Effects of an Aspergillus niger mycelium , antibiotic and probiotic on growth performance , P and Ca retention and Clostridium perfringens ileal counts in broiler chickens

A study was conducted to evaluate effects of an Aspergillus niger mycelium, antibiotic and probiotic on growth performance, P and Ca retention and Clostridium perfringens ileal counts in broiler chickens. Chickens were fed maize-wheat-soyabean meal diets from day 1 to 21. Dietary treatments included: 1. a low phosphorus diet (LP), 0.17% nonphytate phosphorus, 0.6% Ca; 2. LP + virginiamycin; 3. phytase diet (Phyt), LP + 750 phytase units/kg diet; 4. Phyt + virginiamycin; 5. fungal mycelium diet (Myc), LP + 1.5% mycelium; 6. standard phosphorus diet (SP), 0.41% nonphytate phosphorus, 0.87% Ca; 7. SP + virginiamycin; 8. probiotic diet (Pr), SP + probiotic; 9. high phosphorus diet (HP), 0.45% nonphytate phosphorus, 1% Ca; 10. HP + virginiamycin. Each diet was fed to five pens replicates of eight birds each from hatch to 21 day of age. Chickens receiving Myc, Pr, both HP, and both SP diets performed better than birds fed both LP and both Phyt diets and showed the highest bone mineralization. The lowest intestinal viscosity (1.53 mPa·s) and the highest phosphorus retention (62.1%) was observed in birds fed diet supplemented with fungal mycelium. Birds fed Myc, Pr, both SP and both HP diets showed the highest calcium retention that did not differ from each other. Antibiotic was the most effective in reducing C. perfringens counts in ileal digesta. The study confirmed a high efficacy of A. niger mycelium used at the level of 1.5% in enhancing growth performance and P retention in chickens.


INTRODUCTION
In order to achieve maximum phytate phosphorus utilization in poultry, different strategies have been undertaken.Turkeys fed low P diets supplemented with enzymic cocktails performed as well as birds receiving control diets recommended by the NRC and retained more P and Ca (Żyła et al., 1996).Turkeys fed diets containing 5% of an industrial waste Aspergillus niger mycelium performed even better and retained more P than poults fed the enzymic mixture.The 4% addition of an A. niger mycelium biosynthesized on a laboratory scale to the broiler diets resulted in performance, bone mineralization, P and Ca retention superior to values found in birds fed the standard phosphorus diets (Żyła, 2000).In our previous studies (Żyła et al., 1995;Żyła and Gogol, 2002) we have found that the A. niger mycelium is high not only in different dephosphorylating activities (phytase, acid phytase, phosphodiesterase), but also in activities that degrade plant cell walls (xylanase, β-glucanase, cellulase, polygalacturonase and acid protease) and reduce intestinal viscosity in broilers.Moreover, preliminary results (Żyła et al., 2000) suggested that mycelial cells or cells fragments might modulate the immune system of birds and influence the intestinal microflora by colonizing the gut and competing with the host microflora.
Clostridium perfringens is regarded as the causative agent of necrotic enteritis (Paulus and Ruckebusch, 1996) and is one of the main factors causing growth depression in poultry (Stutz and Lawton, 1984a).The application of antibiotics as feed additives in poultry production has been limited lately.Feed industry looking for alternative additives is testing different probiotics, prebiotics, acidifiers and new preparations.
The aim of the present study was to evaluate the effectiveness of a mycelial biocatalyst fed to broilers at 1.5% in respect to birds performance, bone mineralization, retention of P and Ca and digesta viscosity.Another purpose of the study was to learn the efficacy of A. niger mycelium in lowering ileal C. perfringens numbers as compared to virginiamycin and to a probiotic culture.

