acid, 1200 mg choline chloride, 75 mg Fe (as iron-(II)-carbonate), 15 mg Cu (as copper-(II)-sulphate), 90 mg Mn (as manganese-(II)-oxide), 675 lg I (as calcium iodate) and 525 mg Se (as sodium selenite). ‡Analysed concentration of zinc in the basal diet without ZnO supplementation. The final experimental diets contained Zn at 57, 164 or 2425 mg/kg.
had free access to water and feed throughout the experimental period. Sampling procedure
At the age of 32 1, 39 1, 46 1 and 53 1 days, eight piglets per group were killed 543
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such that the resulting age groups were balanced for litter and gender. The piglets were sedated with 20 mg/kg bodyweight of ketamine hydrochloride (Ursotaminâ; Serumwerk Bernburg AG, Bernburg, Germany) and 2 mg/kg bodyweight of azaperone (Stresnilâ; Jansen-Cilag, Neuss, Germany) prior to being killed with an intracardial injection of 10 mg/kg bodyweight of tetracaine hydrochloride, mebezonium iodide and embutramide (T61â; Intervet, Unterschleißheim, Germany). A mid-line abdominal incision was made, and 1 m of mid-jejunum was removed. The jejunum was stripped off from its outer muscle layers, rinsed, placed in a transport buffer solution (containing in mM: 115 NaCl, 25 NaHCO3, 0.4 NaH2PO4, 2.4 Na2HPO4, 5 KCl, 5 glucose, 1.2 CaCl2, 1.2 MgCl2 and 2.8 lM indomethacin), gassed with carbogen (95% O2, 5% CO2) and kept at 38 °C. Incubation procedure
Epithelia were cut into squares (2 cm2) and mounted between the halves of a conventional Ussing chamber giving a buffer-exposed area of 0.95 cm2. Tissue damage resulting from contusion was minimised using rings of silicone rubber at each side of the epithelium. The Ussing chamber technique was as described elsewhere (Martens et al., 1987). Approximately 30–60 min elapsed from piglet dispatch to the final mounting of the tissues into the Ussing chambers. The buffer solutions had an osmolarity of 295 mosM and were adjusted to pH 7.4 by titrating with 500 mM 2-(N-morpholino)ethanesulfonic acid (MES). The temperature was maintained at 38 °C by a thermostat (Haake D1; VWR International GmbH, Darmstadt, Germany). The epithelia were bathed on both sides with 10 ml buffer solution. The buffer solutions for incubation in the Ussing chambers were of the same composition as the transport buffer solution, except that the serosal solution contained 10 (instead of 5) mM glucose plus 2 mM mannitol, whereas the mucosal solution was glucose-free but contained 12 mM mannitol. Both mucosal and serosal buffer solutions contained 1.4 lM indomethacin. During the equilibration period (30 min after mounting), the media were continuously circulated and aerated with carbogen (95% O2, 5% CO2). All reagents were purchased from VWR International GmbH, Sigma Aldrich Chemie GmbH or Bachem AG (Bubendorf, Switzerland), and were of analytical grade. 544
Electrophysiological measurements
Electrophysiological measurements were obtained by a microcomputer-controlled voltage/current clamp (version 2.02 supplied by Dipl.-Ing. K. Mußler, Aachen, Germany). Two KCl agar bridges were placed near to each side of the epithelium and connected to Ag–AgCl electrodes for the measurement of the transepithelial potential difference (PDt). Two distantly placed KCl agar bridges were connected to Ag–AgCl electrodes for transmitting current through the tissue. The junction potential and the fluid resistance of the buffer between the tips of the PD-sensing bridges were determined before the tissue was mounted and were subsequently corrected by the computer-controlled voltage clamp. The tissue was alternatively pulsed with a positive or negative pulse of 100 lA and 200 ms duration. The displacement in PDt caused by the current pulse was measured, and the tissue resistance (Rt) was calculated from the change in PDt due to the law of Ohm. The pulse amplitude PDt (mV), current Isc (lA/cm2) and Rt (Ω cm2) were continuously recorded. After an equilibration period of 10 min under open-circuit conditions, the epithelia were short-circuited. In vitro treatments
A total of 12 Ussing chambers were used per animal. After 15 min under short-circuit conditions, half of the epithelia were pre-treated with ZnSO4 (23 lM; Sigma Aldrich Chemie GmbH) at the serosal side, whereas the other chambers received no ZnSO4. To assess the absorptive capacity of jejunal epithelia, DGlucose or L-glutamine concentrations of 1, 4, 12 mM was added to two chambers, each with or without serosal ZnSO4. D-glucose or L-glutamine was added to the mucosal side of the epithelia, while mannitol at an equivalent amount was added to the serosal side to keep osmolarity equal on both sides. After an exchange of incubation buffer and an equilibration period, the secretagogues were added to separate tissues 30 min after the addition of glucose. For the assessment of secretagogue effects, one chamber with and one without serosal ZnSO4 was used for each concentration of the respective secretagogue. Secretion was activated by serosal application of prostaglandin E2 (0.1, 0.5 or 5 lM) or carbachol (10 lM) or by the mucosal application of Escherichia coli heat-stable enterotoxin (2.5 or 5 lM) (Bachem AG) in the respective incubation solution. The allocation of chambers to substrate and secretagogue additions, as well as the sequence of additions, was equal for all animals. All Journal of Animal Physiology and Animal Nutrition © 2014 Blackwell Verlag GmbH
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given concentrations are final concentrations in the incubation solution. To test the viability of the tissue theophylline, 8 mM was added bilaterally to each tissue at the end of the experiment. Calculations and statistical analysis
The Isc and Rt values at 1 min before the serosal addition of zinc in vitro were treated as baseline data. The effect of the zinc application in vitro was assessed from the Isc and Rt values recorded 15 min after the addition of ZnSO4. To quantify the effects of absorptive or secretory stimulants, the maximum change of Isc (DIsc) was calculated by subtracting the maximum value of Isc measured within 5 min after the addition of D-glucose or L-glutamine, within 10 min after the addition of PGE2 or carbachol or within 45 min after the addition of E. coli heat-stable enterotoxin from the Isc value at 1 min before the application of the respective stimulant. Statistical evaluations and the plotting of graphs were carried out by means of the SPSS program for Windows, version 20 (Jandel, Chicago, IL, USA). Statistical significance of differences was assessed by a linear mixed model with the fixed factors ‘concentration’ (concentration levels of the respective stimulant), ‘feeding’ (50, 150, 2500 ppm), ‘age’ (day 32, 39, 46, 53) and ‘zinc’ (with or without ZnSO4 addition in vitro) and the interaction of ‘age 9 concentration’ with the random factor ‘animal’. In the case of statistically significant effects of a fixed factor, the post hoc test LSD (least significant difference) was conducted. The differences between groups were considered statistically significant for p ≤ 0.05. Unless otherwise stated, results are given as means or least square means SEM. The number of experimental animals is given as N, whereas n refers to the number of epithelial tissues per treatment group. Results Baseline Isc and Rt
Baseline values of Isc and Rt measured before any addition was made in vitro (i.e. 1 min before ZnSO4 addition) did not differ significantly between the age and feeding groups (data not shown). Values recorded before and after the addition of ZnSO4 in vitro did not differ significantly as well.
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Na+-coupled glucose or glutamine transport was stimulated by the mucosal application of increasing concentrations of D-glucose or L-glutamine (1, 4 or 12 mM). The change in the short-circuit current (DIsc) was recorded. Both stimulants caused a dose-dependent increase in Isc (p < 0.001; Table 2). The highest responses were measured at day 32 when the changes in Isc after the addition of glucose or glutamine were approximately twice as high compared with those at days 39, 46 and 53 (p < 0.001; Table 2). No significant effect of feeding was observed. However, DIsc after the application of D-glucose or L-glutamine was numerically highest in the Zn2500 group at almost all ages (with only one exception for D-glucose at 32 d) and lowest in the Zn150 group (Fig. 1; data not shown for L-glutamine).
Table 2 Response of short-circuit current (DIsc in lA/cm2) to L-glutamine, D-glucose, prostaglandin E2, Escherichia coli heat-stable enterotoxin and carbachol Age 32 d (mM) 1 13 (8–18)* 4 37 (31–42) 12 54 (48–59) Age effect a D-glucose (mM) 1 68 (52–83) 4 139 (123–155) 12 165 (149–181) Age effect a PGE2 (lM) 0.1 80 (66–94) 0.5 106 (92–120) 5 136 (122–150) Age effect a E. coli toxin (lM) 2.5 15 (8–22) 5 20 (13–26) Age effect a Carbachol (lM) 10 125 (110–141) Age effect a
39 d
46 d
53 d
6 (1–12) 16 (11–22) 26 (20–31) b
5 (0–10) 12 (7–17) 20 (15–25) b
6 (1–12) 13 (8–19) 26 (21–31) b
L-glutamine
31 (15–47) 28 (12–44) 67 (51–83) 61 (45–77) 85 (69–101) 81 (65–97) b b
34 (18–50) 72 (56–88) 93 (77–109) b
57 (42–72) 54 (39–69) 50 (35–64) 76 (62–90) 76 (62–90) 75 (61–88) 100 (86–114) 106 (92–119) 108 (94–122) b b b 5 (1–12) 7 (1–14) b
9 (2–16) 9 (2–16) ab
16 (9–23) 18 (11–25) a
89 (76–103) 95 (82–109) 97 (84–111) b b b
To assess whether zinc supplementation affected the absorptive capacity of the jejunal epithelium,
*Data represent least square means confidence interval 95%. Different letters within one row indicate significant differences between the age groups (p < 0.05). There was a significant interaction ‘age 9 concentration’ for L-glutamine and D-glucose (p < 0.001). N = 24 animals, n = 87–92 epithelia per age and concentration group for L-glutamine and D-glucose; n = 41–69 epithelia per age and concentration group for PGE2, n = 35–46 epithelia for carbachol per age group; N = 12 animals, n = 22–24 epithelia per age and concentration group for E. coli heat-stable enterotoxin.
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Fig. 1 Effect of dietary Zn concentration and age on the jejunal epithelial response of short-circuit current (DIsc) to mucosal addition of D-glucose. Data are representing the mean values over all glucose concentrations (1, 4 and 12 mM). Only epithelia without in vitro ZnSO4 treatment are included. N = 8 piglets, n = 40–47 epithelia per feeding and age group.
