Effects of copper bearing montmorillonite on the growth performance, intestinal microflora and morphology of weanling pigs

Animal Feed Science and Technology 118 (2005) 307–317

Effects of copper bearing montmorillonite on the growth performance, intestinal microflora and morphology of weanling pigs M.S. Xia∗ , C.H. Hu, Z.R. Xu Animal Science College, Zhejiang University, Hua Jia Chi Campus, Hangzhou 310029, PR China Received 7 April 2004; received in revised form 18 November 2004; accepted 18 November 2004

Abstract A total of 128 weaning pigs (Duroc × Landrace × Yorkshire) at an average initial body weight of 7.5 ± 0.6 kg were used to investigate the effects of copper bearing montmorillonite (Cu-MMT) on the growth performance, intestinal microflora, bacterial enzyme activities, and intestinal morphology. The pigs were allocated to four dietary treatments in a randomized complete block design (four replicates of eight pigs per replicate) for 45 days. The dietary treatments were: (1) basal diet, (2) basal diet + 1.5 g/kg MMT, (3) basal diet + 36 mg/kg copper as CuSO4 (equivalent to the copper in the CuMMT treatment group or (4) basal diet + 1.5 g/kg Cu-MMT. The results showed that supplementation with Cu-MMT improved (P < 0.05) growth performance, reduced (P < 0.05) the total viable counts of intestinal Clostridium and Escherichia coli, depressed (P < 0.05) the activities of intestinal ␤glucosidase and ␤-glucuronidas, and increased (P < 0.05) villus height and the villus height to crypt depth ratio at the small intestinal mucosa as compared with control. Supplementation with MMT also increased (P < 0.05) villus height and the villus height to crypt depth ratio at the jejunum as compared with control. However, supplementation with MMT or CuSO4 had no (P > 0.05) effect on growth performance, intestinal microflora and bacterial enzymes as compared with control. Supplementation with CuSO4 had no (P > 0.05) effect on the small intestinal morphology as compared with control. Supplementation with Cu-MMT increased daily gain, decreased viable counts of Clostridium and E. coli and activity of ␤-glucuronidase in colonic contents (P < 0.05) as compared with MMT or CuSO4 , and increased (P < 0.05) villus height and the villus height to crypt depth ratio at the small intestinal

∗ Corresponding author. Present address: Feed Science Institute, Animal Science College, Zhejiang University, Hangzhou 310029, PR China. Tel.: +86 571 86985607. E-mail address: [email protected] (M.S. Xia).

0377-8401/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.anifeedsci.2004.11.008

308

M.S. Xia et al. / Animal Feed Science and Technology 118 (2005) 307–317

mucosa as compared with CuSO4 . The results indicated that Cu-MMT is more effective than MMT or CuSO4 in enhancing the growth, intestinal microflora and morphology of weanling pigs. © 2004 Elsevier B.V. All rights reserved. Keywords: Bacterial enzyme; Copper bearing montmorillonite; Growth performance; Intestinal microflora; Intestinal morphology; Weanling pigs

1. Introduction Montmorillonite (MMT), aluminosilicate clay, has specific physical–chemical properties such as high surface area, strong adsorptive capacity, high structural stability, and chemical inertia (Borchardt, 1989). Animal feed containing MMT has been shown to promote growth performance, reduce both bacterial colonization of the gut and the detrimental effects of mycotoxin-contaminated diets (Schell et al., 1993; Venglovsky et al., 1999; Tauqir and Nawaz, 2001). Moreover, MMT is a protector of intestinal mucosa (Droy-Lefain et al., 1985). It can adhere to enteric pathogens selectively and excrete them, reinforce intestinal mucosal barrier and help in the regeneration of the epithelium (Albengres et al., 1985; Girardeau, 1987). In human medicine MMT has been applied as an antidiarrhoeal remedy (Ahmed et al., 1993). In vitro study showed that MMT could adsorb Escherichia coli and S. aureus, but showed no bacteriostatic or bactericidal effect (Hu et al., 2002). Non-metallic minerals have been used as antimicrobial carriers for some time. Silver carried on zeolite, montmorillonite and other clays has been reported as an effective antibacterial material (Rivera-Garza et al., 2000). Recent experiments have revealed that Cu-bearing montmorillonite (CuMMT), which is produced through Cu2+ exchange reaction, has antibacterial activity on E. coli, Clostridium and Salmonella (Ye et al., 2003). Previous unpublished results showed that Cu-MMT adsorbed E. coli and reduced bacterial numbers by more than 97% and MMT only produced reductions of 20%. There are no data of the effects of Cu-MMT on pigs. In the present study, the effects of Cu-MMT on growth performance, intestinal microflora, and intestinal morphology of weanling pigs were investigated. Bacterial enzymes may be a sensitive, although less direct, measurement of microbial activity (Nalini et al., 1998; Jin et al., 2000). Therefore, the effects of Cu-MMT on bacterial enzymes activities were also evaluated.

