Aquaculture 270 (2007) 200 206 www.elsevier.

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Effects of Cu 2+ -exchanged montmorillonite on growth performance, microbial ecology and intestinal morphology of Nile tilapia (Oreochromis niloticus)
C.H. Hu a,, Y. Xu a , M.S. Xia b , L. Xiong a , Z.R. Xu a
a

College of Animal Science, Zhejiang University, The Key Laboratory of Molecular Animal Nutrition of Ministry of Education, Hangzhou, 310029, China b Department of Earth Science, Zhejiang University, Hangzhou, 310027, China Received 10 September 2006; received in revised form 19 January 2007; accepted 19 January 2007

Abstract A total of 360 Nile tilapia at an average initial body weight of 3.9 g were randomly allocated to 4 treatments, each of which had three replicates of 30 fish/tank and used to investigate the effects of Cu2+-exchanged montmorillonite (Cu-MMT) on growth performance, microbial ecology of the skin, gill and intestine, and intestinal morphology. The dietary treatments were: 1) basal diet, 2) basal diet + 1.5 g/kg MMT, 3) basal diet + 30 mg/kg copper as CuSO4 (equivalent to the copper in the Cu-MMT treatment group), or 4) basal diet + 1.5 g/kg Cu-MMT. The results showed that supplementation with Cu-MMT significantly improved growth performance as compared to control and fish fed with Cu-MMT had higher growth performance than those fed with MMT or CuSO4. Supplementation with Cu-MMT reduced (P b 0.05) the total intestinal aerobic bacterial counts and affected the composition of intestinal microflora with a tendency of Aeromonas, Vibrio, Pseudomonas, Flavobacterium, Acinetobacter, Alcaligence, Enterobacteriaceae decreasing as compared with control. Measurements of villus and microvillus heights at different sites of the intestinal mucosa indicated that dietary addition of MMT or Cu-MMT improved intestinal mucosal morphology. However, the microbial ecology of the skin and gill was not affected by the addition of CuSO4, MMT or Cu-MMT. Supplementation with CuSO4 had no (P N 0.05) effect on the growth performance, microbial ecology and intestinal morphology. The results showed that Cu-MMT exhibited antibacterial activity in vivo and protected intestinal mucosa from invasion of pathogenic bacterium and toxins, resulting in a positive effect on the growth performance. 2007 Elsevier B.V. All rights reserved.
Keywords: Cu2+-exchanged montmorillonite; Growth performance; Microbial ecology; Intestinal morphology; Nile tilapia

1. Introduction Bacterial disease is a major problem encountered in the rapidly developing fish farming industry. Although
Corresponding author. Feed Science Institute, Animal Science College, Zhejiang University, Hangzhou, 310029, PR China. Tel./fax: +86 571 86985607. E-mail address: chhu@zju.edu.cn (C.H. Hu). 0044-8486/$ - see front matter 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2007.01.027

vaccines are being developed and marketed, they cannot be used alone as a universal disease control measure in aquaculture. Treatment with antibiotics continues to be important disease control measures in the aquaculture industry. However, abuse or overuse of antibiotics causes various side-effects and also results in the emergence and increase of bacteria resistant to antibiotics. Therefore, it is necessary to develop new types of antibacterial agents that can replace antibiotics.