Secretory capacity
Chloride secretion can be activated by at least three different pathways, which we stimulated with the following secretagogues: (i) prostaglandin E2 (PGE2) exerts its impact on chloride secretion by activating the adenylate cyclase and increasing the cAMP level (Blikslager et al., 2001); (ii) E. coli heat-stable enterotoxin induces secretory diarrhoea by activating the cGMP system (Field, 2003); and (iii) carbachol stimulates Cl secretion as a result of an increased intracellular calcium level (Leonhard-Marek et al., 2009). The DIsc measured after the application of PGE2 increased with increasing dose (p < 0.001), whereas the DIsc evoked by E. coli enterotoxin was not different for the factor ‘concentration’ (p = 0.056) (Table 2). Significant age effects were observed for the responses to PGE2 (p = 0.001), E. coli heat-stable enterotoxin (p = 0.027) or carbachol (p = 0.005; Table 2). The change in short-circuit current was highest in the youngest piglets (32 d) compared with the other age groups (39, 46, 53 d), with an exception for E. coli heat-stable enterotoxin for which only the 32nd and the 39th day differed significantly. In general, the effect of rising dietary Zn supply on secretory responses was not significant. However, at days 39 and 46, the Zn50 group tended to have the highest DIsc after stimulation with PGE2, but the response of this feeding group was markedly inconsistent (Fig. 2). With regard to the E. coli toxin-induced chloride secretion, a decrease in secretion from 150 to 546
Fig. 2 Effect of dietary Zn concentration and age on the jejunal epithelial response of short-circuit current (DIsc) to serosal addition of PGE2. Data are representing the mean values over all PGE2 concentrations (0.1, 0.5 and 5 lM). Only epithelia without in vitro ZnSO4 treatment are included. N = 8 piglets, n = 25–32 epithelia per feeding and age group.
2500 ppm zinc supplementation was observed (Fig. 3). The secretory response to carbachol was not consistently altered by the different dietary intake of zinc (data not shown). Effect of serosal ZnSO4 in vitro
The increases in Isc following the addition of D-glucose (p < 0.001) and L-glutamine (p = 0.013) were significantly lower in the tissues pre-treated with serosal
Fig. 3 Effect of dietary Zn concentration and age on the jejunal epithelial response of short-circuit current (DIsc) to serosal addition of Escherichia coli heat-stable enterotoxin (Stp). Data are representing the mean values over all Stp concentrations (2.5 and 5 lM). Only epithelia without in vitro ZnSO4 treatment are included. N = 4 piglets, n = 7–8 epithelia per feeding and age group.
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ZnSO4 in vitro (Fig. 4). The epithelia pre-treated with ZnSO4 also showed significantly reduced secretory reactions to PGE2 (p = 0.027) and Stp (p = 0.042) compared with the respective control groups without ZnSO4 (Fig. 5). The carbachol-induced DIsc was numerically lower in tissues that were pre-incubated with ZnSO4 (Fig. 5). The reduction in Isc was 8.7% of the control value for D-glucose, 8.5% for L-glutamine,
Fig. 4 Jejunal epithelial response of short-circuit current (DIsc) to mucosal addition of D-glucose or L-glutamine comparing epithelia without and with prior ZnSO4 treatment in vitro. Data are means SEM. N = 96 piglets, n = 533–542 epithelia. *Significant differences from the respective group without serosal ZnSO4 addition in vitro are indicated by asterisks.
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4.9% for PGE2, 8.4% for carbachol and 14.5% of the control value for E. coli toxin. Discussion Zinc is well known to support piglet performance around weaning (Hahn and Baker, 1993; Hill et al., 2001; Zhang and Guo, 2009) and to reduce the incidence of post-weaning diarrhoea (Owusu-Asiedu et al., 2003). It has also been shown to reduce bacterial translocation into gut-associated lymph nodes (Huang et al., 1999; Metzler-Zebeli et al., 2010). Furthermore, data concerning performance parameters of the same piglets were published by Martin et al. (2012). They observed a higher average daily gain and feed intake in piglets fed high ZnO levels (2500 ppm) during the first week of feeding compared with the other two feeding groups, but these did not differ thereafter. In the present study, we have examined both the possible effect of chronic dietary zinc supplementation in vivo and the influence of an acute addition of ZnSO4 in vitro on absorptive and secretory transport properties in the jejunum of weaned pigs. In the feeding trial, the diets provided three different concentrations of ZnO. The lowest concentration was 50 ppm, which can be considered a marginal supply based on the recommended Zn concentration of 80–100 ppm in the feed of piglets (NRC, 1998; GfE, 2006). The Zn150 group received the maximal concentration legally allowed within the EU (EC.1334, 2003), whereas the high dietary supplementation with 2500 ppm zinc represented pharmacological levels. For the in vitro studies, ZnSO4 was chosen because of the low solubility of ZnO. The serosal concentration of 23 lM ZnSO4 was applied as it is close to the zinc plasma concentrations of piglets fed with a diet containing 2000 ppm zinc (Carlson et al., 1999, 2007) or 2500 ppm zinc (Pieper et al., 2012). Age effects
Fig. 5 Jejunal response of short-circuit current (DIsc) after serosal addition of carbachol, PGE2 and Escherichia coli heat-stable enterotoxin (Stp) comparing epithelia without and with prior ZnSO4 treatment in vitro. Data are means SEM. N = 96 piglets, n = 354–361 epithelia for PGE2, n = 84–86 epithelia for carbachol; N = 48 piglets, n = 93 epithelia for E. coli heat-stable enterotoxin (Stp). *Significant differences from the respective group without serosal ZnSO4 addition in vitro are indicated by asterisks.
Previous studies have shown that the transport capacity of the intestinal epithelium increases as the intestinal surface increases from the day of birth until the time after weaning. In relation to the intestinal mass, however, the transport rate decreases, perhaps because of a modified density of transport proteins (Buddington and Diamond, 1990; Puchal and Buddington, 1992). In agreement with this concept, the youngest piglets (32 d) showed the highest responses to all added stimulants regarding absorption and secretion (Figs 1–3 and Table 2), whereas significantly decreased transport properties were assessed at day
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39. The decrease in absorptive capacity from 32 to 39 d of age (i.e. from 6 to 13 d after weaning) differs only slightly from the data of Carlson et al. (2004), who have shown that glucose transport is increased until day 15 after weaning. Boudry et al. (2004) have found that jejunal glucose-induced currents are higher at the day of weaning compared with the DIsc at 2 weeks after weaning, assuming that this decrease in glucose transport capacity is attributable to a switch in the diet from milk to cereals. The overall magnitude for the glucose-stimulated DIsc in the present study was in accordance with earlier electrophysiological studies (Lodemann et al., 2006, 2008). Regarding secretion, Miller and Skadhauge (1997) conducted Ussing chamber experiments and found that Cl secretion was not affected by weaning, in accordance with McEwan et al. (1990) who did not find significant differences in the secretory responses of 14-d-old piglets compared with 14-week-old pigs to PGE2 and theophylline. In agreement with our results, however, Carlson et al. (2004) observed a higher secretory capacity at 5–6 days after weaning than at 14–15 days after weaning. Boudry et al. (2004) also measured an increased response to secretagogues at the day of weaning compared with 2 weeks after weaning. The reduced secretory capacity compared with that of younger animals is assumed to result from a decreased sensitivity with age (Grondahl et al., 1996; Erlwanger et al., 1999) and from the maturation of the intestinal tissue (Cummins et al., 1988). As a possible explanation for the decrease in secretory capacity after weaning, Lange et al. (1993) have demonstrated that the antisecretory factor (ASF) increases from negligible levels in piglets younger than 5 week of age to high levels in older pigs. Sensitivity to secretagogues is thus assumed to decrease with age (Grondahl et al., 1996; Erlwanger et al., 1999) and with the maturation of the intestinal tissue (Cummins et al., 1988). The weaning time seems to represent an event, whereby transport capacities increase notably and then further decrease with age.
