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J. Anim Sci. 2006. 84:3310-3320. doi:10.2527/jas.2005-701
© 2006 American Society of Animal Science
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ANIMAL NUTRITION

Influence of dietary zinc and copper on digestive enzyme activity and intestinal morphology in weaned pigs1

M. S. Hedemann2, B. B. Jensen and H. D. Poulsen

Department of Animal Health, Welfare and Nutrition, Danish Institute of Agricultural Sciences, Research Centre Foulum, 8830 Tjele, Denmark


   Abstract
Top
 Abstract
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
The current study was conducted to investigate the effects of high dietary concentrations of Zn as zinc oxide and Cu as copper sulfate on the activity of digestive enzymes in the pancreas and the intestinal mucosa, intestinal morphology, and mucin histochemistry in pigs after weaning. Thirty-two pigs were weaned at 4 wk of age. The pigs were fed standard weaning diets supplemented with Zn (100 or 2,500 ppm) and Cu (0 or 175 ppm) in a 2 x 2 factorial arrangement of treatments for a 14-d period. In pancreatic tissue, the activity of amylase, carboxypeptidase A, chymotrypsin, trypsin, and lipase increased (P < 0.01) in pigs fed 2,500 ppm of Zn, whereas the activity of carboxypeptidase B and carboxylester hydrolase was unaffected. Copper had no effect on the activity of pancreatic enzymes. In small intestinal contents, the total activity of amylase and carboxypeptidase A was greater in pigs fed 100 ppm of Zn (P < 0.05), whereas feeding 2,500 ppm of Zn increased the chymotrypsin activity (P < 0.001). The remaining enzymes were unaffected by dietary Zn concentration. The villi were longer in the cranial small intestine (P < 0.001) in pigs fed 100 ppm of Zn than in pigs fed 2,500 ppm of Zn, but otherwise there were no clear effects of Zn and Cu supplementation on intestinal morphology. In the cranial small intestine, the activity of maltase (P < 0.001), sucrase (P < 0.001), and lactase was greater in pigs fed 100 ppm of Zn, even though there was a Zn x Cu interaction (P < 0.05) in lactase activity. In the middle and caudal small intestine, no clear differences between dietary treatments were observed. The activity of {gamma}-glutamyl transpeptidase in the intestinal mucosa was not affected by dietary Zn or Cu. In pigs fed 100 ppm of Zn, the activity of aminopeptidase N was greater in the caudal small intestine, but dietary Zn or Cu had no effect on aminopeptidase N in the cranial and middle small intestine. No effect of dietary Zn or Cu supplementation was found on carbohydrate histochemistry in the caudal small intestine, whereas high dietary Zn increased the area of neutral, acidic, and sulfomucins in the cecum (P < 0.01) and in the colon (P < 0.001). In summary, high dietary Zn increased the activity of several enzymes in the pancreatic tissue and increased the mucin staining area in the large intestine, whereas Cu had no clear effect on these variables. However, no definite answers were found as to how the growth promoting and diarrhea reducing effects of excess dietary Zn are exerted.

Key Words: copper • digestive enzyme • intestinal morphology • mucin • pig • zinc


   INTRODUCTION
Top
 Abstract
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Feeding high concentrations of Zn and Cu is well recognized to have growth promoting effects in weaned pigs (Poulsen, 1995Go; Hill et al., 2000Go; Case and Carlson, 2002Go) and reduce problems with postweaning diarrhea (Poulsen, 1989Go). However, the modes of action still remain to be fully elucidated (Veum et al., 2004Go; Buff et al., 2005Go).

Zinc is needed for various physiological processes and has been found to be present in over 200 metallo-enzymes (Prasad, 1984Go). Copper is surpassed only by Zn in the number of enzymes that it can activate; thus it is essential for reproduction, bone development, and growth (Underwood and Suttle, 1999Go). Because of their roles in those enzymes, Zn and Cu are required for a wide variety of factors related to tissue growth.