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The metal-carrying inorganic antibacterial agents, being carried by non-metallic minerals, have attracted attention by durable antibacterial properties, nonresistance, and safety. Montmorillonite (MMT) is a sort of aluminum silicate with 2:1 layer structure of tetrahedral and octahedral layers. Between the structural sheets, there are exchangeable cations readily being replaced by other cations or compounds. Taking into account that MMT has specific physicalchemical properties such as high surface area, strong absorptive power, high structural stability, chemical inertia and strong capacity to form stable suspensions, MMT may be suitable as an antimicrobial carrier. Our recent experiments revealed that MMT had no antibacterial activity, while Cu2+-exchanged montmorillonite (CuMMT) has strong antibacterial ability on Aeromonas hydrophila, Pseudomonas fluorescens, Vibrio parahaemolyticus and Escherichia coli K88 (Hu and Xia, 2005; Hu et al., 2006). There are no data of the effects of Cu-MMT on fish in vivo. In the present study, the effects of Cu-MMT on growth performance, microbial ecology of the skin, gill and intestine, and intestinal morphology of Nile tilapia (Oreochromis niloticus) were investigated. 2. Materials and methods 2.1. Materials MMT is a hydrothermal product of volcano sedimentary rocks from the Inner Mongolia Autonomous Region, China. Besides MMT, there were minor amounts of quartz and volcanic glass in the ore. The raw material was dried in oven over night at 80 C and then milled to less than 300 mesh. The milled material was dispersed in water to form a 10% suspension and kept for about 10 min with stirring. Particles larger than 1 m were separated out by sedimentation while the suspension was centrifuged to get refined MMT. The refined MMT was dried at 80 C followed by another milling to less than 300 mesh for use. The formula of the purified MMT was [Na 0.158 K 0.082 Ca 0.256 Mg 0.063 ] [Mg 0.376 Fe 2+ 0.014 Fe 3+ 0.136 Al 1.474 ][Si 3.87 Al 0.13 ]O 10 (OH)2nH2O. Cu-MMT was prepared by Cu2+-exchanged reaction. Ten grams of the refined MMT were mixed with 100 ml of 0.2 mol/L NaCl solution. The dispersion was kept for 5 h with stirring. The Na-MMT were then separated by centrifugation at a speed of 8000 g for about 15 min and washed with deionized water for three times. The washed Na-MMT was then dispersed in 200 ml of 0.05 mol/L CuSO4 solution and pH value was adjusted

to 5.0. The dispension was kept at 60 C with stirring for 6 h. After centrifugation, the sediment was washed with deionized water for three times, dried at 80 C over night, then ground to a size less than 300 mesh. The copper concentration in Cu-MMT was found to be 2.0% measured by atomic absorption spectroscopy. 2.2. Experimental design and fish rearing Nile tilapia (O. niloticus) fingerlings were provided by WuYI fishery breeding field and acclimated to laboratory conditions for 2 weeks, fed with a conventional diet, after which time a total of 360 Nile tilapia at an average initial body weight of 3.9 g were randomly allocated to four dietary treatments for 56 days, each of which had three replicates of 30 fish/tank. The dietary treatments were: 1) basal diet, 2) basal diet +1.5 g/kg MMT, 3) basal diet + 30 mg/kg copper as CuSO4 (equivalent to the copper in the Cu-MMT treatment group), or 4) basal diet +1.5 g/kg Cu-MMT. Diets were formulated to meet nutrient requirements suggested by the NRC (1993) for Nile tilapia. No antibiotic was included in diets (Table 1). The experimental system was a closed recirculation system consisting of 12 self-cleaning aquarium (50 cm wide 100 cm long 50 cm high), sedimentation tanks, a biological filter and a UV filter to prevent crosscontamination of micro-organisms between treatments. The system was installed in an environment-controlled laboratory maintained at 25 C, with a photoperiod of 12 h light and 12 h darkness. The culture system was also provided with continuous aeration through an air compressor and heaters to keep water temperature at

Table 1 Ingredients and nutrient composition of diets Ingredients Fish meal Soybean meal Rapeseed meal Wheat middlings Corn oil Vitamin premix a Mineral premix b Carboxymethyl cellulose
a

% 10 30 25 26 2 1 5 1

Nutrition composition Gross energy (kJ/g) Crude protein Crude lipid Fibre Ash

% 18.1 35.2 2.5 6.8 11.1

Vitamin premix (mg/ kg of diet): thiamine, 10; riboflavin, 20; pyridoxine, 10; cobalamin, 2; retinal, 4; cholecalciferol, 0.4; phylloquinone, 80; folic acid, 5; Calcium pantotheniate, 40; inositol, 400; niacin, 150; tocopherol, 60; choline, 6000; ascorbic acid, 500. b Mineral premix (g/kg of diet): NaCl, 0.25; MgSO4, 3.75; KH2PO4, 8; Ca(H2PO4)2, 5; FeSO4, 0.72; (CH2CHCOO)2Ca.5H20, 0.88; ZnSO4.7H2O, 0.088; MnSO4.4H2O, 0.040; CuSO4.5H2O, 0.008; CoCl2.6H2O, 0.00025; KIO3.6H2O, 0.00075.