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Chronic zinc supplementation in feed did not significantly affect the ion currents linked to glucose or glutamine absorption in the present study. These results are in contrast to observations in rats in which feeding 200 mg ZnCl2/kg bodyweight decreased small-intestinal glucose absorption (Lyall et al., 1979) and, vice versa, zinc deficiency led to
increased glucose absorption in vivo (Southon et al., 1984). As the effect of zinc feeding (chronic zinc supplementation) on these transport pathways is not statistically significant, our results are in agreement with previous findings in the porcine target species in which the supplementation of the diet of piglets with increasing concentrations of zinc had no effect on Na+-dependent glucose transport at 5–7 days after weaning (Carlson et al., 2004). To our knowledge, no study of the effects of zinc feeding on L-glutamine transport has been conducted so far, although Lyall et al. (1979) did not observe an effect on alanine transport in rats after feeding them zinc. These findings are confirmed in the present study for L-glutamine transport. Nonetheless, our results also show that a depressing effect of Zn on both glucose and glutamine absorption occurs in pigs when a high plasma Zn concentration is acutely simulated in vitro. This observation is in agreement with in vitro studies of the small-intestinal epithelium of pigs (Watkins et al., 1989), rabbits (Yoldi et al., 1992; Rodriguez-Yoldi et al., 1994) and rats (Lyall et al., 1979). However, Hoque et al. (2005) have observed no such effect in a study with ileal tissues of rats. Lee et al. (1989) have conducted smallintestinal perfusion studies and shown that acute addition of zinc (0.9 mM) raises the glucose absorption in the human small intestine. For the amino acid L-alanine, Lyall et al. (1979) have found that zinc in vitro does not affect the absorption of this amino acid. However, the present study has revealed that Lglutamine transport is significantly reduced by acute zinc addition in vitro. We should, however, emphasise that the above-mentioned studies involved the use of zinc concentrations between 0.5 and 10 mM, which is far above the zinc concentration used in the present investigation. The underlying mechanisms by which Zn inhibits glucose absorption in vitro are still not known. Authors of relevant studies suggest that heavy metals decrease intestinal sugar transport by binding to chemical groups of the membrane protein and thereby influence the Na+-dependent cotransport system (Rodriguez-Yoldi et al., 1989; Yoldi et al., 1992; Mesonero et al., 1993). As sugar transport is driven by the transmembrane Na+ gradient, zinc might reduce sugar uptake by decreasing the activity of the Na+/K+ATPase (Rodriguez-Yoldi et al., 1994). The latter seems plausible when considering that Na+/glucose and Na+/glutamine cotransport were equally affected in the present study and that the ZnSO4 was applied at the serosal side in which the Na+/K+ATPase is located. An inhibition of basolateral K+ channels by serosal Zn
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application, as described by Hoque et al. (2005), could likewise negatively affect the activity of the Na+/K+ATPase or simply decrease the driving force for Na+-dependent glucose uptake because an inhibition of basolateral K+ channels also depolarises the potential difference of the apical membrane. Secretory studies
Three main intracellular signalling cascades are utilised to activate the epithelial ion channels that drive secretion. These signalling cascades include cAMP, cGMP and intracellular calcium, all of which have been investigated in the present study by applying PGE2, Stp or carbachol. Our study has not confirmed an antisecretory effect of the dietary zinc supplementation on PGE2- and carbachol-activated secretion. The secretion induced by Stp tended to be decreased with higher levels of zinc in the diet, especially in 32-d-old piglets, but a significant feeding effect was also not observed. These results are in contradiction to Carlson et al. (2004) who found that piglets fed a diet containing 2500 ppm ZnO showed a significantly reduced secretory response compared with a group receiving 100 ppm ZnO when secretion was stimulated by adding theophylline and serotonin. No published data are available on the effect of dietary zinc on PGE2-stimulated Cl secretion. Although an effect of Zn pre-feeding on secretory pathways was not delineable in the present study, the acute zinc addition in vitro had significant reducing effects on the Cl secretion induced by PGE2, Stp and, at least numerically, carbachol. This agrees with several previous studies showing that the presence of zinc at the basolateral side reduces the effect of secretagogues such as 5-HT, vasoactive intestinal peptide (VIP), theophylline and carbachol in pigs (Carlson et al., 2006) and carbachol (Berni Canani et al., 2010) and cholera toxin (Canani et al., 2005) in Caco-2 cells. The latter study by Canani et al. (2005) emphasises that the antisecretory effect of serosal Zn application (35 lM) applies only to cholera toxin and not to E. coli heat-stable enterotoxin. However, this might not really be in contrast to the present study in which the inhibition of the Stp effect by serosal Zn application is significant but extremely small indeed. A preferential inhibition of cAMP-activated chloride secretion over carbachol-activated secretion had been demonstrated using extremely high serosal Zn concentrations (1 mM) in the rat ileum (Hoque et al., 2005); this result agrees with the finding of the present study in which only PGE2, but not carbachol, effects reached statistical significance. Some authors (Hoque et al., 2005; Medani et al., 2012) explain the Journal of Animal Physiology and Animal Nutrition © 2014 Blackwell Verlag GmbH
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Zn effect by a selective inhibition of K+ channels which depolarises the potential difference as mentioned above and hence the driving force necessary for cAMP-activated Cl secretion. However, Medani et al. (2012) showed that zinc sulphate not only inhibits PGE2 but also carbachol-induced secretion in human colonic mucosae. While the present study focussed on Zn effects on electrogenic pathways, electroneutral mechanisms may also be involved in positive effects of zinc on diarrhoea. When diarrhoea occurs, cAMP is increased by enterotoxins and neurotransmitters which then lead not only to stimulation of Cl secretion but also to inhibition of electroneutral NaCl absorption (Donowitz and Welsh, 1986). Hoque et al. (2009) studied whether Zn has an effect on the Na+/H+ exchanger NHE3, which is a key player in electroneutral NaCl absorption. In Caco-2 cells, they demonstrated that Zn significantly increases the NHE3 activity and that the inhibitory effect of cAMP on NHE3 activity was significantly blunted by zinc. Based on these findings, the authors supported the use of Zn in the therapy of diarrhoea, for example in oral rehydration solutions. Conclusion This study has confirmed the results of most previous studies showing that the absorptive and secretory capacity of the porcine intestinal epithelium decreases with age after weaning. In a highly systematic approach employing four different age groups, two different absorptive pathways, three different secretory stimulants and three different feeding levels of Zn, our results have further provided conclusive evidence suggesting that chronic zinc supplementation does not alter small-intestinal transport mechanisms sustainably. However, the acute presence of zinc on the basolateral side of the epithelium seems to interfere directly with transport mechanisms. This observation might explain the contradictory effects of chronic and acute zinc treatments of other studies (Carlson et al., 2004, 2006, 2008, 2010). The acute presence of zinc reduces both absorptive and secretory transport processes, a finding that appears to be contra-productive with regard to the absorption of glucose and glutamine. However, over the total length of the intestine, this reduced mid-jejunal absorption might be compensated in distal parts of the intestine. Indeed, performance parameters were not negatively affected by supplementation with zinc oxide in the same pigs (Martin et al., 2012). With regard to secretion, the reducing effect might be beneficial for the animal, as the epithelium seems to be 549
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less susceptible to diarrhoeal diseases in the presence of zinc. We should also acknowledge, however, that the Zn effects on epithelial transport function are moderate from a quantitative point of view, compared with the effects occurring around weaning. This suggests that the benefits of high Zn supplementation for weight gain and diarrhoea incidence after weaning might only partially be explained by the direct influence of Zn on epithelial transport processes, with the main effects of Zn being exerted on other key players, for example the intestinal microbiota or the intestinal immune system or the enteric nervous system (ENS). With regard to the latter, References Berni Canani, R.; Secondo, A.; Passariello, A.; Buccigrossi, V.; Canzoniero, L. M.; Ruotolo, S.; Puzone, C.; Porcaro, F.; Pensa, M.; Braucci, A.; Pedata, M.; Annunziato, L.; Guarino, A., 2010: Zinc inhibits calcium-mediated and nitric oxide-mediated ion secretion in human enterocytes. European journal of pharmacology 626, 266–270. Blikslager, A. T.; Pell, S. M.; Young, K. M., 2001: PGE2 triggers recovery of transmucosal resistance via EP receptor cross talk in porcine ischemia-injured ileum. American Journal of Physiology Gastrointestinal and Liver Physiology 281, G375– G381. Boldizsar, H.; Simon, F., 1981: Effect of zinc sulphate supplementation on amino acid absorption from pig intestine. I. Intestinal loop tests. Acta Veterinaria Academiae Scientiarum Hungaricae 28, 409–418. Boudry, G.; Peron, V.; Le Huerou-Luron, I.; Lalles, J. P.; Seve, B., 2004: Weaning induces both transient and long-lasting modifications of absorptive, secretory, and barrier properties of piglet intestine. Journal of Nutrition 134, 2256–2262. Bruewer, M.; Luegering, A.; Kucharzik, T.; Parkos, C. A.; Madara, J. L.; Hopkins, A. M.; Nusrat, A., 2003: Proinflammatory cytokines disrupt epithelial barrier function by apoptosis-independent mechanisms. Journal of Immunology 171, 6164– 6172. Buddington, R. K.; Diamond, J. M., 1990: Ontogenetic development of monosaccharide and amino acid transporters in rabbit intestine. American Journal of Physiology 259, G544–G555. Canani, R. B.; Cirillo, P.; Buccigrossi, V.; Ruotolo, S.; Passariello, A.; De Luca, P.;
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however, Feng et al. (2006) concluded that zinc attenuates the cAMP-dependent ion secretion mainly due to an effect on epithelial cells rather than affecting mucosal neuronal pathways. Acknowledgements This research was financially supported by the Deutsche Forschungsgemeinschaft, Grant No. SFB 852/1 and by the H.-W.-Schaumann-Stiftung, Hamburg. The help of U. Tietjen, K. Wolf, M. Grunau and U. Ujkani is gratefully acknowledged.
Porcaro, F.; De Marco, G.; Guarino, A., 2005: Zinc inhibits cholera toxininduced, but not Escherichia coli heat-stable enterotoxin-induced, ion secretion in human enterocytes. Journal of Infectious Diseases 191, 1072–1077. Carlson, M. S.; Hill, G. M.; Link, J. E., 1999: Early- and traditionally weaned nursery pigs benefit from phase-feeding pharmacological concentrations of zinc oxide: effect on metallothionein and mineral concentrations. Journal of animal science 77, 1199–1207. Carlson, D.; Damgaard Poulsen, H.; Sehested, J., 2004: Influence of weaning and effect of post weaning dietary zinc and copper on electrophysiological response to glucose, theophylline and 5-HT in piglet small intestinal mucosa. Comparative Biochemistry and Physiology – Part A: Molecular And Integrative Physiology 137, 757–765. Carlson, D.; Sehested, J.; Poulsen, H. D., 2006: Zinc reduces the electrophysiological responses in vitro to basolateral receptor mediated secretagogues in piglet small intestinal epithelium. Comparative Biochemistry and Physiology – Part A: Molecular And Integrative Physiology 144, 514–519. Carlson, D.; Beattie, J. H.; Poulsen, H. D., 2007: Assessment of zinc and copper status in weaned piglets in relation to dietary zinc and copper supply. Journal of Animal Physiology and Animal Nutrition 91, 19–28. Carlson, D.; Sehested, J.; Feng, Z.; Poulsen, H. D., 2008: Serosal zinc attenuate serotonin and vasoactive intestinal peptide induced secretion in piglet small intestinal epithelium in vitro. Comparative Biochemistry and Physiology – Part A: Molecular And Integrative Physiology 149, 51–58.