After weaning, it is well established that villus atrophy is observed in the pig small intestine (Hampson, 1986Go; Hedemann et al., 2003Go), and a decrease in the activity of digestive enzymes in the pancreatic tissue has been reported (Hedemann and Jensen, 2004Go). It is speculated that supplementing pig diets with Zn and Cu may promote the processes of tissue repair in the small intestine and stimulate the synthesis of digestive enzymes, resulting in a better digestion and absorption of nutrients and potentially improving growth performance.

The growth-promoting effects of Zn and Cu have been attributed to effects on intestinal microflora (Katouli et al., 1999Go; Højberg et al., 2005Go). The microflora interacts with the mucus layer covering the gastrointestinal tract (Deplancke and Gaskins, 2001Go), and any changes in the microflora may affect the mucus layer. The mucus layer is believed to play an important role in the protection against infectious diseases (Forstner, 1978Go), and hence the effect of Zn and Cu on postweaning diarrhea may be through changes in mucus layer.

The current study was conducted to investigate the effects of high dietary concentrations of Zn and Cu on the activity of digestive enzymes in the pancreas and the intestinal mucosa, intestinal morphology, and mucin histochemistry in pigs after weaning.


   MATERIALS AND METHODS
Top
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
Animals and Diets
The protocol used in this experiment complied with the guidelines of the Danish Ministry of Justice concerning animal experimentation and care of experimental animals. The experiment was conducted to evaluate the interactive effects of high concentrations of dietary Zn, as ZnO, and Cu, as CuSO4. A total of 32 pigs (Danish Landrace x Yorkshire), 2 females and 2 males from each of 8 litters, were obtained from the herd of the Research Center Foulum, Denmark. Male pigs were castrated at 4 d of age.

All pigs were weighed at weaning and allotted to treatments on the basis of BW and ancestry, with sex equalized across treatments in a randomized complete block design. Pigs were weaned at 28.0 ± 0.7 d of age and had a BW of 8.3 ± 1.0 kg. After weaning, pigs were penned individually in pens (0.7 x 1.5 m) with a slatted plastic flooring that covered two-thirds of the pen and a heated concrete floor in the remaining portion. Each pen had a feeder and a nipple waterer that allowed for ad libitum access to feed and water throughout the experiment. Pigs and feeders were checked twice daily and were weighed at the end of the experiment for calculation of ADG, ADFI, and G:F. The consistency of the feces was visually classified every day on a scale of 0 to 2, with 0 = firm; 1 = soft; and 2 = liquid. Pigs with a score of 2 were classified as having diarrhea.

The basal diet (Table 1Go) was a typical weaner diet, with all nutrients meeting or exceeding estimated nutrient recommendations for 9- to 20-kg pigs (NCPP, 2004Go). The basal diet provided approximately 60 mg of Zn·kg·Dm–1 and 30 mg of Cu·kg·Dm–1, which reflects the contributions from feed ingredients. The 4 dietary treatments (Table 2Go), which comprised a 2 x 2 factorial arrangement, were 1) 100 mg of Zn, 0 mg of Cu, 2) 100 mg of Zn, 175 mg of Cu, 3) 2,500 mg of Zn, 0 mg of Cu, and 4) 2,500 mg of Zn, 175 mg of Cu.


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  		Table 1. Composition of the basal diet (as-fed basis)1
 

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  		Table 2. Amount of Zn as ZnO and Cu as CuSO4 added to the basal diet (mg/kg, as-fed basis) and analyzed content of Zn and Cu in the experimental diets (mg/kg of DM)1
 
Sample Collection
Pigs were killed on d 14 after weaning by i.p. injection of an overdose of sodium pentobarbital (80 mg/kg of BW). The abdominal cavity was opened, and the entire gastrointestinal tract was removed. The pancreas was carefully dissected free, weighed, and stored at –80°C for subsequent analysis. The small intestine (SI) was isolated, and the length was determined. The positions at 10, 50, and 90% of the length of the SI were located at SI10, SI50, and SI90, respectively. To the cranial side, a 5-cm sample was taken for microscopy; and to the caudal side, a 10-cm sample was taken for determination of mucosal enzyme activity. A sample for microscopy was taken from the cecum (Ce), and after determination of the length of the colon, a 5-cm sample for microscopy was taken at 50% of the colon length (Co50). The samples for microscopy were immediately transferred to a neutral buffered formaldehyde solution (4% wt/vol; Bie & Berntsen, Rødovre, Denmark). The samples for enzyme determination were cut open lengthwise, rinsed carefully with ice-cold 0.9% NaCl, blotted dry, and stored at –80°C until enzyme analysis. After taking the tissue samples from the SI, the total contents of the SI were collected, weighed, and stored at –80°C.