2. Materials and methods 2.1. Materials MMT used in the current work was a hydrothermal product of volcanic sedimentary rocks from the Inner Mongolia Autonomous Region, China. Besides MMT, there were minor amounts of quartz and volcanic glass present. To remove the impurities, the raw material was dried in oven at 80 ◦ C for 24 h and then milled to less than 48 ␮m. The milled material was dispersed in water to form a 10% suspension that was stirred for

M.S. Xia et al. / Animal Feed Science and Technology 118 (2005) 307–317

309

about 10 min. Particles larger than 2 ␮m were separated out by sedimentation while the suspension was centrifuged to obtain refined MMT. The refined MMT was dried at 80 ◦ C followed by another milling to less than 48 ␮m. The content of the purified MMT is 99% and the formula was ([Na0.158 K0.082 Ca0.256 Mg0.063 ] [Mg0.376 Fe2+ 0.014 Fe3+ 0.136 Al1.474 ][Si3.87 Al0.13 ]O10 (OH)2 ·nH2 O) with the cation exchange capacity (CEC) of 139.9 mmol/100 g analyzed by National Research Center for GeoAnalysisis, PRC. Cu-bearing montmorillonite (Cu-MMT) was prepared by a Cu2+ cation exchange reaction. Five grams of the refined MMT was mixed with 100 ml of 0.05 mol/l CuSO4 solution to form a suspension by agitation and the pH value of the suspension was adjusted to 5.0 with 0.1 mol/l sodium hydroxide. The suspension was maintained at 60 ◦ C for about 6 h to accelerate the cation exchange. The Cu-MMT was then separated by centrifugation at a speed of 10,000 × g for about 15 min and washed for three times with 30 ml-deionized water each. The washed material was dried at 80 ◦ C over night, and then ground through a screen to a size less than 48 ␮m. The Cu content in Cu-MMT was found to be 24.5 g/kg on the basis of atomic absorption spectral analysis. 2.2. Experimental design and sampling procedure All procedures were approved by the University of Zhejiang Institutional Animal Care and Use Committee. A total of 128 weaning pigs (Duroc × Landrace × Yorkshire) of an average initial body weight of 7.5 ± 0.6 kg were allocated to four dietary treatments in a randomized complete block design for 45 days, each of which was replicated four times with eight pigs per replicate. The dietary treatments were: (1) basal diet, (2) basal diet + 1.5 g/kg MMT, (3) basal diet + 36.75 mg/kg copper as CuSO4 (equivalent to the copper in the CuMMT treatment group or (4) basal diet + 1.5 g/kg Cu-MMT. Diets were formulated to meet or exceed requirements suggested by the NRC (1998) for 10–20 kg pigs. No antibiotic was included in diets (Table 1). All pigs were given ad libitum access to feed and water. Average daily gain (ADG), average daily feed intake (ADFI), and gain/feed ratio were measured. After the feeding trial, eight pigs from each treatment (two pigs per pen) were slaughtered under general anaesthesia and then immediately eviscerated. Samples of the contents from the small intestine (from the distal end of the duodenum to the ileo–caecal junction) and proximal colon were collected into glass containers under CO2 , sealed and put on ice until they were transported to the lab for enumeration of microbial populations. The specimens from the middle part of duodenum, jejunum and ileum segment were excised, flushed with physiological saline and fixed in 10% formalin. 2.3. Intestinal microbial populations Ten grams of mixed contents were blended under CO2 in 90 ml of anaerobic dilution solution (ADS) (Bryant and Allison, 1961). Further, serial dilutions were made in ADS for anaerobic bacterial enumeration (Bryant, 1972). The initial dilution in ADS was also used as a source for serial dilutions in phosphate buffer solution for enumeration of aerobic bacterial populations. Triplicate plates were then inoculated with 0.1 ml samples and incubated at 37 ◦ C aerobically or anaerobically as appropriate. Three dilutions were plated for each medium. Bacteria were enumerated on Wilkins chalgren agar (Oxoid; total anaer-