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Table 2 Effect of Cu-MMT on growth performance of Nile tilapia1 Control BWi5, g BW6, g f SGR7, % PER8 FCR9 SR10, %
ab

CuSO4 3.94 28.60b 3.54 b 1.46b 1.35a 95.56ab

MMT2 3.86 29.65ab 3.64b 1.56ab 1.28ab 97.78ab

Cu-MMT3 3.82 34.70a 3.94a 1.71a 1.20b 98.89a

SEM4 0.11 1.75 0.05 0.08 0.05 1.12

4.08 29.46ab 3.53 b 1.41b 1.38a 94.44b

Means within a row with different letters differ significantly (P b 0.05) when tested with Duncan's new multiple-range test following analysis of variance. l Data represent mean values of three replicate tanks of 30 fish each. 2 MMT = montmorillonite; 3Cu-MMT = Cu2+-exchanged montmorillonite; 4Standard error of the mean; 5BWi = Initial body weight; 6BWf = Final Body weight; 7SGR = Specific growth rate; 8PER = Protein efficiency ratio; 9FCR = feed conversion ratio; 10SR = percent survival.

placed on the bottom of the Petri dish and then 20 ml of molten agar was poured onto and mixed with the inoculum. Total aerobic counts were determined on nutrient agar after incubation at 28 C for 4872 h. Thirty colonies were randomly picked (to pick many different phenotypes) from every sample and restreaked on nutrient agar three times to obtain pure cultures. Isolated micro-organisms were identified by using a scheme based on Bergey's Manual (Holt et al., 1993). The following tests were conducted: Gram reaction by the Hucker method; cell morphology by phase-contrast microscopy (Gerhardt, 1981); flagella type by the Nishizawa and Sugawara method (Aoyama, 1976); catalase formation by dropping a 3% H2O2 solution directly onto each colony; oxidase test by the Kovac method; and O/F-test by the Hugh and Leifson method (Harrigan, 1998). 2.4. Intestinal mucosal morphology Light microscopy: Segments were removed from the proximal, mid and distal intestine and flushed with physiological saline and fixed in 10% formalin. Three cross-sections for each intestinal sample (total of 9 samples for each of the three intestinal segments per dietary treatment) were then prepared after staining with hematoxylin and eosin using standard paraffin embedding procedures. A total of 10 intact villus were selected in triplicate for each intestinal cross-section (30 measurements for each sample, total of 270 measurements for each of the three intestinal segments per dietary treatment). Villus height were determined using image processing and analysis system (Version 1, Leica Imaging Systems Ltd, Cambridge, England).

28 C. For water quality control, temperature and dissolved oxygen were measured daily, and weekly analyses were done of total ammonium, nitrite, nitrate and pH levels. The following values (S.D.), appropriate for tilapia cultivation, were used: temperature, 28.7 0.5 C; dissolved oxygen, 6.3 1.5 mg/l; pH, 8.02 0.18; ammonia, 0.05 0.01 mg/l; nitrite, 0.04 0.02 mg/l; nitrate, 6.15 1.12 mg/l. The fish were fed at a rate of 4% (wet weight basis) of their total biomass per day in 2 portion. The daily ration was divided into two equal feedings and fed at 9:00 and 15:00. Fish were weighed biweekly and the daily ration was adjusted accordingly. Growth performance indicators measured were specific growth rate (SGR), feed conversion ratio (FCR), protein efficiency ratio (PER) and percent survival. These indicators were calculated as: SGR = 100(ln(Final Body weight) ln(Initial body weight)) / time (days); FCR = dry feed intake (g) / wet weight gain (g); PER = wet weight gain (g) / protein intake (g); percent survival (%) = Final fish number / initial fish number 100. 2.3. Isolation and identification of tilapia bacteria At the 56th day of the feeding trial, 9 fish per treatment (3 fish per tank) were anesthetized with tricaine methanesulfonate (MS-222, Sigma, St. Louis, MO) and then killed by cervical separation. Total counts of aerobic bacteria were determined from tilapia skin by placing a 4 cm 4 cm sterile template on the fish surface (in the central region) and swabbing the area enclosed by the template. Under aseptic conditions, samples of gill and intestine were also taken by sterile knife. The skin, gill and intestine samples were homogenized with sterilized 0.85% NaCl saline solution at room temperature. Poured plates were prepared and that a 1.0 ml inoculum was