Sample Preparation
For the homogenization of the pancreas, as well as for mixing the digesta, a homogenizer (Ultra Turrax T 25, Janke & Kunkel GMBH & Co. KG, Staufen, Germany, equipped with a S25N-18G probe) was used. The pancreas was thawed and homogenized in 4 vol of ice-cold 154 mM NaCl. The homogenate was centrifuged (13,800 x g for 20 min at 4°C), and aliquots of the supernatant were stored at –80°C. Digesta were mixed and centrifuged (13,800 x g for 20 min at 4°C), and the supernatant was collected for analysis. This procedure results in less than 10% of the activity of trypsin, chymotrypsin, lipase, and carboxylester hydrolase (CEH) remaining in the pellet (M. S. Hedemann, unpublished data). However, the activity of amylase remaining in the pellet was substantial. To wash the amylase from the digesta, samples were diluted in 3 vol of 154 mM NaCl, mixed, and centrifuged (13,800 x g for 20 min at 4°C), and the supernatants were collected for analysis. The pellet was dissolved in the same volume of 154 mM NaCl, and centrifugation was repeated. The activity of amylase was determined in both supernatants.

After thawing, mucosa of the intestinal segment was scraped from the underlying muscular layers. The mucosa was homogenized in aqueous Triton X-100 (1%, vol/vol; 6 mL/g of mucosa) using a homogenizer (Ultra Turrax T 25 equipped with a S25N-8G probe, Janke & Kunkel GMBH & Co. KG; Sangild et al., 1995Go). After centrifugation (20,000 x g for 60 min at 4°C), this procedure was repeated with the sediment. The activity of aminopeptidase N was determined in both supernatants, whereas the remaining enzymes were determined only in the supernatant from the first centrifugation because the activity was below the detection limit in the second supernatant (M. S. Hedemann, unpublished data).

Enzyme Analyses
Trypsinogen and chymotrypsinogen in pancreatic homogenates were activated as previously described (Jensen et al., 1997Go), whereas trypsin (EC 3.4.21.4) and chymotrypsin (EC 3.4.21.1) in digesta were determined without activation. The substrate used for trypsin determination was benzoyl DL-arginine p-nitroanilide (B 4875, Sigma, St. Louis, MO), and succinyl ala-ala-pro-phe p-nitroanilide (S 7388, Sigma) was used as a substrate to measure chymotrypsin activity (Jensen et al., 1997Go). Amylase (EC 3.2.1.1) activity was determined using the Phadebas Amylase Test kit (Pharmacia Diagnostics, Uppsala, Sweden). In pancreatic tissue, the activity of lipase (EC 3.1.1.3) was determined by a titremetric method, in which the hydrolysis of tributyrin by lipase in the presence of bile salts and excess colipase was followed (Erlanson-Albertsson et al., 1987Go). In small intestinal contents, the activity of lipase was determined in the presence of bile salts but without addition of colipase. The activity determined was a result of the relative content of lipase and colipase in the sample (Borgström and Hildebrand, 1975Go). Activation of procarboxypeptidase A and procarboxypeptidase B in homogenized pancreatic tissue was performed as described by Hedemann et al. (1998)Go. The activity of carboxypeptidase A (CPA; EC 3.4.17.1) and carboxypeptidase B (CPB; EC 3.4.17.2) in pancreatic homogenate and digesta was measured using hip-puryl- DL-phenyllactic acid (H 9755, Sigma) and hip-puryl-arginine (H 2508, Sigma) as substrates, respectively (Hedemann et al., 1998Go). Carboxylester hydrolase (EC 3.1.1.1) was determined using p-nitrophenyl acetate (N 8130, Sigma) as a substrate, as previously described (Jensen et al., 1997Go).

