Effects of Modified Montmorillonite Nanocomposite on Growing / Finishing Pigs during Aflatoxicosis

Experiments were conducted to determine the efficacy of modified montmorillonite nanocomposite (MMN) to reduce the toxic effects of aflatoxin (AF) in growing/finishing pigs. 96 weaned pigs were assigned to four dietary treatment groups (0 g of MMN and 0 mg of AF/kg feed, 3 g of MMN/kg feed, 0.1 mg of AF/kg feed, and 3 g of MMN plus 0.1 mg of AF/kg feed). Body weight gain (BW gain), feed/gain ratio, serum biochemical values and enzyme activities were evaluated. Compared with the control, AF alone markedly reduced BW gain and resulted in a significantly higher feed/gain ratio. There were no differences in BW gain and feed/gain ratio between 0.3% MMN or 0.3% MMN plus AF and the control. These results suggested that the deleterious effects of AF were ameliorated by MMN addition. AF intake markedly increased relative organ weights of liver, kidney, spleen and pancreas, and resulted in significant alterations of serum parameters. However, these parameters for pigs fed diets containing MMN and AF returned to normal values, indicating that MMN had the ability to recover the AF-decreased performance, organ damage and to correct aberrations in serum parameters. These findings in our study suggested that MMN can effectively modulate the toxicity of AF in growing/finishing pigs and may offer a novel approach to the preventive management of aflatoxicosis in animals. (Asian-Aust. J. Anim. Sci. 2005. Vol 18, No. 9 :


INTRODUCTION
The mycotoxin, aflatoxin (AF) B 1 , G 1 , B 2 and G 2 , are highly toxic, mutagenic, teratogenic and carcinogenic compounds produced by species of A. flavus and A. parasiticus (Smela et al., 2001;Mishra et al., 2003).Liver damage, gastrointestinal dysfunction, lower reproductivity, reduced feed utilization and efficiency, anemia, and jaundice are toxic manifestations linked to aflatoxicosis in various species of animals (Huff et al., 1986;Harvey et al., 1988;Robens et al., 1992).Pigs are highly sensitive to the effects of AF, and exposure to AF-contaminated feed seriously affects the economy of the swine industry.Therefore, practical and cost-effective methods to detoxify AF-containing feedstuffs are in great demand.One of the more encouraging approaches is the addition of nonnutritive sorptive materials to feedstuffs with the consequent reduction of the gastrointestinal absorption of these fungal metabolites.Studies showed that dietary addition of montmorillonite, bentonite and hydrated sodium calcium aluminosilicate (HSCAS) can reduce detrimental effects on farm animals of several mycotoxins like AF and T-2 toxin (Bonna et al., 1991;Harvey et al., 1991Harvey et al., , 1994;;Kubena et al., 1990Kubena et al., , 1991Kubena et al., , 1993;;Abdel-Wahhab et al., 1999).But natural montmorillonite is always congregated with some impurities, which make their adsorptive effect difficult to exert and make their addition level high.
Modified montmorillonite nanocomposite (MMN) is a new sorptive additive developed by our institute.
We made preparation to natural montmorillonite via separation, purification, depolymerization and dispersion and got nanoparticles which size was 10-60 nm and purity was above 98%, and then made modification to these nanoparticles by pillar composition, intercalation composition and spot polymerization (Vaia et al., 1997a, b;Ishida et al., 2000), finally constructed a new inorganic nanocomposite (MMN).
Because MMN possess sizable surface areas, high porosity, and variable cation exchange activities along with active sites, it can interact with and immobilize AF molecules via macro quantum tunnel effect or weak electrostatic forces or through the formation of strong bonds (Dale et al., 1991), then reduce gastrointestinal absorption of toxins and ameliorate adverse effects of AF on animals.
The objective of this experiment was to evaluate the efficacy of MMN to reduce bioavailability and to diminish the toxic effects of AF in pigs.