Table 3 Effect of Cu-MMT on total aerobic bacterial counts of the skin, gill and intestinel Skin log10CFU per cm2 Control CuSO4 MMT2 Cu-MMT3 SEM4
ab

Gill log10CFU per gram 5.24 5.17 4.78 4.65 0.25

Intestine log10CFU per gram 6.47a 6.24a 5.94ab 5.25b 0.30

3.94 3.98 3.78 3.80 0.21

Means within a line with different letters differ significantly (P b 0.05) when tested with Duncan's new multiple-range test following analysis of variance. l Data represent mean values of three replicate tanks of three fish per replicate. 2 MMT = montmorillonite; 3Cu-MMT = Cu2+-exchanged montmorillonite; 4Standard error of the mean.

C.H. Hu et al. / Aquaculture 270 (2007) 200206 Table 4 Effect of Cu-MMT on the composition of microflora from the skin l Aer. Control CuSO4 MMT2 Cu-MMT3 SEM4 36.1 31.2 32.5 33.1 2.1 Pse. 5.2 4.2 3.3 3.8 0.8 Fla. 30.9 30.2 30.1 30.1 1.5
l

203

Mor. 6.2 5.1 6.5 6.4 0.9

Aci. 9.4 12.1 10.8 9.2 1.0

Ent. 5.3 6.8 6.2 4.8 0.7

Mic. 6.1 8.5 9.2 10.5 1.5

Oth. 0.8 1.9 1.4 2.1 0.5

Effect of Cu-MMT on the composition of microflora from the gill Aer. Control CuSO4 MMT2 Cu-MMT3 SEM4 11.6 11.1 10.9 11.3 0.8 Pse. 11.7 11.2 10.8 10.1 0.8 Fla. 34.2 31.4 32.5 34.1 1.4 Mor. 11.2 10.9 10.6 9.7 0.7
l

Aci. 9.7 9.9 9.4 9.4 0.6

Ent. 4.4 5.1 5.2 4.2 0.4

Alc 6.6 5.9 6.5 6.2 0.4

Bac. 5.6 8.1 8.9 8.5 1.2

Cor. 3.5 5.2 4.5 5.5 0.7

Oth. 1.5 1.2 0.7 1.0 0.3

Effect of Cu-MMT on the composition of intestinal microflora Aer. Control CuSO4 MMT2 Cu-MMT3 SEM4
ac

Pse.
a

Fla.
a

Vib. 5.3 4.9ab 3.8b 4.1b 0.4

a

Aci. 4.1 3.9a 2.6b 1.6b 0.4

a

Ent. 18.6 17.9ab 15.7ab 15.0b 1.1

a

Alc. 6.6 6.2a 3.6b 3.3b 0.6

a

Bac. 1.5 3.4b 8.8a 10.4a 0.8

b

Cor. 1.2 1.8c 7.2b 10.8a 1.0

c

Mic 2.5 3.3b 5.3a 5.7a 0.5

b

Oth. 1.5 2.5 2.6 2.8 0.5

39.2 37.3ab 36.8ab 33.5b 1.7

10.4 9.9a 8.5ab 7.2 b 0.8

9.1 8.9a 5.1b 5.6b 0.7

Means within a line with different letters differ significantly (P b 0.05) when tested with Duncan's new multiple-range test following analysis of variance. l Data represent mean values of three replicate tanks of three fish per replicate. The units are %. Aeromonas, Aer; Pseudomonas, Pse.; Flavobacterium, Fla.; Acinetobacter, Aci.; Alcaligence, Alc.; Enterobacteriaceae, Ent.; Vibrio, Vib.; Bacillus, Bac.; Corynebacterium, Cor.; Moraxella, Mor.; Micrococcus, Mic.; Other, Oth. 2 MMT = montmorillonite; 3Cu-MMT = Cu2+-exchanged montmorillonite; 4Standard error of the mean.