Carlson, D.; Sehested, J.; Poulsen, H. D., 2010: Serosal Zn2+ inhibits 8-Br-cAMP stimulated chloride secretion in piglet small intestinal epithelium in vitro. Livestock Science 134, 30–32. Cummins, A. G.; Steele, T. W.; LaBrooy, J. T.; Shearman, D. J., 1988: Maturation of the rat small intestine at weaning: changes in epithelial cell kinetics, bacterial flora, and mucosal immune activity. Gut 29, 1672–1679. Donowitz, M.; Welsh, M. J., 1986: Ca2+ and cyclic AMP in regulation of intestinal Na, K, and Cl transport. Annual review of physiology 48, 135–150. EC.1334, 2003: Commission Regulation (EC) No. 1334/2003. Official Journal of the European Union. Erlwanger, K. H.; Unmack, M. A.; Grondahl, M. L.; Skadhauge, E.; Thorboll, J. E., 1999: Effect of age on vasoactive intestinal polypeptide-induced shortcircuit current in porcine jejunum. Comparative Biochemistry and Physiology. Part A, Molecular & Integrative Physiology 124, 29–33. Feng, Z.; Carlson, D.; Poulsen, H. D., 2006: Zinc attenuates forskolin-stimulated electrolyte secretion without involvement of the enteric nervous system in small intestinal epithelium from weaned piglets. Comparative Biochemistry and Physiology – Part A: Molecular and Integrative Physiology 145, 328–333. Field, M., 2003: Intestinal ion transport and the pathophysiology of diarrhea. Journal of Clinical Investigation 111, 931– 943. GfE, 2006: Empfehlungen zur Energie- und N€ ahrstoffversorgung von Schweinen. Ausschuss f€ ur Bedarfsnormen der Gesellschaft f€ ur Ern€ ahrungsphysiologie, DLG-Verlag, Frankfurt/M., Germany, p. 132.
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Grondahl, M. L.; Hansen, M. B.; Larsen, I. E.; Skadhauge, E., 1996: Age and segmental differences in 5-hydroxytryptamine-induced hypersecretion in the pig small intestine. Journal of Comparative Physiology B Biochemical Systemic and Environmental Physiology 166, 21–29. Hahn, J. D.; Baker, D. H., 1993: Growth and plasma zinc responses of young pigs fed pharmacologic levels of zinc. Journal of animal science 71, 3020–3024. Hill, G. M.; Mahan, D. C.; Carter, S. D.; Cromwell, G. L.; Ewan, R. C.; Harrold, R. L.; Lewis, A. J.; Miller, P. S.; Shurson, G. C.; Veum, T. L., 2001: Effect of pharmacological concentrations of zinc oxide with or without the inclusion of an antibacterial agent on nursery pig performance. Journal of animal science 79, 934–941. Hoque, K. M.; Rajendran, V. M.; Binder, H. J., 2005: Zinc inhibits cAMP-stimulated Cl secretion via basolateral Kchannel blockade in rat ileum. American Journal of Physiology Gastrointestinal and Liver Physiology 288, G956–G963. Hoque, K. M.; Sarker, R.; Guggino, S. E.; Tse, C. M., 2009: A new insight into pathophysiological mechanisms of zinc in diarrhea. Annals of the New York Academy of Sciences 1165, 279–284. Huang, S. X.; McFall, M.; Cegielski, A. C.; Kirkwood, R. N., 1999: Effect of dietary zinc supplementation on Escherichia coli septicemia in weaned piglets. Swine Health Production 7, 109–111. Lall es, J.-P.; Boudry, G.; Favier, C.; Floc’h, N. L.; Luron, I.; Montagne, L.; Oswald, I. P.; Pi e, S.; Piel, C.; S eve, B., 2004: Gut function and dysfunction in young pigs: physiology. Animal Research 53, 301– 316. Lange, S.; Martinsson, K.; Lonnroth, I.; Goransson, L., 1993: Plasma level of antisecretory factor (ASF) and its relation to post-weaning diarrhoea in piglets. Zentralblatt fuer Veterinaermedizin. Reihe B 40, 113–118. Lee, H. H.; Prasad, A. S.; Brewer, G. J.; Owyang, C., 1989: Zinc absorption in human small intestine. American Journal of Physiology 256, G87–G91. Leonhard-Marek, S.; Hempe, J.; Schroeder, B.; Breves, G., 2009: Electrophysiological characterization of chloride secretion across the jejunum and colon of pigs as affected by age and weaning. Journal of Comparative Physiology B Biochemical Systemic and Environmental Physiology 179, 883–896.
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Lodemann, U.; Hubener, K.; Jansen, N.; Martens, H., 2006: Effects of Enterococcus faecium NCIMB 10415 as probiotic supplement on intestinal transport and barrier function of piglets. Archives of Animal Nutrition 60, 35–48. Lodemann, U.; Lorenz, B. M.; Weyrauch, K. D.; Martens, H., 2008: Effects of Bacillus cereus var. toyoi as probiotic feed supplement on intestinal transport and barrier function in piglets. Archives of Animal Nutrition 62, 87–106. Lyall, V.; Nath, R.; Mahmood, A., 1979: Inhibition of D-glucose uptake by zinc in rat intestine. Biochemical Medicine 22, 192–197. Martens, H.; Gabel, G.; Strozyk, H., 1987: The effect of potassium and the transmural potential difference on magnesium transport across an isolated preparation of sheep rumen epithelium. Quarterly Journal of Experimental Physiology 72, 181–188. Martin, L.; Pieper, R.; Vahjen, W.; Zentek, J., 2012: Influence of high levels of dietary zinc oxide on performance and small intestinal gene expression in weaned piglets. XIIth International Symposium on Digestive Physiology in Pigs, Keystone, US Abstract No.1104. McEwan, G. T.; Schousboe, B.; Nielsen, C. G.; Skadhauge, E., 1990: Effect of age on the secretory capacity of pig small intestine in vivo and in vitro. American Journal of Physiology 259, G474–G480. Medani, M.; Bzik, V. A.; Rogers, A.; Collins, D.; Kennelly, R.; Winter, D. C.; Brayden, D. J.; Baird, A. W., 2012: Zinc sulphate attenuates chloride secretion in human colonic mucosae in vitro. European journal of pharmacology 696, 166–171. Mesonero, J. E.; Rodriguez Yoldi, M. C.; Rodriguez Yoldi, M. J., 1993: Effect of cadmium on D-galactose transport across the small intestine of rabbits. Research in Veterinary Science 54, 189– 194. Metzler-Zebeli, B. U.; Caine, W. R.; McFall, M.; Miller, B.; Ward, T. L.; Kirkwood, R. N.; Mosenthin, R., 2010: Supplementation of diets for lactating sows with zinc amino acid complex and gastric nutriment-intubation of suckling pigs with zinc methionine on mineral status, intestinal morphology and bacterial translocation in lipopolysaccharidechallenged weaned pigs. Journal of Animal Physiology and Animal Nutrition (Berl) 94, 237–249.
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Miller, B. G.; Skadhauge, E., 1997: Effect of weaning in the pig on ileal ion transport measured in vitro. Zentralblatt f€ ur Veterin€ armedizin. Reihe A 44, 289–299. Monteilh-Zoller, M. K.; Zonno, V. V.; Storelli, C.; Ahearn, G. A., 1999: Effects of zinc on l-proline uptake by lobster hepatopancreatic brush border membrane vesicles. Journal of Experimental Biology 202, 3003–3010. NRC, 1998: Nutrient Requirements of Swine, 10th edn. National Research Council, National Academy Press, Washington, DC. Owusu-Asiedu, A.; Nyachoti, C. M.; Marquardt, R. R., 2003: Response of early-weaned pigs to an enterotoxigenic Escherichia coli (K88) challenge when fed diets containing spray-dried porcine plasma or pea protein isolate plus egg yolk antibody, zinc oxide, fumaric acid, or antibiotic. Journal of animal science 81, 1790–1798. Pieper, R.; Vahjen, W.; Neumann, K.; Van Kessel, A. G.; Zentek, J., 2012: Dosedependent effects of dietary zinc oxide on bacterial communities and metabolic profiles in the ileum of weaned pigs. Journal of Animal Physiology and Animal Nutrition 96, 825–833. Puchal, A. A.; Buddington, R. K., 1992: Postnatal development of monosaccharide transport in pig intestine. American Journal of Physiology 262, G895–G902. Rodriguez-Yoldi, M. J.; Lugea, A.; Barber, A.; Lluch, M.; Ponz, F., 1989: Inhibition of sugar and amino acid transport across rat jejunum by cadmium, copper and mercury. Revista Espanola de Fisiologia 45 (Suppl), 207–214. Rodriguez-Yoldi, M. C.; Mesonero, J. E.; Rodriguez-Yoldi, M. J., 1994: Action of zinc on enzymatic digestion and intestinal transport of sugar in the rabbit. Research in Veterinary Science 57, 15–20. Southon, S.; Gee, J. M.; Johnson, I. T., 1984: Hexose transport and mucosal morphology in the small intestine of the zinc-deficient rat. British Journal of Nutrition 52, 371–380. Stevens, J. B.; Gyles, C. L.; Barnum, D. A., 1972: Production of diarrhea in pigs in response to Escherichia coli enterotoxin. American Journal of Veterinary Research 33, 2511–2526. Vallee, B. L.; Falchuk, K. H., 1993: The biochemical basis of zinc physiology. Physiological Reviews 73, 79–118. Watkins, D. W.; Chenu, C.; Ripoche, P., 1989: Zinc inhibition of glucose uptake
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ORIGINAL ARTICLE
Effects of age and zinc supplementation on transport properties in the jejunum of piglets E. M. Gefeller1, H. Martens1, J. R. Aschenbach1, S. Klingspor1, S. Twardziok2, P. Wrede2, R. Pieper3 and U. Lodemann1 1 Faculty of Veterinary Medicine, Institute of Veterinary Physiology, Freie Universit€at Berlin, Berlin, Germany – Universit€atsmedizin Berlin, Berlin, Germany, and 2 Molecular Biology and Bioinformatic, Charite 3 Faculty of Veterinary Medicine, Institute of Animal Nutrition, Freie Universit€at Berlin, Berlin, Germany
Summary Zinc is effective in the prevention and treatment of post-weaning diarrhoea and in promoting piglet growth. Its effects on the absorption of nutrients and the secretory capacity of the intestinal epithelium are controversial. We investigated the effects of age, dietary pharmacological zinc supplementation and acute zinc exposure in vitro on small-intestinal transport properties of weaned piglets. We further examined whether the effect of zinc on secretory responses depended on the pathway by which chloride secretion is activated. A total of 96 piglets were weaned at 26 days of age and allocated to diets containing three different levels of zinc oxide (50, 150 and 2500 ppm). At the age of 32, 39, 46 and 53 days, piglets were killed, and isolated epithelia from the mid-jejunum were used for intestinal transport studies in conventional Ussing chambers, with 23 lM ZnSO4 being added to the serosal side for testing acute effects. Absorptive transport was stimulated by mucosal addition of D-glucose or L-glutamine. Secretion was activated by serosal addition of prostaglandin E2, carbachol or by mucosal application of Escherichia coli heat-stable enterotoxin (Stp). Jejunal transport properties showed significant age-dependent alterations (p < 0.03). Both absorptive and secretory responses were highest in the youngest piglets (32 d). The dietary zinc supplementation had no significant influence on jejunal absorptive and secretory responses. However, the pre-treatment of epithelia with ZnSO4 in vitro led to a small but significant decrease in both absorptive and secretory capacities (p < 0.05), with an exception for carbachol (p = 0.07). The results showed that, in piglets, chronic supplementation with zinc did not sustainably influence the jejunal transport properties in the post-weaning phase. Because transport properties are influenced by the addition of zinc in vitro, we suggest that possible epithelial effects of zinc depend on the acute presence of this ion. Keywords epithelium, ion transport, pig, small intestine, Ussing chamber, chloride secretion Correspondence Dr. Ulrike Lodemann, Institute of Veterinary Physiology, Freie Universit€at Berlin, Oertzenweg 19b, 14163 Berlin, Germany. Tel: +49 30 83862592; Fax: +49 30 83862610; E-mail: [email protected] Received: 25 April 2013; accepted: 30 June 2014
Introduction Around weaning, piglets are exposed to many stress factors that manifest themselves in increased intestinal permeability (Bruewer et al., 2003) and make piglets highly susceptible to pathogenic bacteria and their enterotoxins (Stevens et al., 1972; Lall es et al., 2004). Zinc supplementation at pharmacological levels has been shown to improve performance parameters in weaned piglets (Hahn and Baker, 1993; Hill et al., 2001; Zhang and Guo, 2009). The incidence of post-weaning diarrhoea can also be reduced by high doses of zinc (Owusu-Asiedu 542
et al., 2003), but the underlying mechanisms are poorly understood. Evidence has been provided for the importance of zinc in sustaining intestinal barrier function (Vallee and Falchuk, 1993). The influence of zinc on the intestinal absorption of nutrients is, however, controversial as it has been shown to both decrease (Lyall et al., 1979; Watkins et al., 1989; Yoldi et al., 1992; Rodriguez-Yoldi et al., 1994) and increase (Lee et al., 1989) glucose absorption. Similarly, contradictory results have been obtained regarding the intestinal transport of amino acids in the presence of zinc (Boldizsar and Simon, 1981; Monteilh-Zoller et al., 1999).