Materials
AF was produced via fermentation of milled corn by A. parasiticus NRRL 2999.The sterile substrate was inoculated with 100 ml of the mold aqueous suspension containing 10 6 spores -1 ml, cultures were allowed to grow for 15 d at 28°C in darkness.On the fifteenth day, culture materials were autoclaved and dried.The AF content was measured by HPLC according to AOAC (1995).The AF

ABSTRACT :
Experiments were conducted to determine the efficacy of modified montmorillonite nanocomposite (MMN) to reduce the toxic effects of aflatoxin (AF) in growing/finishing pigs.96 weaned pigs were assigned to four dietary treatment groups (0 g of MMN and 0 mg of AF/kg feed, 3 g of MMN/kg feed, 0.1 mg of AF/kg feed, and 3 g of MMN plus 0.1 mg of AF/kg feed).Body weight gain (BW gain), feed/gain ratio, serum biochemical values and enzyme activities were evaluated.Compared with the control, AF alone markedly reduced BW gain and resulted in a significantly higher feed/gain ratio.There were no differences in BW gain and feed/gain ratio between 0.3% MMN or 0.3% MMN plus AF and the control.These results suggested that the deleterious effects of AF were ameliorated by MMN addition.AF intake markedly increased relative organ weights of liver, kidney, spleen and pancreas, and resulted in significant alterations of serum parameters.However, these parameters for pigs fed diets containing MMN and AF returned to normal values, indicating that MMN had the ability to recover the AF-decreased performance, organ damage and to correct aberrations in serum parameters.These findings in our study suggested that MMN can effectively modulate the toxicity of AF in growing/finishing pigs and may offer a novel approach to the preventive management of aflatoxicosis in animals.(Asian-Aust.J. Anim.Sci.2005.Vol 18, No. 9 : 1305-1309) content in the milled corn was 84% AFB 1 , 8% AFG 1 , 6% AFB 2 and 2% AFG 2 .The milled corn was incorporated into the basal diet to provide the desired level of 0.1 mg AFB 1 /kg of diet.MMN was obtained from Feed Science Institute, Zhejiang University, Hangzhou, China.

Animals and diets
A total of 96 crossbred (Yorkshire×Landrace× Hampshire) weaned barrows and gilts (mean body weight, 29.8±1.32 kg) were identified by individual ear tags and tattoos, and randomly assigned to four treatment groups by sex (three replicates per treatment with eight pigs per replicate), housed in concrete-floored indoor pens, and provided with corn/soybean diets and water ad libitum.The diets either met or exceeded critical nutrient concentrations, as recommended by the National Research Council (1998).Four dietary treatments of 0 g of MMN and 0 mg of AF/kg feed (control), 3 g of MMN/kg feed (0.3% MMN), 0.1 mg of AF/kg feed, and 3 g of MMN plus 0.1 mg of AF/kg feed were fed to treatment groups for 90 days (Table 1).
Pigs were weighed and group feed consumption were recorded weekly.Blood samples were taken via jugular venipuncture for serum biochemical and enzymatic measurements at the end of the trial.Serum analysis for glutamic-pyruvic transaminase (GPT), glutamic-oxalacetic transaminase (GOT), alkaline phosphatase (ALP), lactic dehydrogenase (LDH) activities and for total protein, albumin (ALB), globulin (GLOB), urea nitrogen, cholesterol, triglycerides were accomplished by described methods using an biochemical autoanalyzer (Beckman Instruments, Inc.).

Histopathology
At the end of the trial, pigs were euthanatized and necropsied.Liver, kidney, spleen and pancreas were weighed, and organ weight was calculated as a percentage of body weight (g/100 g).Liver and kidney Specimens were fixed in 10% neutral buffered formalin.Fixed tissues were trimmed, embedded in paraffin, and stained with hematoxylin and eosin for histopathological examination.

Statistical analysis
All data were subjected to statistical analysis using the General Linear Models Procedure of the Statistical Analysis System (SAS Institute, 1985).The significance of the differences among treatment groups with variable means was determined by Duncan's new multiple range test.All statements of significance were based on probability p≤ 0.05.