Transmission electron microscopy (TEM): Specimens of the proximal, mid and distal intestine were collected from the same sites as those used for tissues examined with Light microscopy. Samples were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.0). The fixed samples were washed in the buffer, dehydrated with graded ethanol and embedded in Spurr's resin. Three ultrathin sections for each intestinal sample (total

of 9 samples for each of the 3 intestinal segments per dietary treatment) were cut and stained with 2% uranyl acetate, post-stained with 0.2% lead citrate and examined in a Jeol JEM-1200 TEM at 60 kV. Mid-villus regions were identified under low magnification. After mid-villus regions were located, micrographs of the microvillus border were taken at 20,000 magnification. A total of 10 well-oriented longitudinal microvilli were

Table 5 Effect of Cu-MMT on villus and microvillus height at different sites of the intestine of Nile tilapia1 Site Villus height (m) Proximal intestine Mid-intestine Distal intestine Microvillus height (m) Proximal intestine Mid-intestine Distal intestine
ac

Control 350.29b 637.04c 361.37c 7.24b 7.89c 3.56bc

CuSO4 372.55b 664.57bc 379.48bc 7.15b 8.01c 3.40c

MMT2 398.11ab 718.42ab 411.13ab 8.05ab 9.06ab 4.28a

Cu-MMT3 435.15a 758.68a 430.81a 8.53a 9.84a 4.49a

SEM4 17.40 27.02 16.04 0.36 0.30 0.22

Means within a line with different letters differ significantly (P b 0.05) when tested with Duncan's new multiple-range test following analysis of variance. l Data represent mean values of three replicate tanks of three fish per replicate. 2 MMT = montmorillonite; 3Cu-MMT = Cu2+-exchanged montmorillonite; 4Standard error of the mean.

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assessed for each intestinal section (30 measurements for each sample, total of 270 measurements for each of the 3 intestinal segments per dietary treatment). 2.5. Statistical analysis Data were analyzed as a randomized complete block using the general linear model procedure (SAS Institute Inc., 1989., Cary, NC). A tank of fish 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 b 0.05. 3. Results 3.1. Growth performance The growth performance of Nile tilapia is presented in Table 2. As compared to control, supplementation with Cu-MMT improved (P b 0.05) specific growth rate, protein efficiency ratio, survival and decreased (P b 0.05) feed conversion ratio. While fish fed with MMT had slightly greater growth performance than the control, the tendency was not significant. Supplementation with CuSO4 had no (P N 0.05) effect on growth performance. 3.2. Microbial ecology of the skin, gill and intestine 3.2.1. Total aerobic bacterial counts of the skin, gill and intestine The total aerobic bacterial counts of the skin, gill and intestine of Nile tilapia is presented in Table 3. Supplementation with Cu-MMT reduced (P b 0.05) the total intestinal aerobic bacterial counts as compared with the control. While supplementation with MMT or CuSO4 had no influence (P N 0.05) on intestinal bacterial counts. The total aerobic bacterial counts of the skin and gill was not affected by the addition of CuSO4, MMT or Cu-MMT. 3.2.2. The composition of microflora The composition of microflora of the skin, gill and intestine is presented in Table 4. The results indicated that the dominant organisms were found to be Aeromonas and Flavobacterium in skin, Flavobacterium in gill, and Aeromonas and Enterobacteriaceae in intestine. Aeromonas constituted the largest group in both skin and intestine, and was followed by Flavobacterium and Enterobacteriaceae in skin and intestine, respectively. Meanwhile Flavobacterium constituted the largest group in gill. The composition of microflora of the skin and gill was not affected by the addition of CuSO4, MMT or Cu-MMT. Supplementation with