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As a possible explanation for the antidiarrhoeal effect of zinc, decreased secretory responses to several secretagogues have been observed when zinc has been applied at the basolateral side of isolated intestinal epithelia (Feng et al., 2006; Carlson et al., 2008). However, even this information is contradictory (Canani et al., 2005; Hoque et al., 2005; Berni Canani et al., 2010). Given the inconsistent and sometimes inconclusive results of previous studies, this study aimed at a very systematic and broad-based approach to assess the effect of dietary zinc supplementation at different dosages on both absorptive and secretory transport properties in the jejunum of piglets of various age groups after weaning. To dissociate long-term effects of zinc feeding from short-term effects attributable to the actual presence of zinc, intestinal epithelia from all zinc supplementation levels and age groups were additionally tested for the effects of an acute serosal application of zinc in vitro. Finally, the secretory response and the mode of activation of chloride secretion were also studied. Material and methods The experiments were conducted in accordance to German law, and legal permission was granted by the Landesamt f€ ur Gesundheit und Soziales (G 0347/09). Animals and diets
The experiment included 96 purebred Landrace piglets that were weaned at 26 1 days (d) of age with a mean bodyweight of 7.2 1.2 kg. Animals were housed in pens (n = 2 animals per pen) with straw bedding. From 12 d of age, piglets had free access to the same non-medicated pre-starter diet formulated to meet the requirements of piglets with an average bodyweight of 5–10 kg (GfE, 2006). After being weaned, the piglets were assigned randomly to one of three feeding groups balanced for gender, litter and bodyweight. The piglets received a mash starter diet (Table 1) until the 53th d of life; this diet provided zinc at final concentrations of 50 (Zn50), 150 (Zn150) or 2500 (Zn2500) mg/kg (ppm) by replacing corn starch with analytical grade zinc oxide (ZnO) (Sigma Aldrich, Taufkirchen, Germany). All diets were mixed in the feed mill at the Institute of Animal Nutrition of the Freie Universit€at Berlin. Dietary zinc levels were determined by atomic absorption spectrometry in an AAS vario 6 spectrometer (Analytik Jena, Jena, Germany) to be 57, 164 and 2425 ppm in the Zn50, Zn150 and Zn2500 feeding groups respectively. The piglets Journal of Animal Physiology and Animal Nutrition © 2014 Blackwell Verlag GmbH
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Table 1 Ingredients and chemical composition of the starter diet g/kg Ingredients Wheat Barley Soybean meal Corn starch/ zinc oxide* Limestone Monocalcium phosphate Mineral and Vitamin Premix† Soy oil Salt Lysine HCl Methionine Chemical composition Dry matter ME (MJ/kg) Crude ash Crude protein Crude fibre Ether extract Starch Lysine Methionine Threonine Tryptophan Calcium Phosphorus Sodium Magnesium Zinc mg/kg‡ Iron mg/kg Manganese mg/kg Copper mg/kg
380 300 232 10 20 20 15 17.5 2.0 2.5 1.0 879 13.0 81 194 36 34 376 11.7 4.0 7.2 2.4 11.0 8.0 3.1 2.2 34 309 81 18
*Corn starch in the basal diet was partially replaced in the diets containing 50, 150 and 2500 mg/kg zinc with analytical grade zinc oxide (Sigma Aldrich, Taufkirchen, Germany) to adjust for the desired zinc level. †Mineral and Vitamin Premix (Spezialfutter Neuruppin, Neuruppin, Germany) providing per kg feed: 1.95 g Na (as sodium chloride), 0.83 g Mg (as magnesium oxide), 10 500 IU vitamin A, 1800 IU vitamin D3, 120 mg vitamin E, 4.5 mg vitamin K3, 3.75 mg thiamine, 3.75 mg riboflavin, 6.0 mg pyridoxine, 30 lg cobalamine, 37.5 nicotinic acid, 1.5 mg folic acid, 375 lg biotin, 15 mg pantothenic acid, 1200 mg choline chloride, 75 mg Fe (as iron-(II)-carbonate), 15 mg Cu (as copper-(II)-sulphate), 90 mg Mn (as manganese-(II)-oxide), 675 lg I (as calcium iodate) and 525 mg Se (as sodium selenite). ‡Analysed concentration of zinc in the basal diet without ZnO supplementation. The final experimental diets contained Zn at 57, 164 or 2425 mg/kg.
had free access to water and feed throughout the experimental period. Sampling procedure
At the age of 32 1, 39 1, 46 1 and 53 1 days, eight piglets per group were killed 543
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such that the resulting age groups were balanced for litter and gender. The piglets were sedated with 20 mg/kg bodyweight of ketamine hydrochloride (Ursotaminâ; Serumwerk Bernburg AG, Bernburg, Germany) and 2 mg/kg bodyweight of azaperone (Stresnilâ; Jansen-Cilag, Neuss, Germany) prior to being killed with an intracardial injection of 10 mg/kg bodyweight of tetracaine hydrochloride, mebezonium iodide and embutramide (T61â; Intervet, Unterschleißheim, Germany). A mid-line abdominal incision was made, and 1 m of mid-jejunum was removed. The jejunum was stripped off from its outer muscle layers, rinsed, placed in a transport buffer solution (containing in mM: 115 NaCl, 25 NaHCO3, 0.4 NaH2PO4, 2.4 Na2HPO4, 5 KCl, 5 glucose, 1.2 CaCl2, 1.2 MgCl2 and 2.8 lM indomethacin), gassed with carbogen (95% O2, 5% CO2) and kept at 38 °C. Incubation procedure
Epithelia were cut into squares (2 cm2) and mounted between the halves of a conventional Ussing chamber giving a buffer-exposed area of 0.95 cm2. Tissue damage resulting from contusion was minimised using rings of silicone rubber at each side of the epithelium. The Ussing chamber technique was as described elsewhere (Martens et al., 1987). Approximately 30–60 min elapsed from piglet dispatch to the final mounting of the tissues into the Ussing chambers. The buffer solutions had an osmolarity of 295 mosM and were adjusted to pH 7.4 by titrating with 500 mM 2-(N-morpholino)ethanesulfonic acid (MES). The temperature was maintained at 38 °C by a thermostat (Haake D1; VWR International GmbH, Darmstadt, Germany). The epithelia were bathed on both sides with 10 ml buffer solution. The buffer solutions for incubation in the Ussing chambers were of the same composition as the transport buffer solution, except that the serosal solution contained 10 (instead of 5) mM glucose plus 2 mM mannitol, whereas the mucosal solution was glucose-free but contained 12 mM mannitol. Both mucosal and serosal buffer solutions contained 1.4 lM indomethacin. During the equilibration period (30 min after mounting), the media were continuously circulated and aerated with carbogen (95% O2, 5% CO2). All reagents were purchased from VWR International GmbH, Sigma Aldrich Chemie GmbH or Bachem AG (Bubendorf, Switzerland), and were of analytical grade. 544
Electrophysiological measurements
Electrophysiological measurements were obtained by a microcomputer-controlled voltage/current clamp (version 2.02 supplied by Dipl.-Ing. K. Mußler, Aachen, Germany). Two KCl agar bridges were placed near to each side of the epithelium and connected to Ag–AgCl electrodes for the measurement of the transepithelial potential difference (PDt). Two distantly placed KCl agar bridges were connected to Ag–AgCl electrodes for transmitting current through the tissue. The junction potential and the fluid resistance of the buffer between the tips of the PD-sensing bridges were determined before the tissue was mounted and were subsequently corrected by the computer-controlled voltage clamp. The tissue was alternatively pulsed with a positive or negative pulse of 100 lA and 200 ms duration. The displacement in PDt caused by the current pulse was measured, and the tissue resistance (Rt) was calculated from the change in PDt due to the law of Ohm. The pulse amplitude PDt (mV), current Isc (lA/cm2) and Rt (Ω cm2) were continuously recorded. After an equilibration period of 10 min under open-circuit conditions, the epithelia were short-circuited. In vitro treatments
A total of 12 Ussing chambers were used per animal. After 15 min under short-circuit conditions, half of the epithelia were pre-treated with ZnSO4 (23 lM; Sigma Aldrich Chemie GmbH) at the serosal side, whereas the other chambers received no ZnSO4. To assess the absorptive capacity of jejunal epithelia, DGlucose or L-glutamine concentrations of 1, 4, 12 mM was added to two chambers, each with or without serosal ZnSO4. D-glucose or L-glutamine was added to the mucosal side of the epithelia, while mannitol at an equivalent amount was added to the serosal side to keep osmolarity equal on both sides. After an exchange of incubation buffer and an equilibration period, the secretagogues were added to separate tissues 30 min after the addition of glucose. For the assessment of secretagogue effects, one chamber with and one without serosal ZnSO4 was used for each concentration of the respective secretagogue. Secretion was activated by serosal application of prostaglandin E2 (0.1, 0.5 or 5 lM) or carbachol (10 lM) or by the mucosal application of Escherichia coli heat-stable enterotoxin (2.5 or 5 lM) (Bachem AG) in the respective incubation solution. The allocation of chambers to substrate and secretagogue additions, as well as the sequence of additions, was equal for all animals. All Journal of Animal Physiology and Animal Nutrition © 2014 Blackwell Verlag GmbH