RESULTS
Pigs of the control group, 0.3% MMN, and 0.3% MMN plus AF treatment groups did not have clinical signs of disease or abnormal behavior, whereas AF-treated pigs were listless, had rough coat, and were noticeably smaller than pigs of the other groups.The effects of MMN and AF diets on growth performance were presented in Table 2.The results showed that 0.3% MMN in the diet significantly diminished the inhibitory effects of feeding 0.1 mg AF/kg diet.Mortality did not occur.Compared with the control group, a significant decrease in BW gain was observed in pigs fed AF alone (p<0.05), but no differences in BW gain were noted in other diets.The feed/gain ratio was significantly increased by AF intake (p<0.05), the other treatments did not show significant differences.
The effects of dietary treatments on serum parameters  4 Supplied per kg of diet: 180 mg of Zn; 150 mg of Cu; 50 mg of Mn; 0.3 mg of I; 0.3 mg of Se; 0.3 mg of Co; 6,500 IU of vitamin A; 750 IU of vitamin D3; 20 IU of vitamin E; 3.5 mg of vitamin K3; 2.8 mg of vitamin B1; 6.2 mg of vitamin B2; 33 mg of niacin; 18 mg of dpantothenic acid; 3.5 mg of vitamin B6; 0.85 mg of folic acid; 60.0 µg of biotin; 35 µg of vitamin B12; and 600 mg choline chloride.Premix also provided 80 mg of chlortetracycline and 1,000 mg L-lysine HCL (purity, 98%) per kg of diet.were shown in Table 3 and 4. Toxicity of AF expressed through significant changes in serum biochemical values and enzymatic activities.When compared to the control, Levels of serum total protein, ALB, GLOB, urea nitrogen, cholesterol and triglycerides were markedly decreased in pigs fed AF alone (p<0.05),whereas GPT, GOT, ALP and LDH activities were significantly increased in pigs fed AF alone (p<0.05).But there were no significant differences in these parameters between 0.3% MMN or 0.3% MMN plus AF and the control.This demonstrated that addition of 0.3% MMN made the levels of these parameters return to the normal.
Table 5 showed the effects of MMN and AF on relative organ weights.Compared with the control, the diet containing AF alone significantly increased relative liver, kidney, spleen and pancreas weights (p<0.05).However, addition of 0.3% MMN to the AF-contaminated diet prevented an increase in weight of these organs.The significant increases in relative organ weights were prevent by the MMN addition, that is, there was no significant difference between 0.3% MMN plus AF and the control.Microscopically, hepatic lesions in pigs of the AF-alone treatment group were characterized as severe diffuse cytoplasmatic vacuolization accompanied by centrilobular and peripheral-lobular lipidosis and bile duct hyperplasia (Figure 1A).When AF fed to pigs, renal damage including localized degeneration and vacuolization in cytoplasm of convoluted tubular epithelia were observed (Figure 2A).Microscopic lesions of the liver and kidney were not observed in pigs of the control, MMN, or MMN plus AF treatment groups (Figure 1B, 2B).