MMT or Cu-MMT affected the composition of intestinal microflora with Aeromonas, Vibrio, Pseudomonas, Flavobacterium, Acinetobacter, Alcaligence, Enterobacteriaceae decreasing and Bacillus, Corynebacterium, Micrococcus increasing as compared with control. 3.3. Intestinal mucosal morphology Morphological measurements of the intestinal mucosa of Nile tilapia are presented in Table 5. Supplementation with Cu-MMT had higher (P b 0.05) villus and microvillus height at the proximal, mid and distal intestine mucosa as compared with the control or CuSO4. Supplementation with MMT also increased (P b 0.05) villus and microvillus height at the mid and distal intestine mucosa as compared with the control. Supplementation with CuSO4 had no (P N 0.05) effect on the intestinal morphology as compared with control. 4. Discussion Traditionally, MMT has been incorporated in animal diets as a technological additive (lubricant or agglomerant) to improve feed manufacture. However, recently a role as enhancer of the nutritive value of diets in animals has been also proposed. Animal feed containing MMT (1030 g/kg) has been shown to promote growth performance in chickens and swine (Tauqir and Nawaz, 2001; Venglovsky et al., 1999). In the present study, while tilapia fed with MMT had slightly greater growth performance than the control, the tendency was not significant. The reason of the lack of effects of MMT supplementation on tilapia growth performance in this study may be because the concentration of MMT (1.5 g/kg) was not adequate. It is well known that MMT can adhere to bacteria. Adsorption was the main interaction between MMT and bacteria. Copper sulfate is one of the traditional inorganic antibacterial materials with wide usage. However, Cu2+ is difficult to contact with bacterium in water by colliding so that the usage amount must be large for the purpose of antimicrobial. In order to increase the antibacterial activity of Cu, we consider to carry the Cu2+ in/on MMT with large specific surface area, which can adsorb bacterium rapidly after dispersed in water. Copper tended to enter interlayer position of MMT dominantly as [Cu(AlO)n(H2O)4n]x+ or locate in the ditrigonal intra-crystal hole surrounded by SiO tetrahedron, or take a position in AlO octahedron in MMT (Mosser et al., 1997). This made the mineral have surplus positive charge. On the other hand, the cellular wall of the bacteria was negatively charged, so that Cu-

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MMT particles would attract bacteria, due to the opposite static charge. The Zeta potentials of MMT, Cu-MMT, A. hydrophila, V. parahaemolyticus and P. fluorescens at pH 6.0 are 21 mV, 11.8 mV, 18 mV, 16 mV and 22 mV, respectively. The results of in vitro bacterialmineral adsorptive trial showed that the percent of bacteria adsorbed onto MMT and Cu-MMT was 37.446.5% and 94.499.4%, respectively. The antimicrobial trial in vitro indicated that MMT had no antibacterial activity while Cu-MMT has strong antibacterial ability on the tested aquacultural pathogenic bacteria (Hu and Xia, 2005). Surplus positive charge of Cu-MMT was most probably an important factor for their antibacterial capability (Hu et al., 2005a; Hu and Xia, 2006). In this case, the released Cu2+ would act directly on the bacteria adsorbed on the surface of CuMMT, 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. The high concentration of Cu2+ will act directly on the attracted bacteria. Summary, two possible mechanisms for the antibacterial activity of Cu-MMT were proposed. One involved the adsorption of the bacteria from solution and immobilization on the surface of the Cu-MMT. Alternatively, Cu2+ could disassociate from the clay surface and directly exerts its antibacterial effect on the bacteria (Hu et al., 2005a, 2006). The present study showed that supplementation with Cu-MMT not only exhibited antibacterial activity in vivo but also affected the composition of intestinal microflora with Aeromonas, Vibrio, Pseudomonas, Flavobacterium, Acinetobacter, Alcaligence, Enterobacteriaceae decreasing while Bacillus, Corynebacterium, Micrococcus increasing as compared with control. Wang and Fang (1995) also reported that the intestinal bacteria counts of Bifidobacteria were significantly increased while those of E. coli were decreased after diarrhoeal children had been treated with MMT for 5 days. The changes of the composition of intestinal microflora caused by Cu-MMT may be related to the characteristics of MMT. It was reported that MMT adsorbed bacteria selectively and only adhere to the pathogenic E. coli strains producing surface antigen designated CS31A (Girardeau, 1987). Hu et al. (2006) used Caco-2 cell line to investigate the adhesion of probiotic or pathogenic strains to intestina epithelial cells and the effects of MMT on the bacterial adhesion. The results showed that the ability of MMT to inhibit adhesion of pathogenic strains to Caco-2 cells was higher than that of probiotic strains. The percentages of MMT against adhesion of E. coli, Salmonella typhimur-