310

M.S. Xia et al. / Animal Feed Science and Technology 118 (2005) 307–317

Table 1 Ingredient and chemical composition of the basal diet on an as-fed basis Ingredients Maize Soya bean meal (480 g CP/kg) Wheat middling Extruded full-fat Soya bean Fish meal Dried whey Dicalcium phosphate Limestone Sodium chloride l-Lysine HCl Vitamin–mineral premixa Analyzed chemical composition as feed DE (MJ/kg)b Crude protein (g/kg) Lysine (g/kg) Methionine + cystine (g/kg) Calcium (g/kg) Phosphorus (g/kg)

g/kg 58.0 17.0 5.0 7.0 4.5 4.5 1.5 1.1 0.25 0.15 1.0 13.9 201 11.2 6.5 9.0 7.5

a

The vitamin–mineral premix provided (per kg feed): 1500 IU Vitamin A, 200 IU Vitamin D3 , 10 IU Vitamin E, 0.5 mg Vitamin K3 , 0.05 mg biotin, 0.3 mg folic acid, 10 mg niacin, 10 mg d-pantothenic acid, 3.6 mg riboflavin, 1.0 mg thiamine, 1.5 mg pyridoxine, 15 mg cobalamin, 3 mg Mn, 80 mg Zn, 80 mg Fe, 5.0 mg Cu, 0.14 mg I and 0.15 mg Se. b DE was based on calculated values.

obes), brain heart infusion agar (Oxoid; total aerobes), MRS agar (Oxoid; Lactobacillus), reinforced clostridial agar plus supplements (Munoa and Pares, 1988; Bifidobacterium), sulphite-polymyxin milk agar (Mevissen-Verhage et al., 1987; Clostridium), and MacConkey’s no. 2 (Oxoid; E. coli). Single colonies were removed from selective media plates and grown in peptone yeast glucose (PYG) broth (Holdeman et al., 1977). Subsequently, the bacteria were characterized to genus level on the basis of colonial appearance, gram reaction, spore production, cell morphology and fermentation end-product formation (Holdeman et al., 1977). 2.4. Bacterial enzyme assay ␤-Glucosidase (EC 3.2.1.21) and ␤-glucuronidase (EC 3.2.1.31) activities in the small intestine and proximal colon were analyzed under anaerobic conditions by using the method of Jin et al. (2000). ␤-Glucosidase activity unit was expressed as micromole of p-nitrophenol released from p-nitrophenyl-␤-d-glucopyranoside per hour per gram of intestinal digesta protein. The amount of nitrophenol released was determined by comparison with a standard nitrophenol curve. One ␤-glucuronidase activity unit will liberate 1 ␮mol of phenolphthalein from phenolphthalein glucuronic acid per hour per gram of intestinal digesta protein. The amount of phenolphthalein released was determined by comparison with a standard phenolphthalein curve. The intestinal digesta protein concentrations were determined by the method of Lowry et al. (1951). Bovine serum albumin was used as a standard.

M.S. Xia et al. / Animal Feed Science and Technology 118 (2005) 307–317

311

2.5. Histomorphometry Three cross-sections for each intestinal sample were prepared after staining with hematoxylin and eosin using standard paraffin embedding procedures (Xu et al., 2003). A total of 10 intact, well-oriented crypt-villus units were selected in triplicate for each intestinal cross-section (30 measurements for each sample, total of 240 measurements per dietary treatment). Villus height and crypt depth were determined using image processing and analysis system (version 1, Leica Imaging Systems Ltd., Cambridge, England). 2.6. Statistical analysis Data were analyzed as a randomized complete block using the general linear model procedure (SAS Inst. Inc., 1989, Cary, NC). A pen of pigs served as the experimental unit for all data. Differences among means were tested using Duncan’s multiple range tests. Effects were considered significant at P < 0.05.