The activity of lactase (EC 3.2.1.23-62), maltase (EC 3.2.1.20), and sucrase (EC 3.2.48-10) was determined according to Dahlqvist (1968Go, using a glucose kit (166 391, Boehringer Mannheim, Mannheim, Germany) to determine the amount of liberated glucose. Aminopeptidase N (APN; EC 3.4.11.2) and {gamma}-glutamyl transpeptidase (GTP; EC 2.3.2.2) were determined using L-alanine-4-nitroanilide (Merck 101014; Darmstadt, Germany) and {gamma}- L-glutamic acid 3-carboxy-4-nitroanilide (G 5008, Sigma) as substrates, respectively (Hedemann et al., 2003Go). One unit of enzyme activity was defined as the hydrolysis of 1 µmol of substrate in 1 min.

Morphology and Carbohydrate Histochemistry
After 24 h in 4% neutral buffered formaldehyde, tissue samples were carefully cleaned of remaining digesta using saline (154 mM NaCl) and then were transferred to a fresh solution of 4% neutral buffered formaldehyde. Subsequently, samples were dehydrated and infiltrated with paraffin wax. Three slides were prepared from each sample, and each slide contained a minimum of 4 sections cut at 4 µm, at least 50 µm apart. The slides were processed for carbohydrate histochemistry using either the periodic acid-Schiff’s (PAS) reaction or the Alcian blue (AB) reaction at pH 2.5 (AB2.5) or pH 1.0 (AB1.0; Kiernan, 1990Go). The PAS reaction stains for neutral mucins, the AB2.5 stains for carboxylated or sulfated types of acidic mucins, and the AB1.0 stains for sulfomucins (Kiernan, 1990Go).

Morphological characteristics and carbohydrate histochemistry on the PAS- and AB-stained samples were evaluated as described previously (Hedemann et al., 2005Go) using a computer-integrated microscope and an image analysis system (Quantimet 500MC, Leica, Cambridge, UK) with a monitor. Based on results from a previous study in which marked effects on carbohydrate histochemistry were observed in the colon of pigs fed diets containing antibiotics and 2,500 mg of Zn/kg of diet (T. Thymann, Royal Veterinary and Agricultural University, Copenhagen, Denmark, personal communication), we chose to use cranial (SI10) and middle (SI50) SI for the determination of morphological characteristics, and the caudal SI (SI90), the cecum (Ce), and the middle colon (Co50) for the determination of morphological characteristics and carbohydrate histochemistry.

Statistical Analyses
The effects of Zn and Cu on the activity of digestive enzymes in pancreatic homogenate and small intestinal digesta, activity of mucosal enzymes, morphological characteristics, and histochemistry were analyzed with the MIXED procedure (SAS Inst. Inc., Cary, NC; Littell et al., 1996Go). The following model was used:


Formula

where {alpha}z is the concentration of Zn (z = 100, 2,500), ßc is the concentration of Cu (c = 0, 175), ({alpha}ß)zc is the interaction between Zn and Cu, Ul is the random effect of litter (l = 1, ...., 8), and {varepsilon}zcl ~ N(0,{sigma}2) represents the unexplained random error. Regarding the activity of enzymes in pancreatic homogenate, the GROUP option of PROC MIXED was used to account for any differences in variance between dietary treatments.

Results are presented as least squares means with their SEM. To obtain normality, data on the activity of amylase, chymotrypsin, trypsin, lipase, and CEH in pancreatic homogenate were analyzed on a logarithmic scale. Because confidence intervals on the original scale are not symmetric around the parameter estimates, the confidence intervals rather than the SE are presented for those responses. Differences were considered significant at P < 0.05.