Animals and sample collection
The experiment was conducted on 400 Ross 208 broilers.Day-old chickens were randomly allotted to 10 groups in 5 replication of 8 chickens (4 males and 4 females) each and kept in cages with mesh floors.The birds were housed under standard conditions and fed three weeks ad libitum with mash diets (Table 1).Over the entire experimental period feed consumption and chickens growth were measured.On day 21 of age after 4 h of feed deprivation, the birds were weighed, feed intake measured and feeding of the experimental diets resumed.
During the 3rd week (days 15 to 20), a total collection of excreta from each pen was carried out.Excreta were stored in plastic bags at -20 o C and then, after thawing were dried in an oven at 50 o C to a constant weight.Prior to analysis, excreta were ground to pass a 1 mm sieve.On day 21, three broilers from every replicate were killed by cervical dislocation.Toe samples of three chickens (selected at random) from each replicate were collected for ash analysis.The samples were dried at 100 o C for 24 h, weighed, and ashed at 600 o C overnight for determination of toe ash.After dissection, the abdominal cavity was opened and the contents of the ileum was collected.Digesta from 3 birds were pooled, then centrifuged at 10.000 g for 10 min and the viscosity of the supernatant was immediately measured on a capillary viscometer.Nine chickens from tested treatments were sacrificed for enumeration of C. perfringens in the ilea.The cut section of the ileum, that portion of the small intestine from the yolk stalk to the ileocaecal junction, was ligated with a nylon suture.

Chemical and microbiological assays
Duplicate samples of feed were digested by wet ash procedure, which was validated by including standard reference material 1572 (Citrus leaves) from the National Institute of Standards and Technology (USA).Phosphorus concentration was determined colorimetrically by the molybdeno-vanadate method (AOAC, 1995).Calcium was analysed by flame atomic absorption spectrophotometry.P and Ca retention were calculated relative to P and Ca intake.The quantitative analysis and the typing of the intestine contents for C. perfringens were carried out as described by Stutz and Lawton (1984b) and expressed as a decimal logarithm from the most probable counts number (MNP) per 1 g of digesta.

Statistical analysis
Data were analysed statistically by general linear models procedure of Statgraphics Plus for Windows (1996).Mean differences were determined by GOGOL D. ET AL.
Fischer's least significant difference test at P<0.05.Correlation coefficients was calculated and statistical significance determined between body weight gains and C. perfringens counts.Two-way Anova (factors: treatment, antibiotic) was performed on data excluding the Myc and Pr groups.

RESULTS
There were significant effects of dietary additions on feed intake, body weight, toe ash, intestinal viscosity, P and Ca retention, and the number of C. perfringens (Tables 2 and 3).Chickens fed the low P (LP) diets with and without antibiotic consumed less feed and gained less weight than those assigned to Phyt, Myc, standard phosphorus (SP), Pr and high phosphorus (HP) dietary treatments.Phytase supplementation of LP diet resulted in significant increases in feed intake and body weight gain.Feed intake in birds fed the Myc, SP, Pr, values within the same column with different letters differ significantly (P<0.05) 1 treatments were as followed: 1. a low phosphorus diet (LP); 2. LP + 10 mg virginiamycin/kg diet; 3.
phytase diet (Phyt), LP + 750 phytase units/kg diet; 4. Phyt + 10 mg virginiamycin/kg diet; 5. fungal mycelium diet (Myc), LP + 15 g mycelium/kg diet; 6. standard phosphorus diet (SP); 7. SP + 10 mg virginiamycin/kg diet; 8. probiotic diet (Pr), SP + probiotic added to the drinking water 1,5 ml/L; 9. high phosphorus diet (HP), 0.45% nonphytate phosphorus, 1% Ca; 10.HP + 10 mg virginiamycin/kg diet 2 two-way Anova (factors: treatment, antibiotic) was performed on data excluding the Myc and Pr diets HP diets were similar but significantly higher than birds fed the LP and Phyt diets.The influence of phytase, probiotic and fungal mycelium on body weight parallelled that of feed intake, except the standard phosphorus group, where the antibiotic addition significantly increased the body weight, but did not affect feed intake.There were no significant differences among treatments in respect to feed conversion ratio.The phytase addition to the LP diet, as well as to the LP diet containing antibiotic, resulted in significant increase in toe ash.Chickens receiving the Myc diet showed the highest percentage of ash in the toe (12.7%).Toe ash found in birds of the Myc, SP, Pr, HP diets were similar but significantly higher than in birds fed the LP and Phyt diets.The highest intestinal viscosities were found in birds fed the low phosphorus diet without antibiotic (2.18 mPa•s).Supplementation of antibiotic to the LP diet significantly reduced digesta viscosity.The same downtrend was observed in groups receiving phytase without and with antibiotic.However, in the standard phosphorus diet that did not contain antibiotic lower viscosity (1.85 mPa•s) was found than in the diet enriched with antibiotic (2.12 mPa•s).The lowest intestinal viscosity was observed in birds consuming diet supplemented with fungal mycelium (1.53 mPa•s).The highest P retention was observed in birds fed Myc diet, significantly lower values were found in the SP group without antibiotic and the Pr group.Ca retention in Myc, Pr, both SP and both HP diets was the highest and did not differ from each other.The numbers of C. perfringens, for the birds that did not receive antibiotic (2.04 log 10 cfu/g digesta), were higher than Clostridium counts in other tested dietary treatments.Addition of fungal mycelium or probiotic to the diets significantly reduced the population of C. perfringens in the ileum (1.37 and 1.45 log 10 cfu/g digesta, respectively) but was less effective than virginiamycin (0.93 log 10 cfu/g digesta).
Two-way Anova (Tables 2 and 3) demonstrated that the influence of treatment on the growth performance parameters, toe ash, intestinal viscosity and Ca retention was significant (P<0.05).There was no influence of the treatments on P retention, however.Antibiotic did not significantly influence the parameters tested except for digesta viscosity.Linear regression analysis revealed inverse correlation (r = -0.307),at P = 0.069 between body weight of broilers and C. perfringens counts in the ilea.The reciprocal-X model produced even stronger relationship (r = -0.381;P=0.022).