3. Results and discussion 3.1. Effect of dietary Cu-MMT on growth performance of pigs The growth performance of weaning pigs is presented in Table 2. Pigs fed with CuMMT had higher (P < 0.05) ADG than those fed with control or MMT or CuSO4 . Pigs fed with Cu-MMT had higher feed conversion than those fed with control (P < 0.05). However, supplementation with MMT or CuSO4 had no (P > 0.05) effect on growth performance as compared with the control treatment. Feed intake was not affected by dietary treatments. The results of previous experiments on the effects of clays on animal performance were generally inconsistent (Poulsen and Oksbjerg, 1995; Ouhida et al., 2000), although animal feed containing MMT (10–30 g/kg) has been shown to promote weight gain and feed efficiency of chickens and pigs (Tauqir and Nawaz, 2001). In the present study, supplementation with MMT had no significant effect on growth performance as compared with the Table 2 Effect of dietary Cu-MMT on growth performance of weaning pigsa Item

Control

MMTb

CuSO4

Cu-MMTc

S.E.M.d

ADG (g) ADFI (g) Gain:feed

382b 741 0.52b

394b 745 0.53ab

400b 748 0.54ab

428a 749 0.57a

9 16 0.016

Means within a row with different letters (a, b) differ significantly (P < 0.05) when tested with Duncan’s new multiple-range test following analysis of variance. a Data are means of four replicate pens of eight pigs each. Average initial and final body weight were 7.5 and 25.6 kg, respectively. The trial lasted 45 days. b MMT: montmorillonite. c Cu-MMT: copper bearing montmorillonite. d Standard error of the mean.

312

M.S. Xia et al. / Animal Feed Science and Technology 118 (2005) 307–317

Table 3 Effect of dietary Cu-MMT on intestinal microflora of pigsa,b Site and microflora

Control

MMTc

CuSO4

Small intestine Total aerobes Total anaerobes Bifidobacterium Lactobacillus Clostridium Escherichia coli

8.4 9.4 6.9 7.9 6.5a 7.8a

8.2 9.3 7.4 8.2 5.8ab 7.4ab

8.2 9.5 6.9 8.0 6.4ab 7.3ab

Proximal colon Total aerobes Total anaerobes Bifidobacterium Lactobacillus Clostridium Escherichia coli

9.2 10.6 7.8 8.9 7.9a 8.8a

9.1 10.4 8.2 9.4 7.7a 8.6a

9.2 10.2 8.0 8.9 7.7a 8.6a

Cu-MMTd

S.E.M.e

7.9 9.6 7.2 8.4 5.6b 6.7b

0.24 0.35 0.23 0.33 0.26 0.26

8.9 10.1 8.2 9.3 6.8b 8.0b

0.30 0.22 0.23 0.28 0.27 0.17

Means within a row with different letters (a, b) differ significantly (P < 0.05). a Bacterial numbers are expressed as log colony-forming units per gram of DM. 10 b Data are the means of four replicates of two pigs per replicate. c MMT: montmorillonite. d Cu-MMT: copper bearing montmorillonite. e Standard error of the mean.