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given concentrations are final concentrations in the incubation solution. To test the viability of the tissue theophylline, 8 mM was added bilaterally to each tissue at the end of the experiment. Calculations and statistical analysis
The Isc and Rt values at 1 min before the serosal addition of zinc in vitro were treated as baseline data. The effect of the zinc application in vitro was assessed from the Isc and Rt values recorded 15 min after the addition of ZnSO4. To quantify the effects of absorptive or secretory stimulants, the maximum change of Isc (DIsc) was calculated by subtracting the maximum value of Isc measured within 5 min after the addition of D-glucose or L-glutamine, within 10 min after the addition of PGE2 or carbachol or within 45 min after the addition of E. coli heat-stable enterotoxin from the Isc value at 1 min before the application of the respective stimulant. Statistical evaluations and the plotting of graphs were carried out by means of the SPSS program for Windows, version 20 (Jandel, Chicago, IL, USA). Statistical significance of differences was assessed by a linear mixed model with the fixed factors ‘concentration’ (concentration levels of the respective stimulant), ‘feeding’ (50, 150, 2500 ppm), ‘age’ (day 32, 39, 46, 53) and ‘zinc’ (with or without ZnSO4 addition in vitro) and the interaction of ‘age 9 concentration’ with the random factor ‘animal’. In the case of statistically significant effects of a fixed factor, the post hoc test LSD (least significant difference) was conducted. The differences between groups were considered statistically significant for p ≤ 0.05. Unless otherwise stated, results are given as means or least square means SEM. The number of experimental animals is given as N, whereas n refers to the number of epithelial tissues per treatment group. Results Baseline Isc and Rt
Baseline values of Isc and Rt measured before any addition was made in vitro (i.e. 1 min before ZnSO4 addition) did not differ significantly between the age and feeding groups (data not shown). Values recorded before and after the addition of ZnSO4 in vitro did not differ significantly as well.
Effects of zinc on pig jejunum
Na+-coupled glucose or glutamine transport was stimulated by the mucosal application of increasing concentrations of D-glucose or L-glutamine (1, 4 or 12 mM). The change in the short-circuit current (DIsc) was recorded. Both stimulants caused a dose-dependent increase in Isc (p < 0.001; Table 2). The highest responses were measured at day 32 when the changes in Isc after the addition of glucose or glutamine were approximately twice as high compared with those at days 39, 46 and 53 (p < 0.001; Table 2). No significant effect of feeding was observed. However, DIsc after the application of D-glucose or L-glutamine was numerically highest in the Zn2500 group at almost all ages (with only one exception for D-glucose at 32 d) and lowest in the Zn150 group (Fig. 1; data not shown for L-glutamine).
Table 2 Response of short-circuit current (DIsc in lA/cm2) to L-glutamine, D-glucose, prostaglandin E2, Escherichia coli heat-stable enterotoxin and carbachol Age 32 d (mM) 1 13 (8–18)* 4 37 (31–42) 12 54 (48–59) Age effect a D-glucose (mM) 1 68 (52–83) 4 139 (123–155) 12 165 (149–181) Age effect a PGE2 (lM) 0.1 80 (66–94) 0.5 106 (92–120) 5 136 (122–150) Age effect a E. coli toxin (lM) 2.5 15 (8–22) 5 20 (13–26) Age effect a Carbachol (lM) 10 125 (110–141) Age effect a
39 d
46 d
53 d
6 (1–12) 16 (11–22) 26 (20–31) b
5 (0–10) 12 (7–17) 20 (15–25) b
6 (1–12) 13 (8–19) 26 (21–31) b
L-glutamine
31 (15–47) 28 (12–44) 67 (51–83) 61 (45–77) 85 (69–101) 81 (65–97) b b
34 (18–50) 72 (56–88) 93 (77–109) b
57 (42–72) 54 (39–69) 50 (35–64) 76 (62–90) 76 (62–90) 75 (61–88) 100 (86–114) 106 (92–119) 108 (94–122) b b b 5 (1–12) 7 (1–14) b
9 (2–16) 9 (2–16) ab
16 (9–23) 18 (11–25) a
89 (76–103) 95 (82–109) 97 (84–111) b b b
To assess whether zinc supplementation affected the absorptive capacity of the jejunal epithelium,
*Data represent least square means confidence interval 95%. Different letters within one row indicate significant differences between the age groups (p < 0.05). There was a significant interaction ‘age 9 concentration’ for L-glutamine and D-glucose (p < 0.001). N = 24 animals, n = 87–92 epithelia per age and concentration group for L-glutamine and D-glucose; n = 41–69 epithelia per age and concentration group for PGE2, n = 35–46 epithelia for carbachol per age group; N = 12 animals, n = 22–24 epithelia per age and concentration group for E. coli heat-stable enterotoxin.
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Effects of zinc on pig jejunum
Fig. 1 Effect of dietary Zn concentration and age on the jejunal epithelial response of short-circuit current (DIsc) to mucosal addition of D-glucose. Data are representing the mean values over all glucose concentrations (1, 4 and 12 mM). Only epithelia without in vitro ZnSO4 treatment are included. N = 8 piglets, n = 40–47 epithelia per feeding and age group.
Secretory capacity
Chloride secretion can be activated by at least three different pathways, which we stimulated with the following secretagogues: (i) prostaglandin E2 (PGE2) exerts its impact on chloride secretion by activating the adenylate cyclase and increasing the cAMP level (Blikslager et al., 2001); (ii) E. coli heat-stable enterotoxin induces secretory diarrhoea by activating the cGMP system (Field, 2003); and (iii) carbachol stimulates Cl secretion as a result of an increased intracellular calcium level (Leonhard-Marek et al., 2009). The DIsc measured after the application of PGE2 increased with increasing dose (p < 0.001), whereas the DIsc evoked by E. coli enterotoxin was not different for the factor ‘concentration’ (p = 0.056) (Table 2). Significant age effects were observed for the responses to PGE2 (p = 0.001), E. coli heat-stable enterotoxin (p = 0.027) or carbachol (p = 0.005; Table 2). The change in short-circuit current was highest in the youngest piglets (32 d) compared with the other age groups (39, 46, 53 d), with an exception for E. coli heat-stable enterotoxin for which only the 32nd and the 39th day differed significantly. In general, the effect of rising dietary Zn supply on secretory responses was not significant. However, at days 39 and 46, the Zn50 group tended to have the highest DIsc after stimulation with PGE2, but the response of this feeding group was markedly inconsistent (Fig. 2). With regard to the E. coli toxin-induced chloride secretion, a decrease in secretion from 150 to 546
Fig. 2 Effect of dietary Zn concentration and age on the jejunal epithelial response of short-circuit current (DIsc) to serosal addition of PGE2. Data are representing the mean values over all PGE2 concentrations (0.1, 0.5 and 5 lM). Only epithelia without in vitro ZnSO4 treatment are included. N = 8 piglets, n = 25–32 epithelia per feeding and age group.
2500 ppm zinc supplementation was observed (Fig. 3). The secretory response to carbachol was not consistently altered by the different dietary intake of zinc (data not shown). Effect of serosal ZnSO4 in vitro
The increases in Isc following the addition of D-glucose (p < 0.001) and L-glutamine (p = 0.013) were significantly lower in the tissues pre-treated with serosal
Fig. 3 Effect of dietary Zn concentration and age on the jejunal epithelial response of short-circuit current (DIsc) to serosal addition of Escherichia coli heat-stable enterotoxin (Stp). Data are representing the mean values over all Stp concentrations (2.5 and 5 lM). Only epithelia without in vitro ZnSO4 treatment are included. N = 4 piglets, n = 7–8 epithelia per feeding and age group.
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ZnSO4 in vitro (Fig. 4). The epithelia pre-treated with ZnSO4 also showed significantly reduced secretory reactions to PGE2 (p = 0.027) and Stp (p = 0.042) compared with the respective control groups without ZnSO4 (Fig. 5). The carbachol-induced DIsc was numerically lower in tissues that were pre-incubated with ZnSO4 (Fig. 5). The reduction in Isc was 8.7% of the control value for D-glucose, 8.5% for L-glutamine,
Fig. 4 Jejunal epithelial response of short-circuit current (DIsc) to mucosal addition of D-glucose or L-glutamine comparing epithelia without and with prior ZnSO4 treatment in vitro. Data are means SEM. N = 96 piglets, n = 533–542 epithelia. *Significant differences from the respective group without serosal ZnSO4 addition in vitro are indicated by asterisks.