DISCUSSION
AF is very harmful to the swine industry because of its toxicity and occurrence in feedstuffs.In our study, aflatoxicosis was induced by a diet of 0.1 mg of AF/kg feed.The toxic effects of AF were expressed as reduced BW gain, higher feed/gain ratio, increased relative organ weights and serum biochemical alterations.These results agreed with    previous reports by Harvey et al. (1989aHarvey et al. ( , 1990)).No significant difference was found between pigs fed the control diet and fed the diet containing MMN alone, indicating that the adsorbent was inert and nontoxic.The better growth performance of pigs fed the diet of 3 g of MMN plus 0.1 mg of AF/kg feed demonstrated that the ability of MMN to ameliorate the inhibitory effects produced by AF.AF has been reported to cause inhibition of protein synthesis (Tung et al., 1975;Yu, 1977).As a result, hypoproteinemia is a common effect of aflatoxicosis.Reduced levels of serum total protein, ALB, and GLOB are indictors of aflatoxicosis.Serum biochemical values and enzymatic activities of GPT, GOT, ALP and LDH are sensitive serological indicators of liver and kidney toxicity.These parameters significantly altered in our study, suggesting that AF caused critical injury to these organs.Serum profiles in the trial were similar to those obtained by Harvey et al. (1989b), who reported that AF reduced indicators of protein synthesis such as serum total protein, ALB, GLOB and urea nitrogen.The high levels of serum enzymes found in the study were consistent with the results of Harvey et al. (1990) and Lindemann et al. (1993), who reported high levels of AST, GGT and ALP in the serum of pigs fed AF-contaminated diets.However, these parameters for pigs fed diets containing MMN and AF returned to normal values, showing beneficial effects of MMN on aflatoxicosis.The results were in agreement with the study by Kececi et al. (1998) who reported that some serum biochemical changes could be ameliorated by bentonite administration to the diet at doses of 5 mg AF/kg diet.Schell et al. (1993a, b) also reported that sodium bentonite or HSCA were effective in reducing the high levels of some serum enzymes.
The relative weights of liver, kidney, spleen and pancreas were significantly increased in our study.AF has been known to irritate gastrointestinal tract and gut and make them dysfunction, thus causing an increase in the relative weights of these organs, the target organs were liver and kidney (Huff et al., 1981).However, there was no significant difference between 0.3% MMN plus AF and the control, which showed that MMN addition diminished the adverse effects of AF and organ damage were prevented.
The histological changes observed in the maternal liver and kidney induced by AF have been documented previously (Heathcote et al., 1978;Ikegwuonu et al., 1980).We also observed bile duct proliferation and periportal hepatocellular vacuolar degeneration in the livers of pigs treated with AF alone.Arora et al. (1978) and Grosman et al. (1983) reported that rats were acutely sensitive to the nephrotoxic effects of AF.In our study, the kidney from pigs fed AF alone showed localized degeneration and vacuolization in convoluted tubules at the corticomedullary junction.However, the liver and kidney of pigs fed MMN plus AF diet were normal.These findings indicated that organ lesions induced by AF were eliminated by the addition of MMN in the feed, and apparently, MMN was effective in preventing the maternal and developmental toxicity of AF in pigs.The results obtained were in agreement with previous reported by Mayura et al. (1998) and Harvey et al. (1989b).
In our study, on the basis of performance data, serum biochemical values and enzyme activities, as well as histopathologic findings, it was apparent that MMN had the ability to recover the AF-decreased performance and to correct aberrations in serum parameters, and 0.3% MMN in the diet protected pigs from the deleterious effects of AF.The serum parameters reflected the growth performance data.It should be noted that because montmorillonite has been demonstrated to bind AF in aqueous solution (Ramos et al., 1996;Grant et al., 1998), the presumed mechanism of action of MMN is to bind with the toxin in the gut and form stable sorption complex, thereby to prevent absorption of the toxin across the gastrointestinal wall, then the recovery, or restoration of performance were obtained.

IMPLICATIONS
When diets containing AF are fed to growing/finish pigs, there are reductions in growth rate and metabolic alterations reflected in an altered serum profile.The addition of MMN can ameliorate the toxic effects of AF (mainly via sequestration and reduced bioavailability in vivo).Our work suggests that MMN is a high affinity adsorbent for AF in pigs, and that the use of MMN may offer a novel approach to the preventive management of aflatoxicosis in animals.
a row with the different superscript letters differ significantly (p<0.05).

Figure 1 .
Figure 1.Photomicrographs of sections of porcine liver specimens.(A) AF group showing diffuse vacuolization and hepatic steatosis.(B) MMN plus AF group showing the lack of lesions.Hematoxylin and eosin stain, ×200.

Figure 2 .
Figure 2. Photomicrographs of sections of porcine kidney specimens.(A) AF group showing many vacuoles of varying sizes in cytoplasm of convoluted tubules.(B) MMN plus AF group showing normal kidney.Hematoxylin and eosin stain, ×200.

Table 1 .
The composition of normal and AF-contaminated corn/ Contaminated corn contained 1,000 µg/kg of aflatoxin B 1 . 3

Table 2 .
Effects of MMN and AF on growth performance in pigs 1

Table 3 .
Effects of MMN and AF on serum biochemical values of pigs 1

Table 5 .
Effects of MMN and AF on relative organ weights of pigs 1