control treatment. The feeding value of MMT may be affected by the addition concentration, sanitary condition, level performance, diet composition, and so on. 3.2. Effect of dietary Cu-MMT on intestinal microflora of pigs Intestinal microflora of weaning pigs is presented in Table 3. Supplementation with CuMMT reduced (P < 0.05) the total viable counts of Clostridium and E. coli in the small intestine and proximal colon of weanling pigs as compared with the control. Supplementation with MMT or CuSO4 had no (P > 0.05) effect on intestinal microflora as compared with control. Pigs fed with Cu-MMT had lower (P < 0.05) viable counts of Clostridium and E. coli in colonic contents than those fed with MMT or CuSO4 . The total aerobes, total anaerobes, Bifidobacterium and Lactobacillus in the small intestinal and colonic contents of pigs were not affected by the dietary treatments. It is well known that MMT can adhere to bacteria selectively (Albengres et al., 1985; Girardeau, 1987). Adsorption was the main interaction between MMT and bacteria. The adsorption effect was related to layer charge density of MMT. In human medicine MMT has been applied as antidiarrheal remedy (Ahmed et al., 1993; Wang and Fang, 1995). Wu et al. (1999) used MMT as antidiarrheic to treat the diseases of colon bacillus and diarrhoeal syndrome in piglets and they found that the curative effects of MMT were higher than sulphate gentamicin in treating the diarrhoeal syndrome of early weaning piglets. Wang and Fang (1995) reported that, after diarrhoeal children had been treated with MMT for 5 days, the population of Bifidobacteria increased and those of E. coli decreased significantly.

M.S. Xia et al. / Animal Feed Science and Technology 118 (2005) 307–317

313

Recent experiments have revealed that Cu-MMT has antibacterial activity on E. coli, Clostridium and Salmonella (Ye et al., 2003). Hu et al. (2002) showed in vitro that MMT adsorbed E. coli and S. aureus while had no bacteriostatic or bactericidal effect; however, Cu-MMT did show these activities. Stadler and Schindler (1993) found that Cu2+ in aqueous solution with pH > 4.5 tends to enter the interlayer position of MMT and forms [Cu(AlO)n (H2 O)4−n ]x+ . When Na+ or Ca2+ was replaced by [Cu(AlO)n (H2 O)4−n ]x+ , or Cu2+ entered the tetrahedron and octahedron, MMT lost its electrical balance. This made the mineral has surplus positive charge. On the other hand, the bacterial cell wall is negatively charged due to functional groups such as carboxylates present in lipoproteins at the surface (Breen et al., 1995), so that Cu-MMT particles would attract bacteria, due to the opposite static charge. Previous unpublished results showed that Cu-MMT adsorbed E. coli and reduced bacterial numbers by more than 97% and MMT only produced reductions of 20%. A similar phenomenon was reported by Herrera et al. (2000). In their work, MMT was treated with cytylpyridinium. The product CP-MMT (cytylpyridinium- exchanged montmorillonite) also has a surplus positive charge on surface similar to Cu-MMT. Using scanning electron microscopy, they found that a large quantity of Salmonella enteritidis accumulated on CP-MMT surface, but untreated MMT was not attracted to S. enteritidis. Surplus positive charge of Cu-MMT and CP-MMT was most probably an important factor for their antibacterial capability. In this case, the released Cu2+ would act directly on the attracted bacteria, instead of into the medium and indirectly on the bacteria. In other words, the active Cu2+ density on mineral surface was much higher than its concentration in the solution. In summary, electrostatic attraction and the antibacterial effect of Cu2+ ion on bacteria are two ways of the antimicrobial action of Cu-MMT. 3.3. Effect of Cu-MMT on the bacterial enzyme activities in the intestine of pigs As compared with the control, supplementation with Cu-MMT depressed (P < 0.05) the activities of ␤-glucosidase and ␤-glucuronidase in the small intestinal and colonic contents (Table 4). Supplementation with MMT or CuSO4 had no (P > 0.05) effect on bacterial enzymes as compared with control. Supplementation with Cu-MMT decreased colonic ␤glucuronidase (P < 0.05) as compared with MMT or CuSO4 and lowed ␤-glucuronidase (P < 0.05) in the small intestine as compared with CuSO4 . ␤-Glycosidase is involved in the carcinogenicity of the naturally occurring nontoxic glycosides. It has been postulated that amygdaline is hydrolyzed in the gut by bacterial ␤-glucosidase to yield mandelonitrile, which is unstable and is readily hydrolyzed to release toxic cyanide (Goldin and Gorbach, 1976). ␤-Glucuronidase is believed to be largely responsible for the hydrolysis of glucuronides in the lumen of the gut. This reaction is potentially important in the generation of toxic and carcinogenic substances as many compounds are detoxified by glucuronide formation in the liver and subsequently enter the bowel via the bile. In this way, toxic aglycones can be regenerated in the bowel by bacterial ␤-glucuronidase (Nalini et al., 1998; Jin et al., 2000). Although these potential harmful metabolites may not cause disease to pigs, they may hinder the growth performance or reduce feed utilization. The reduction of ␤-glucosidase and ␤-glucuronidase activities in pigs fed Cu-MMT may be attributed to the lower numbers of E. coli and Clostridium in the intestine. Hawksworth