   RESULTS
Top
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
Growth Performance
The total feed intake during the 14-d experimental period and ADG were not affected by the dietary concentration of Zn (feed intake, 3.90 ± 0.40 vs. 3.59 ± 0.41 kg, P = 0.60; ADG, 169 ± 25 vs. 156 ± 26 g·d–1, P = 0.71). The dietary concentration of Cu did not affect feed intake during the 14-d experimental period and ADG either (ADFI, 3.64 ± 0.40 vs. 3.86 ± 0.41 kg, P = 0.70; ADG 156 ± 24 vs. 170 ± 26 g·d–1, P = 0.70). Diarrhea was observed in 4 pigs: 1 fed the diet supplemented with 100 ppm of Zn and 0 ppm of Cu, 1 fed the diet supplemented with 100 ppm of Zn and 175 ppm of Cu, and 2 fed the diet supplemented with 2,500 ppm of Zn and 175 ppm of Cu. One pig fed the diet containing 100 ppm of Zn and 0 ppm of Cu died shortly after the initiation of the experiment. The cause of death is unknown, but no signs of diarrhea were observed.

Enzyme Activity in Pancreatic Tissue and Intestinal Contents
The addition of a high concentration of Zn to the weaning diet resulted in a greater activity of 5 of 7 measured enzymes in pancreatic tissue homogenate (Table 3Go). The high concentration of Cu did not affect enzyme activity in pancreatic tissue, and no interaction between Zn and Cu was observed. The amylase activity in pancreatic homogenates increased (P = 0.001) when feeding 2,500 ppm of Zn compared with 100 ppm of Zn. The activity of CPA in pancreatic tissue was increased in pigs fed the high concentration of Zn (P = 0.01), whereas the activity of CPB was unaffected by dietary Zn concentration. The activity of chymotrypsin (P = 0.001) and trypsin (P = 0.002) in pancreatic tissue homogenates was elevated after feeding a high dietary concentration of Zn. Lipase activity was increased in pancreatic tissue homogenates from pigs fed 2,500 ppm of Zn (P = 0.001), whereas the activity of CEH in pancreatic tissue was unaffected by the Zn concentration of the diet.


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  		Table 3. Enzyme activity of pancreatic tissue (U/g of tissue) of weaned pigs fed ZnO or CuSO4 for 14 d postweaning1
 
An interaction occurred between Zn and Cu in the total amount of digesta in the small intestine (Table 4Go). The amount of digesta decreased when feeding the high-Zn diet and increasing the Cu addition from 0 to 175 ppm, but it increased slightly as the Cu supplementation increased from 0 to 175 ppm in pigs fed the low-Zn diet (Zn x Cu, P < 0.01). The total activity of amylase and CPA in small intestinal contents was greater (P < 0.05) in pigs fed 100 ppm of Zn compared with pigs fed 2,500 ppm of Zn. Total CPB, trypsin, lipase, and CEH activities in small intestinal contents were not affected by the dietary Zn concentration. The total chymotrypsin activity was increased (P = 0.001) in pigs fed 2,500 ppm of Zn when compared with pigs fed 100 ppm of Zn.


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  		Table 4. Weight of the intestinal contents (g) and total enzyme activity (U, thousands) of small intestinal contents of weaned pigs fed ZnO or CuSO4 for 14 d postweaning1
 
Gut Morphology and Mucosal Enzyme Activities
Pigs fed diets containing 100 ppm of Zn had longer villi in the SI10 than pigs the diet with 2,500 ppm added Zn (P = 0.001; Table 5Go). In the SI50 and SI90, no effect of the dietary concentrations of Zn and Cu on the villus height was observed. The crypt depth was reduced in pigs fed the diet with 175 ppm added Cu in the SI10 and SI90 compared with those fed the diet with 0 ppm added Cu (P = 0.05). In the SI50, the high concentration of Zn reduced the crypt depth (P = 0.03), but no effect of the high concentration of Zn or Cu on the crypt depth was observed in the Ce and Co50. The high concentration of Zn or Cu had no effect on the thickness of the muscularis externa.