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4.9% for PGE2, 8.4% for carbachol and 14.5% of the control value for E. coli toxin. Discussion Zinc is well known to support piglet performance around weaning (Hahn and Baker, 1993; Hill et al., 2001; Zhang and Guo, 2009) and to reduce the incidence of post-weaning diarrhoea (Owusu-Asiedu et al., 2003). It has also been shown to reduce bacterial translocation into gut-associated lymph nodes (Huang et al., 1999; Metzler-Zebeli et al., 2010). Furthermore, data concerning performance parameters of the same piglets were published by Martin et al. (2012). They observed a higher average daily gain and feed intake in piglets fed high ZnO levels (2500 ppm) during the first week of feeding compared with the other two feeding groups, but these did not differ thereafter. In the present study, we have examined both the possible effect of chronic dietary zinc supplementation in vivo and the influence of an acute addition of ZnSO4 in vitro on absorptive and secretory transport properties in the jejunum of weaned pigs. In the feeding trial, the diets provided three different concentrations of ZnO. The lowest concentration was 50 ppm, which can be considered a marginal supply based on the recommended Zn concentration of 80–100 ppm in the feed of piglets (NRC, 1998; GfE, 2006). The Zn150 group received the maximal concentration legally allowed within the EU (EC.1334, 2003), whereas the high dietary supplementation with 2500 ppm zinc represented pharmacological levels. For the in vitro studies, ZnSO4 was chosen because of the low solubility of ZnO. The serosal concentration of 23 lM ZnSO4 was applied as it is close to the zinc plasma concentrations of piglets fed with a diet containing 2000 ppm zinc (Carlson et al., 1999, 2007) or 2500 ppm zinc (Pieper et al., 2012). Age effects
Fig. 5 Jejunal response of short-circuit current (DIsc) after serosal addition of carbachol, PGE2 and Escherichia coli heat-stable enterotoxin (Stp) comparing epithelia without and with prior ZnSO4 treatment in vitro. Data are means SEM. N = 96 piglets, n = 354–361 epithelia for PGE2, n = 84–86 epithelia for carbachol; N = 48 piglets, n = 93 epithelia for E. coli heat-stable enterotoxin (Stp). *Significant differences from the respective group without serosal ZnSO4 addition in vitro are indicated by asterisks.
Previous studies have shown that the transport capacity of the intestinal epithelium increases as the intestinal surface increases from the day of birth until the time after weaning. In relation to the intestinal mass, however, the transport rate decreases, perhaps because of a modified density of transport proteins (Buddington and Diamond, 1990; Puchal and Buddington, 1992). In agreement with this concept, the youngest piglets (32 d) showed the highest responses to all added stimulants regarding absorption and secretion (Figs 1–3 and Table 2), whereas significantly decreased transport properties were assessed at day
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39. The decrease in absorptive capacity from 32 to 39 d of age (i.e. from 6 to 13 d after weaning) differs only slightly from the data of Carlson et al. (2004), who have shown that glucose transport is increased until day 15 after weaning. Boudry et al. (2004) have found that jejunal glucose-induced currents are higher at the day of weaning compared with the DIsc at 2 weeks after weaning, assuming that this decrease in glucose transport capacity is attributable to a switch in the diet from milk to cereals. The overall magnitude for the glucose-stimulated DIsc in the present study was in accordance with earlier electrophysiological studies (Lodemann et al., 2006, 2008). Regarding secretion, Miller and Skadhauge (1997) conducted Ussing chamber experiments and found that Cl secretion was not affected by weaning, in accordance with McEwan et al. (1990) who did not find significant differences in the secretory responses of 14-d-old piglets compared with 14-week-old pigs to PGE2 and theophylline. In agreement with our results, however, Carlson et al. (2004) observed a higher secretory capacity at 5–6 days after weaning than at 14–15 days after weaning. Boudry et al. (2004) also measured an increased response to secretagogues at the day of weaning compared with 2 weeks after weaning. The reduced secretory capacity compared with that of younger animals is assumed to result from a decreased sensitivity with age (Grondahl et al., 1996; Erlwanger et al., 1999) and from the maturation of the intestinal tissue (Cummins et al., 1988). As a possible explanation for the decrease in secretory capacity after weaning, Lange et al. (1993) have demonstrated that the antisecretory factor (ASF) increases from negligible levels in piglets younger than 5 week of age to high levels in older pigs. Sensitivity to secretagogues is thus assumed to decrease with age (Grondahl et al., 1996; Erlwanger et al., 1999) and with the maturation of the intestinal tissue (Cummins et al., 1988). The weaning time seems to represent an event, whereby transport capacities increase notably and then further decrease with age.
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Chronic zinc supplementation in feed did not significantly affect the ion currents linked to glucose or glutamine absorption in the present study. These results are in contrast to observations in rats in which feeding 200 mg ZnCl2/kg bodyweight decreased small-intestinal glucose absorption (Lyall et al., 1979) and, vice versa, zinc deficiency led to
increased glucose absorption in vivo (Southon et al., 1984). As the effect of zinc feeding (chronic zinc supplementation) on these transport pathways is not statistically significant, our results are in agreement with previous findings in the porcine target species in which the supplementation of the diet of piglets with increasing concentrations of zinc had no effect on Na+-dependent glucose transport at 5–7 days after weaning (Carlson et al., 2004). To our knowledge, no study of the effects of zinc feeding on L-glutamine transport has been conducted so far, although Lyall et al. (1979) did not observe an effect on alanine transport in rats after feeding them zinc. These findings are confirmed in the present study for L-glutamine transport. Nonetheless, our results also show that a depressing effect of Zn on both glucose and glutamine absorption occurs in pigs when a high plasma Zn concentration is acutely simulated in vitro. This observation is in agreement with in vitro studies of the small-intestinal epithelium of pigs (Watkins et al., 1989), rabbits (Yoldi et al., 1992; Rodriguez-Yoldi et al., 1994) and rats (Lyall et al., 1979). However, Hoque et al. (2005) have observed no such effect in a study with ileal tissues of rats. Lee et al. (1989) have conducted smallintestinal perfusion studies and shown that acute addition of zinc (0.9 mM) raises the glucose absorption in the human small intestine. For the amino acid L-alanine, Lyall et al. (1979) have found that zinc in vitro does not affect the absorption of this amino acid. However, the present study has revealed that Lglutamine transport is significantly reduced by acute zinc addition in vitro. We should, however, emphasise that the above-mentioned studies involved the use of zinc concentrations between 0.5 and 10 mM, which is far above the zinc concentration used in the present investigation. The underlying mechanisms by which Zn inhibits glucose absorption in vitro are still not known. Authors of relevant studies suggest that heavy metals decrease intestinal sugar transport by binding to chemical groups of the membrane protein and thereby influence the Na+-dependent cotransport system (Rodriguez-Yoldi et al., 1989; Yoldi et al., 1992; Mesonero et al., 1993). As sugar transport is driven by the transmembrane Na+ gradient, zinc might reduce sugar uptake by decreasing the activity of the Na+/K+ATPase (Rodriguez-Yoldi et al., 1994). The latter seems plausible when considering that Na+/glucose and Na+/glutamine cotransport were equally affected in the present study and that the ZnSO4 was applied at the serosal side in which the Na+/K+ATPase is located. An inhibition of basolateral K+ channels by serosal Zn
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application, as described by Hoque et al. (2005), could likewise negatively affect the activity of the Na+/K+ATPase or simply decrease the driving force for Na+-dependent glucose uptake because an inhibition of basolateral K+ channels also depolarises the potential difference of the apical membrane. Secretory studies
Three main intracellular signalling cascades are utilised to activate the epithelial ion channels that drive secretion. These signalling cascades include cAMP, cGMP and intracellular calcium, all of which have been investigated in the present study by applying PGE2, Stp or carbachol. Our study has not confirmed an antisecretory effect of the dietary zinc supplementation on PGE2- and carbachol-activated secretion. The secretion induced by Stp tended to be decreased with higher levels of zinc in the diet, especially in 32-d-old piglets, but a significant feeding effect was also not observed. These results are in contradiction to Carlson et al. (2004) who found that piglets fed a diet containing 2500 ppm ZnO showed a significantly reduced secretory response compared with a group receiving 100 ppm ZnO when secretion was stimulated by adding theophylline and serotonin. No published data are available on the effect of dietary zinc on PGE2-stimulated Cl secretion. Although an effect of Zn pre-feeding on secretory pathways was not delineable in the present study, the acute zinc addition in vitro had significant reducing effects on the Cl secretion induced by PGE2, Stp and, at least numerically, carbachol. This agrees with several previous studies showing that the presence of zinc at the basolateral side reduces the effect of secretagogues such as 5-HT, vasoactive intestinal peptide (VIP), theophylline and carbachol in pigs (Carlson et al., 2006) and carbachol (Berni Canani et al., 2010) and cholera toxin (Canani et al., 2005) in Caco-2 cells. The latter study by Canani et al. (2005) emphasises that the antisecretory effect of serosal Zn application (35 lM) applies only to cholera toxin and not to E. coli heat-stable enterotoxin. However, this might not really be in contrast to the present study in which the inhibition of the Stp effect by serosal Zn application is significant but extremely small indeed. A preferential inhibition of cAMP-activated chloride secretion over carbachol-activated secretion had been demonstrated using extremely high serosal Zn concentrations (1 mM) in the rat ileum (Hoque et al., 2005); this result agrees with the finding of the present study in which only PGE2, but not carbachol, effects reached statistical significance. Some authors (Hoque et al., 2005; Medani et al., 2012) explain the Journal of Animal Physiology and Animal Nutrition © 2014 Blackwell Verlag GmbH