314

M.S. Xia et al. / Animal Feed Science and Technology 118 (2005) 307–317

Table 4 ␤-Glucosidase and ␤-glucuronidase activities in the small intestinal and colonic contents of pigs as affected by Cu-MMTa,b Control

MMTc

CuSO4

Cu-MMTd

S.E.M.e

Small intestine ␤-Glucosidase activity ␤-Glucuronidase activity

76a 128a

56ab 102ab

64ab 116a

43b 81b

7.6 10.6

Proximal colon ␤-Glucosidase activity ␤-Glucuronidase activity

51a 186a

36ab 149a

45ab 162a

28b 112b

6.8 12.0

Means within a row with different letters (a, b) differ significantly (P < 0.05). a Bacterial enzyme activities are expressed as ␮mol/h per g protein. b Data are the means of four replicates of two pigs per replicate. c MMT: montmorillonite. d Cu-MMT: copper bearing montmorillonite. e Standard error of the mean.

et al. (1971) reported that E. coli produced significantly more ␤-glucuronidase per strain than any other genera tested. Furthermore, over 90% of E. coli strains are able to produce ␤-glucuronidase, whereas only less than 40% of Lactobacillus strains show an ability to produce glucosidase (Drasar and Hill, 1974). 3.4. Effect of dietary Cu-MMT on the small intestinal morphology Morphological measurements of the small intestinal mucosa of pigs are presented in Table 5. Supplementation with Cu-MMT had higher (P < 0.05) villus height and the villus Table 5 Effects of Cu-MMT on the morphology of the intestinal mucosa in different sites of the small intestinea Site

Control

MMTb

CuSO4

Cu-MMTc

S.E.M.d

Villus height (␮m) Duodenum Jejunum Ileum

695b 810b 585b

736ab 890a 640ab

702b 821b 579b

786a 939a 681a

26 26 21

Crypt depth (␮m) Duodenum Jejunum Ileum

484 536a 440

472 509ab 423

481 539a 435

459 484b 391

15 14 23

Villus height:crypt depth Duodenum Jejunum Ileum

1.4b 1.5c 1.3b

1.6ab 1.8b 1.5ab

1.5b 1.5c 1.3b

Means within a row with different letters (a–c) differ significantly (P < 0.05). a Data are the means of four replicates of two pigs per replicate. b MMT: montmorillonite. c Cu-MMT: copper bearing montmorillonite. d Standard error of the mean.

1.7a 1.9a 1.7a

0.06 0.06 0.12

M.S. Xia et al. / Animal Feed Science and Technology 118 (2005) 307–317

315

height to crypt depth ratio at the small intestinal mucosa as compared with the control or CuSO4 . Supplementation with MMT also increased (P < 0.05) villus height and the villus height to crypt depth ratio at the jejunum as compared with the control. Supplementation with CuSO4 had no (P > 0.05) effect on the small intestinal morphology as compared with control. The structure of the intestinal mucosa can reveal some information on gut health. Stressors that are present in the digesta can lead relatively quickly to changes in the intestinal mucosa due to the close proximity of the mucosal surface and the intestinal content. Changes in intestinal morphology such as shorter villus and deeper crypts have been associated with the presence of toxins (Xu et al., 2003). A shortening of the villus decreases the surface area for nutrient absorption. The crypt is the area where stem cells divide to permit the renewal of the villus, and a large crypt indicates fast tissue turnover and a high demand for new tissue. In the present study, an increase in villus height and villus height to crypt depth ratio at the small intestinal mucosa of the pigs supplemented with Cu-MMT were observed. Such improved intestinal mucosal morphology may be explained by the lower numbers of E. coli and Clostridium and the lower activities of bacterial enzymes. The addition of MMT to the diet also produced a positive effect on the intestinal mucosa. It is reported that MMT, a mucus stabilizer, effectively acts by attaching to the mucus to reinforce intestinal mucosal barrier and helps in the regeneration of the epithelium (Albengres et al., 1985; Girardeau, 1987). MMT effectively improves gastrointestinal mucus resistance to various aggressions by interacting closely with the mucous glycoproteins (Droy-Lefain et al., 1985).