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  		Table 5. Intestinal villus height, crypt depth, and thickness of the muscularis externa (µm) of weaned pigs fed ZnO or CuSO4 for 14 d postweaning1
 
An interaction between Zn and Cu was observed for the lactase activity in SI10 (P = 0.03; Table 6Go). When feeding pigs diets containing 100 ppm of Zn, the lactase activity increased when Cu concentration was increased to 175 ppm, whereas in pigs fed diets containing 2,500 ppm of Zn, no effect of Cu concentration was observed. Maltase and sucrase activities in the mucosa from SI10 were greater (P < 0.001) in pigs fed 100 ppm of Zn than those fed 2,500 ppm of Zn. There was no effect of Zn or Cu on the activity of the disaccharidases in the SI50. In the SI90, an interaction occurred between Zn and Cu for the activity of maltase (P = 0.03). In pigs fed 100 ppm of Zn, the addition of high concentration of Cu reduced the maltase activity, but in pigs fed 2,500 ppm of Zn, no such effect was observed. The activity of GTP was unaffected by the high concentrations of Zn and Cu at all positions. The activity of APN in the SI90 was elevated in pigs fed the low-Zn diet (P = 0.03) but no effects of the Zn concentration were observed in the SI10 and SI50.


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  		Table 6. Mucosal enzyme activity (U/g of mucosa) of lactase, maltase, sucrase, {gamma}-glutamyl transpeptidase, and aminopeptidase N in small intestinal segments of weaned pigs fed ZnO or CuSO4 for 14 d postweaning1
 
Carbohydrate Histochemistry
The staining areas of neutral, acidic, and sulfomucins on the villi and in the crypts in the SI90 were not affected by Zn or Cu, and no interaction between Zn and Cu was observed (Table 7Go). In the Ce and Co50, feeding pigs the diet with 2,500 ppm of Zn resulted in greater neutral, acidic, and sulfomucin areas (P < 0.01). When expressing the mucin staining area as percentage of the villus or crypt area, the only effect observed in the SI90 was an interaction between Zn and Cu for the area of neutral mucins in the crypts (P = 0.004; Table 8Go). In pigs fed 2,500 ppm of Zn, the area of neutral mucins increased when the inclusion of Cu increased from 0 to 175 ppm, but no effect of the Cu concentration was observed when pigs were fed 100 ppm of Zn. The area of all 3 mucin categories (neutral, acidic, and sulfomucins) relative to the crypt area in the cecum and colon increased when pigs were fed 2,500 ppm of Zn compared with pigs fed 100 ppm of Zn for 2 wk postweaning (P < 0.01). Furthermore, the addition of 175 ppm of Cu reduced the area of acidic mucins in the colon (P = 0.05).


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  		Table 7. Staining area (µm2) of mucins in the intestinal villi and crypts of weaned pigs fed ZnO or CuSO4 for 14 d postweaning1
 

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  		Table 8. Mucin staining area of intestinal total villi or crypt area (%) of weaned pigs fed ZnO or CuSO4 for 14 d postweaning1
 

   DISCUSSION
Top
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
The lack of effect on feed intake and ADG in piglets receiving the high Zn dose is in disagreement with observations from animal performance studies (Hill et al., 2000Go; Case and Carlson, 2002Go). In contrast to other studies (Cromwell et al., 1998Go; Hill et al., 2000Go), our results indicate no effect of Cu amendment on animal performance. It should, however, be emphasized that effects on animal performance can only be evaluated thoroughly by including a larger number of animals than we did in the current study. Furthermore, piglets in our experiment were housed individually, which has previously been shown to decrease the performance responses to Zn (Buff et al., 2005Go). Individual housing may affect feed consumption because of the lack of social facilitation (Brumm and Gonyou, 2001Go). Moreover, individual housing decreases the exposure of piglets to various infections, and a greater health status may decrease the response to high concentrations of Zn and Cu.