ium, A. hydrophila, V. parahaemolyticus to Caco-2 cell were 54.22%, 48.41%, 60.53% and 50.64%, respectively, while against probiotic strains 21.4925.64%. The structure of the intestinal mucosa can reveal some information on gut health. Stressors that are present in the digesta can lead to relatively quickly changes in the intestinal mucosa due to the close proximity of the mucosal surface and the intestinal content. The addition of MMT and Cu-MMT to the diet in the present study both produced a positive effect on the intestinal mucosa. Hu et al. (2005b) studied the effects of Cu-MMT on A. hydrophila adhesion to epithelial cells of Nile tilapia. The results showed that Cu-MMT showed a strong ability to inhibition adhesion of A. hydrophila to fish skin, gill and intestinal epithelial cells and significantly reduced cell membrane injury caused by the adhesion of A. hydrophila. It is well known that MMT is a protector of intestinal mucosa and can reinforce intestinal mucosal barrier and help in the regeneration of the epithelium. MMT effectively improves gastrointestinal mucus resistance to various aggressions by interacting closely with the mucous glycoproteins (Droy-Lefain et al., 1985). The improved intestinal mucosal morphology caused by CuMMT may be explained by the antibacterial activity of Cu-MMT and the MMT enhancing mucosa function. Since copper is an essential element for all organisms including fish, it is often supplemented to commercial feeds in concentrations which may reach the upper limit of toxicity. Supplementation of Cu to fish feed is therefore a balance between fulfilling the Cu requirement and avoiding Cu toxicity. The requirement and the maximal tolerable levels of dietary copper vary widely for different studies on fish. Murai et al. (1981) found reduced growth and feed conversion in channel catfish (Ictalurus punctatus) fingerlings exposed to 16 mg Cu kg 1 diet for 16 weeks. In contrast, Galtin and Wilson (1986) found no growth inhibition in channel catfish fingerlings exposed to 40 mg Cu kg 1 diet for 13 weeks. For rainbow trout (Oncorhynchus mykiss), growth suppression was not apparent at dietary Cu concentrations up to 350684 mg Cu kg 1 diet (Miller et al., 1993). In Atlantic salmon (Salmo salar L.) parr, based on the efficiency of Cu uptake from the feed, the recommended upper limit for Cu supplementation could be set at 35 mg Cu kg 1. However, based on the reduced growth it seemed possible to permit up to 500 mg Cu kg 1 (Berntssen et al., 1999). Shiau and Ning (2003) found that weight gain of juvenile tilapia was highest in fish given diets supplemented with 2 mg Cu per kg diet, followed by the group given 1 mg Cu per kg diet, then the unsupplemented control group, and was lowest in the 20 mg Cu per kg diet group. They estimated the

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adequate dietary Cu concentration in growing tilapia was about 4 mg Cu per kg diet. In the present study supplementation with 30 mg Cu kg 1 diet had no significant effect on the growth performance of Nile tilapia. Differences in the duration of the experimental period, husbandry, and life stage of the fish make it difficult to compare these studies. Hu et al. (2005a) investigated the Cu release from Cu-MMT in Tryptic soy broth and found the saturated Cu concentration in the supernatant reached only 1.222.27% of the overall Cu in Cu-MMT, suggesting the Cu nutritive value of Cu-MMT was little. Acknowledgements The authors gratefully acknowledge H. X. Sun, H. M. Zhang, J. M. Pan, and X. Y. Zhu for their skilled technical assistance. References
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