4. Implications The improvement of growth performance, the reduction of the intestinal E. coli and Clostridium and the bacterial enzyme activities, and the positive effect on the intestinal mucosa in weaning pigs fed Cu-MMT may be of importance to the pig industry. The electrostatic attraction due to the surplus positive charge of Cu-MMT and the antibacterial effect of Cu2+ ion on bacteria may be the important factors for its higher antibacterial activity compared to MMT or CuSO4 . Acknowledgements The authors gratefully acknowledge to L. Xiong, H.X. Sun, X.L. Hu, N.Q. Chen, and G.L. Wu for their skilled technical assistance. The financial provided by National Natural Science Foundation of China (Project 30471255) is gratefully acknowledged.

References Ahmed, A.M., Ekram, M., Madina, H., Amer, M.A., Abbass, T., 1993. Smectite in acute diarrhea in children: a double-blind placebo-controlled clinical trial. J. Pediatr. Gastrenterol. Nutr. 17, 176–181. Albengres, E., Urien, S., Tillement, J.P., Oury, P., Decourt, S., Flouvat, B., Drieu, K., 1985. Interactions between smectite, a mucus stabilizer, and acidic and basic drugs. Eur. J. Clin. Pharmacol. 28, 601–605.

316

M.S. Xia et al. / Animal Feed Science and Technology 118 (2005) 307–317

Borchardt, G., 1989. Smectites. In: Dixon, J.B., Weed, S.B. (Eds.), Minerals in Soil Environments. Soil Science of America, Madison, WI, pp. 675–727. Breen, P.J., Compadre, C.M., Fifer, E.K., Salari, H., Serbus, D.C., Lattin, D.L., 1995. Quaternary ammonium compounds inhibit and reduce the attachment of viable Salmonella typhimurium to poultry tissues. J. Food Sci. 60, 1191–1196. Bryant, M.P., Allison, I.M., 1961. An improved non-selective culture medium for animal bacterial and its use in determining diurnal variation in numbers of bacteria in the rumen. J. Dairy Sci. 44, 1446–1453. Bryant, M.P., 1972. Commentary on the Hungate technique for culture for anaerobic bacteria. Am. J. Clin. Nutr. 25, 1324–1330. Drasar, B.S., Hill, M.J., 1974. Human Intestinal Flora. Academic Press, London, UK. Droy-Lefain, M.T., Drouet, Y., Schatz, B., 1985. Sodium glycodeoxycholate and spinability of gastrointestinal mucus: Protective effect of smectite. Gastroenterol 88 (Suppl. 2), 1369. Girardeau, J.P., 1987. Smectite aggregation by Escherichia coli. Acta Gastroenterol. Belg. 50, 181–192. Goldin, B.R., Gorbach, S.L., 1976. The relationship between diet and rat fecal bacterial enzymes implicated in colon cancer. J. Natl. Cancer Inst. 57, 371–377. Hawksworth, G.M., Drasar, B.S., Hill, M.J., 1971. Intestinal bacteria and the hydrolysis of glycosidic bonds. J. Med. Microbiol. 4, 451–459. Herrera, P., Burghardt, R.C., Phillp, T.D., 2000. Adsorption of salmonella enteritidis by cytylpyridinimu-exchanged montmorillonite clays. Vet. Microbiol. 