Supplementing the weaning diet with 2,500 mg of Zn/kg of diet resulted in marked increases in the activity of enzymes in the pancreatic tissue homogenate, whereas the high concentration of Cu did not affect the enzyme activities. After weaning, a decrease in the enzymatic activity in pancreatic tissue has been observed (Owsley et al., 1986Go; Hedemann and Jensen, 2004Go), and enzyme activities adjust to a new feed composition and changes in the eating pattern during the postweaning period (Cranwell, 1995Go). The enzyme activities observed in pigs fed 100 ppm of Zn are within the range observed 2 wk postweaning in pigs weaned at 4 wk of age and fed a similar standard weaning diet (Jensen et al., 1997Go). In contrast to the present results, Carlson (2003)Go found that the activity of CPA, trypsin, and lipase in pancreatic tissue was lower in pigs fed 2,500 ppm of Zn compared with pigs fed 100 ppm of Zn during d 5 to 6 postweaning, whereas CPB and amylase were unaffected by Zn concentration. In rats, supplementation of the diet with high concentrations of Zn resulted in an increased enzyme activity in pancreatic tissue (Szabo et al., 2004Go). In chicks, however, feeding 2,000 ppm of Zn resulted in a marked decrease in the activity of amylase, lipase, trypsin, and chymotrypsin in pancreatic tissue, and the pancreas was suggested to be a target organ of Zn toxicity in chicks (Lü and Combs, 1988Go). In pigs, the tolerable level of Zn is dependent on the source of the element (Szabo et al., 2004Go), and toxicity of high concentrations of ZnO used for growth promotion has never been reported. The lack of effect of Cu on the enzyme activities in pancreatic homogenate is in agreement with Luo and Dove (1996)Go.

The activity of pancreatic enzymes in intestinal contents is a rough estimate of exocrine pancreatic secretion (Hedemann and Jensen, 2004Go). The enzyme activities in the intestinal contents did not reflect the activities measured in pancreatic tissue, indicating that high dietary Zn concentration stimulated the synthesis of pancreatic enzymes without stimulating secretion. The lack of effect of Zn on enzyme activity in digesta is in contrary to the results obtained in rats where feeding 1,000 to 5,000 ppm of Zn resulted in increased enzyme activity in pancreatic tissue as well as intestinal contents (Szabo et al., 2004Go). Excess or toxic amounts of Zn have been shown to decrease pancreatic flow and enzyme secretions in sheep (Smith and Embling, 1984Go). No signs of toxicity were, however, observed in the current study. Luo and Dove (1996)Go observed that high concentrations of Cu increased the activity of lipase in intestinal contents. This effect was not seen in the current study where lipase activity in intestinal contents was unaffected by dietary Cu concentration. The current study, using measurements in pancreatic tissue homogenate and intestinal contents, provides a steady-state view on the exocrine pancreatic secretion, but it does not give any information on the exocrine pancreatic secretion over time. Further studies are needed to elucidate the effect of high concentrations of Zn on synthesis and secretion of digestive enzymes from the pancreas. But the current study indicates that the growth-promoting effect of high concentrations of Zn is not via stimulation of exocrine pancreatic secretion.

There was no consistent effect of Zn supplementation on villus height, which is in agreement with Mavromichalis et al. (2000)Go who investigated the effect of Zn during the 21-d postweaning period in pigs weaned at 21 d of age. In contrast to their study, it has been shown that supplementing Zn in starter diets increased the villus height and reduced the crypt depth on d 11 postweaning in pigs weaned at 21 d of age (Li et al., 2001Go). Changes in gut morphology of pigs after weaning, which include villus atrophy and crypt hyperplasia, are well documented (Hampson, 1986Go; Spreeuwenberg et al., 2001Go; Hedemann et al., 2003Go). These morphological changes are more prominent when weaning takes place earlier (Pluske et al., 1997Go). In rats, it has been shown that Zn deficiency is accompanied by reduced jejunal villus height, but after a short period of Zn supplementation, the morphology is returned to normal (Southon et al., 1986Go). As villus height has been shown to increase from d 5 postweaning (Hampson, 1986Go) and reach preweaning values on d 9 postweaning (Hedemann et al., 2003Go), it is possible that Zn has no effect on intestinal morphology or that the examination was made after the intestinal conditions had stabilized after postweaning alterations or both.

Crypt depths were decreased in pigs fed high Cu, which is in contrast to previous studies that have shown increased crypt depths (Shurson et al., 1990Go) or no effect on crypt depths (Radecki et al., 1992Go) when feeding high Cu. Crypt depth is an indicator of cell proliferation, and less energy may thus be spent on cell renewal in pigs fed high Cu.