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Zn effect by a selective inhibition of K+ channels which depolarises the potential difference as mentioned above and hence the driving force necessary for cAMP-activated Cl secretion. However, Medani et al. (2012) showed that zinc sulphate not only inhibits PGE2 but also carbachol-induced secretion in human colonic mucosae. While the present study focussed on Zn effects on electrogenic pathways, electroneutral mechanisms may also be involved in positive effects of zinc on diarrhoea. When diarrhoea occurs, cAMP is increased by enterotoxins and neurotransmitters which then lead not only to stimulation of Cl secretion but also to inhibition of electroneutral NaCl absorption (Donowitz and Welsh, 1986). Hoque et al. (2009) studied whether Zn has an effect on the Na+/H+ exchanger NHE3, which is a key player in electroneutral NaCl absorption. In Caco-2 cells, they demonstrated that Zn significantly increases the NHE3 activity and that the inhibitory effect of cAMP on NHE3 activity was significantly blunted by zinc. Based on these findings, the authors supported the use of Zn in the therapy of diarrhoea, for example in oral rehydration solutions. Conclusion This study has confirmed the results of most previous studies showing that the absorptive and secretory capacity of the porcine intestinal epithelium decreases with age after weaning. In a highly systematic approach employing four different age groups, two different absorptive pathways, three different secretory stimulants and three different feeding levels of Zn, our results have further provided conclusive evidence suggesting that chronic zinc supplementation does not alter small-intestinal transport mechanisms sustainably. However, the acute presence of zinc on the basolateral side of the epithelium seems to interfere directly with transport mechanisms. This observation might explain the contradictory effects of chronic and acute zinc treatments of other studies (Carlson et al., 2004, 2006, 2008, 2010). The acute presence of zinc reduces both absorptive and secretory transport processes, a finding that appears to be contra-productive with regard to the absorption of glucose and glutamine. However, over the total length of the intestine, this reduced mid-jejunal absorption might be compensated in distal parts of the intestine. Indeed, performance parameters were not negatively affected by supplementation with zinc oxide in the same pigs (Martin et al., 2012). With regard to secretion, the reducing effect might be beneficial for the animal, as the epithelium seems to be 549
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less susceptible to diarrhoeal diseases in the presence of zinc. We should also acknowledge, however, that the Zn effects on epithelial transport function are moderate from a quantitative point of view, compared with the effects occurring around weaning. This suggests that the benefits of high Zn supplementation for weight gain and diarrhoea incidence after weaning might only partially be explained by the direct influence of Zn on epithelial transport processes, with the main effects of Zn being exerted on other key players, for example the intestinal microbiota or the intestinal immune system or the enteric nervous system (ENS). With regard to the latter, References Berni Canani, R.; Secondo, A.; Passariello, A.; Buccigrossi, V.; Canzoniero, L. M.; Ruotolo, S.; Puzone, C.; Porcaro, F.; Pensa, M.; Braucci, A.; Pedata, M.; Annunziato, L.; Guarino, A., 2010: Zinc inhibits calcium-mediated and nitric oxide-mediated ion secretion in human enterocytes. European journal of pharmacology 626, 266–270. Blikslager, A. T.; Pell, S. M.; Young, K. M., 2001: PGE2 triggers recovery of transmucosal resistance via EP receptor cross talk in porcine ischemia-injured ileum. American Journal of Physiology Gastrointestinal and Liver Physiology 281, G375– G381. Boldizsar, H.; Simon, F., 1981: Effect of zinc sulphate supplementation on amino acid absorption from pig intestine. I. Intestinal loop tests. Acta Veterinaria Academiae Scientiarum Hungaricae 28, 409–418. Boudry, G.; Peron, V.; Le Huerou-Luron, I.; Lalles, J. P.; Seve, B., 2004: Weaning induces both transient and long-lasting modifications of absorptive, secretory, and barrier properties of piglet intestine. Journal of Nutrition 134, 2256–2262. Bruewer, M.; Luegering, A.; Kucharzik, T.; Parkos, C. A.; Madara, J. L.; Hopkins, A. M.; Nusrat, A., 2003: Proinflammatory cytokines disrupt epithelial barrier function by apoptosis-independent mechanisms. Journal of Immunology 171, 6164– 6172. Buddington, R. K.; Diamond, J. M., 1990: Ontogenetic development of monosaccharide and amino acid transporters in rabbit intestine. American Journal of Physiology 259, G544–G555. Canani, R. B.; Cirillo, P.; Buccigrossi, V.; Ruotolo, S.; Passariello, A.; De Luca, P.;
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however, Feng et al. (2006) concluded that zinc attenuates the cAMP-dependent ion secretion mainly due to an effect on epithelial cells rather than affecting mucosal neuronal pathways. Acknowledgements This research was financially supported by the Deutsche Forschungsgemeinschaft, Grant No. SFB 852/1 and by the H.-W.-Schaumann-Stiftung, Hamburg. The help of U. Tietjen, K. Wolf, M. Grunau and U. Ujkani is gratefully acknowledged.
Porcaro, F.; De Marco, G.; Guarino, A., 2005: Zinc inhibits cholera toxininduced, but not Escherichia coli heat-stable enterotoxin-induced, ion secretion in human enterocytes. Journal of Infectious Diseases 191, 1072–1077. Carlson, M. S.; Hill, G. M.; Link, J. E., 1999: Early- and traditionally weaned nursery pigs benefit from phase-feeding pharmacological concentrations of zinc oxide: effect on metallothionein and mineral concentrations. Journal of animal science 77, 1199–1207. Carlson, D.; Damgaard Poulsen, H.; Sehested, J., 2004: Influence of weaning and effect of post weaning dietary zinc and copper on electrophysiological response to glucose, theophylline and 5-HT in piglet small intestinal mucosa. Comparative Biochemistry and Physiology – Part A: Molecular And Integrative Physiology 137, 757–765. Carlson, D.; Sehested, J.; Poulsen, H. D., 2006: Zinc reduces the electrophysiological responses in vitro to basolateral receptor mediated secretagogues in piglet small intestinal epithelium. Comparative Biochemistry and Physiology – Part A: Molecular And Integrative Physiology 144, 514–519. Carlson, D.; Beattie, J. H.; Poulsen, H. D., 2007: Assessment of zinc and copper status in weaned piglets in relation to dietary zinc and copper supply. Journal of Animal Physiology and Animal Nutrition 91, 19–28. Carlson, D.; Sehested, J.; Feng, Z.; Poulsen, H. D., 2008: Serosal zinc attenuate serotonin and vasoactive intestinal peptide induced secretion in piglet small intestinal epithelium in vitro. Comparative Biochemistry and Physiology – Part A: Molecular And Integrative Physiology 149, 51–58.
Carlson, D.; Sehested, J.; Poulsen, H. D., 2010: Serosal Zn2+ inhibits 8-Br-cAMP stimulated chloride secretion in piglet small intestinal epithelium in vitro. Livestock Science 134, 30–32. Cummins, A. G.; Steele, T. W.; LaBrooy, J. T.; Shearman, D. J., 1988: Maturation of the rat small intestine at weaning: changes in epithelial cell kinetics, bacterial flora, and mucosal immune activity. Gut 29, 1672–1679. Donowitz, M.; Welsh, M. J., 1986: Ca2+ and cyclic AMP in regulation of intestinal Na, K, and Cl transport. Annual review of physiology 48, 135–150. EC.1334, 2003: Commission Regulation (EC) No. 1334/2003. Official Journal of the European Union. Erlwanger, K. H.; Unmack, M. A.; Grondahl, M. L.; Skadhauge, E.; Thorboll, J. E., 1999: Effect of age on vasoactive intestinal polypeptide-induced shortcircuit current in porcine jejunum. Comparative Biochemistry and Physiology. Part A, Molecular & Integrative Physiology 124, 29–33. Feng, Z.; Carlson, D.; Poulsen, H. D., 2006: Zinc attenuates forskolin-stimulated electrolyte secretion without involvement of the enteric nervous system in small intestinal epithelium from weaned piglets. Comparative Biochemistry and Physiology – Part A: Molecular and Integrative Physiology 145, 328–333. Field, M., 2003: Intestinal ion transport and the pathophysiology of diarrhea. Journal of Clinical Investigation 111, 931– 943. GfE, 2006: Empfehlungen zur Energie- und N€ ahrstoffversorgung von Schweinen. Ausschuss f€ ur Bedarfsnormen der Gesellschaft f€ ur Ern€ ahrungsphysiologie, DLG-Verlag, Frankfurt/M., Germany, p. 132.
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Grondahl, M. L.; Hansen, M. B.; Larsen, I. E.; Skadhauge, E., 1996: Age and segmental differences in 5-hydroxytryptamine-induced hypersecretion in the pig small intestine. Journal of Comparative Physiology B Biochemical Systemic and Environmental Physiology 166, 21–29. Hahn, J. D.; Baker, D. H., 1993: Growth and plasma zinc responses of young pigs fed pharmacologic levels of zinc. Journal of animal science 71, 3020–3024. Hill, G. M.; Mahan, D. C.; Carter, S. D.; Cromwell, G. L.; Ewan, R. C.; Harrold, R. L.; Lewis, A. J.; Miller, P. S.; Shurson, G. C.; Veum, T. L., 2001: Effect of pharmacological concentrations of zinc oxide with or without the inclusion of an antibacterial agent on nursery pig performance. Journal of animal science 79, 934–941. Hoque, K. M.; Rajendran, V. M.; Binder, H. J., 2005: Zinc inhibits cAMP-stimulated Cl secretion via basolateral Kchannel blockade in rat ileum. American Journal of Physiology Gastrointestinal and Liver Physiology 288, G956–G963. Hoque, K. M.; Sarker, R.; Guggino, S. E.; Tse, C. M., 2009: A new insight into pathophysiological mechanisms of zinc in diarrhea. Annals of the New York Academy of Sciences 1165, 279–284. Huang, S. X.; McFall, M.; Cegielski, A. C.; Kirkwood, R. N., 1999: Effect of dietary zinc supplementation on Escherichia coli septicemia in weaned piglets. Swine Health Production 7, 109–111. Lall es, J.-P.; Boudry, G.; Favier, C.; Floc’h, N. L.; Luron, I.; Montagne, L.; Oswald, I. P.; Pi e, S.; Piel, C.; S eve, B., 2004: Gut function and dysfunction in young pigs: physiology. Animal Research 53, 301– 316. Lange, S.; Martinsson, K.; Lonnroth, I.; Goransson, L., 1993: Plasma level of antisecretory factor (ASF) and its relation to post-weaning diarrhoea in piglets. Zentralblatt fuer Veterinaermedizin. Reihe B 40, 113–118. Lee, H. H.; Prasad, A. S.; Brewer, G. J.; Owyang, C., 1989: Zinc absorption in human small intestine. American Journal of Physiology 256, G87–G91. Leonhard-Marek, S.; Hempe, J.; Schroeder, B.; Breves, G., 2009: Electrophysiological characterization of chloride secretion across the jejunum and colon of pigs as affected by age and weaning. Journal of Comparative Physiology B Biochemical Systemic and Environmental Physiology 179, 883–896.
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