74, 259–272. Holdeman, L.V., Cato, E.P., Moore, W.E.C. (Eds.), 1977. Anaerobic Laboratory Mannual, fourth ed. Virginia Polytechnic Institute and State University, Blacksburg, pp. 51–70. Hu, X.R., Lu, G.L., Chen, L.S., Gu, J.M., Zhang, Y., 2002. Study on the mechanism of the interaction between montmorillonite and bacterium. Acta Pharmaceutica Sinica 37, 718–720 (in Chinese). Jin, J., Ho, Y.W., Abdullah, N., Jalaludin, S., 2000. Digestive and Bacterial enzyme activities in broilers fed diets supplemented with lactobacillus cultures. Poult. Sci. 79, 886–891. Lowry, O.H., Rosenbrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265–275. Mevissen-Verhage, E.A.E., Marcelis, J.H., de Vos, N.M., Verhoef, J., 1987. Bifidobacterium, Bacteroides and Clostridium spp. in faecal samples from breast-fed and bottle-fed infants with and without iron supplement. J. Clin. Microbiol. 25, 285–289. Munoa, F.J., Pares, R., 1988. Selective medium for the isolation and enumeration of Bifidobacterium spp. Appl. Environ. Microbiol. 54, 1715–1718. Nalini, N., Sabitha, K., Viswanathan, P., Menon, V.P., 1998. Influence of spices on the bacterial (enzyme) activity in experimental colon cancer. J. Ethnopharmacol. 62, 15–24. National Research Council, 1998. Nutrient Requirements of Swine, 10th ed. National Academy Press, Washington, DC. Ouhida, I., Perez, J.F., Piedrafita, J., Gasa, J., 2000. The effects of sepiolite in broiler chicken diets of high, medium and low viscosity. Productive performance and nutritive value. Anim. Feed Sci. Technol. 85, 183–194. Poulsen, H.D., Oksbjerg, N., 1995. Effects of dietary inclusion of a zeolite (clinoptilolite) on performance and protein metabolism of young growing pigs. Anim. Feed Sci. Technol. 53, 297–303. Rivera-Garza, M., Olguin, M.T., Alc´antara, D., Rodr´ıguez-Fuentes, G., 2000. Silver supported on natural Mexican zeolite as an antibacterial material. Micropor. Mesopor. Mater. 39, 431–444. SAS Institute Inc., 1989. SAS/STAT user’s guide, version 6. SAS Institute Inc., Cary, North Carolina. Schell, T.C., Lindemann, M.D., Kornegay, E.T., Blodgett, D.J., Doerr, J.A., 1993. Effectiveness of different types of clay for reducing the detrimental effects of aflatoxin-contaminated diets on performance and serum profiles of weanling pigs. J. Anim. Sci. 71, 1226–1231. Stadler, M., Schindler, P.W., 1993. Modeling of H+ and Cu2+ adsorption on calcium-montmorillonite. Clays Clay Miner. 41, 288–296. Tauqir, N.A., Nawaz, H., 2001. Performance and economics of broiler chicks fed on rations supplemented with different levels of sodium bentonite. Int. J. Agric. Biol. 3, 149–150. Venglovsky, J., Pacajova, Z., Sasakova, N., Vucemilo, M., 1999. Adsorption properties of natural zeolite and bentonite in pig slurry from the microbiological point of view. Veterinarni Medicine 44, 339–344.

M.S. Xia et al. / Animal Feed Science and Technology 118 (2005) 307–317

317

Wang, J.X., Fang, H.S., 1995. Intestinal microecological observation of smectite in the treatment of diarrhea in children. Chin. J. Clin. Pharmacol. 11, 134–137 (in Chinese). Wu, X.X., Gong, D.C., Jin, S.Z., 1999. Research of the curative effects of smectite rich in magnesium on diarrhea in piglet. J. Hubei Agric. Coll. 19, 137–139 (in Chinese). Xu, Z.R., Hu, C.H., Xia, M.S., Zhan, X.A., Wang, M.Q., 2003. Effects of dietary fructooligosaccharide on digestive enzyme activities, intestinal microflora and morphology of male broilers. Poult. Sci. 82, 648–654. Ye, Y., Zhou, Y.H., Xia, M.S., Hu, C.H., 2003. A new type of inorganic antibacterial material: Cu-bearing montmorillonite and discussion on its mechanism. J. Inorg. Mater. 18, 569–574 (in Chinese).