The activity of disaccharidases in the cranial small intestine mucosa was greater in pigs fed 100 ppm of Zn. These pigs also had the longest villi. Peptidase activity did not show the same relation to villus height. The lack of effect of Zn on villus height in the middle and caudal small intestine was also reflected in the activity of mucosal enzymes where only minor differences were observed. Similarly, Carlson (2003)Go found no effect of high Zn or Cu on mucosal enzyme activity on d 5 to 6 postweaning. In rabbits, it has been reported that Zn does not alter the activity of sucrase and APN (Rodriguez-Yoldi et al., 1994Go; Yoldi et al., 1996Go). In rats, however, conflicting results have been observed. No effect of Zn deficiency on lactase, maltase, and sucrase activity was reported by some researchers (Zarling et al., 1985Go; Naveh et al., 1990Go), whereas others found reduced mucosal enzyme activities following Zn deficiency (Gebhard et al., 1983Go; Park et al., 1985Go; Tamada et al., 1992Go).

The area of neutral, acidic, and sulfomucins in the goblet cells was increased in the cecum and colon of pigs fed high Zn, whereas no effect was observed in the caudal small intestine. The thickness of the mucus layer and the amount of mucin in intestinal contents was not determined. A greater goblet cell area is, however, an expected indication of greater mucus production and secretion (Brunsgaard, 1998Go). In the stomach, Zn has been shown to increase the presence of mucus on the gastric surface (Esplugues et al., 1985Go; Bravo et al., 1992Go). How Zn stimulates the mucus secretion in the stomach remains to be elucidated. But, because Zn is a potent regulator of gene expression in the small intestine (Blanchard and Cousins, 1996Go), it is possible that Zn stimulates mucin secretion through a regulation of the mucin genes. Another aspect of mucin dynamics is the continuous interaction between the mucus layer and the microflora. The intestinal microbiota can stimulate mucin gene expression (Mack et al., 2003Go) and the production of mucin degrading enzymes (Corfield et al., 1992Go). It has been shown in chickens that supplementation with an antibiotic growth promoter increased the mucin mRNA expression, and it was suggested that the effect was mediated through changes in intestinal bacterial populations (Smirnov et al., 2005Go). As the effect of Zn on the intestinal microflora resembles the working mechanism suggested for antibiotic growth promoters (Højberg et al., 2005Go), it is implied that the increased goblet cell area is a result of altered microbial activity in the gastrointestinal tract.

The significance of a greater mucin production is not clear, and in the current study, the increased mucin area observed in the cecum and the colon is probably not important in context with postweaning diarrhea caused by E. coli that colonizes the small intestine (Nagy and Fekete, 1999Go). But, the amount of mucin produced in the cecum and the colon may be of importance in preventing intestinal infections located in the large intestine (Brunsgaard, 1998Go).

In summary, the current study demonstrated that a high dietary concentration of Zn affected some physiological responses in the gastrointestinal tract, whereas Cu had no effect on the responses studied. It was shown that Zn had a marked effect on the activity of enzymes in pancreatic tissue. Further studies are needed to elucidate how Zn regulates the synthesis of pancreatic enzymes and whether Zn influences pancreatic secretion. Feeding pigs the high dietary concentration of Zn elicited increased areas of mucins in the cecum and colon, which might be a consequence of altered microbial activity in the large intestine, but this warrants further studies. In spite of the marked changes observed in the gastrointestinal tract when feeding the high concentration of Zn, the current study does not give any definite answers on how the growth promoting and diarrhea reducing effects of Zn are exerted.


   Footnotes
 
1 This work was financially supported by the Danish Ministry of Agriculture, Food and Fisheries, the Research Secretariat and the National Committee for Pig Breeding, Health and Production, the Federation of Danish Pig Producers and Slaughterhouses. The authors wish to thank M. L. Nielsen and L. Märcher for excellent technical assistance. Back

2 Corresponding author: Mette.Hedemann{at}agrsci.dk

Received for publication December 8, 2005. Accepted for publication July 21, 2006.


   LITERATURE CITED
Top
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 


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