DISCUSSION
In the study presented here low phosphorus diets were supplemented with commercial phytase or with A. niger mycelium and fed to growing chickens.Phytase addition to the low phosphorus diet improved both performance and bone mineralization of chickens and significantly decreased digesta viscosity.Birds fed diets supplemented with phytase had higher P and Ca retention than birds fed low phosphorus diets.This finding is consistent with results from our previous studies (Żyła et al., 1996, 2000).The experiment was designed to find whether there are any interactions between antibiotic and phytase added to diets in enhancing broilers growth performance.Antibiotic did not affect chicken performance except for the standard phosphorus group where it increased significantly body weight.Engberg et al. (2000) observed significant growth promoting effect in chickens fed diet supplemented with combination of antibiotics: zinc bacitracin with salinomycin.Studies conducted by Mohan et al. (1996) proved that broiler chickens fed probiotic and antibiotic supplemented diets for four weeks had the highest weight gain, followed by those receiving antibiotic alone, probiotic alone and controls.In our experiment antibiotic added to the low phosphorus diet and to the phytase diet caused significant decrease in digesta viscosity in relation to diets without antibiotic.However, in the case of standard phosphorus group antibiotic increased intestinal viscosity.Moreover, in birds fed the two HP diets no significant differences in digesta viscosity were found.It seems possible therefore that interactions between antibiotic and phytase are related to the level of available phosphorus and calcium in the diet.
A. niger mycelium fed at 1.5% proved its highly effective dephosphorylating abilities (Żyła et al., 1996, 2000).Chickens fed mycelium supplemented diet performed similarly to chickens fed the standard phosphorus and HP diets.They showed enhanced bone mineralization, and high retention of P and Ca.The mycelium addition to a diet caused 28% reduction in digesta viscosity as compared to the standard phosphorus diet containing antibiotic.In the previous study the same fungal mycelium used at 4% caused 40% reduction of intestinal viscosity (Żyła, 2000).Several preparations have been tested to find their efficacies in enhancing poultry performance and reducing C. perfringens counts in the ileum.Nurit et al. (2001) checked non-antibiotic growth promoters Crina Poultry and More Yeast 100E against antibiotic growth promoter virginiamycin used at level of 20 ppm.Both tested preparations improved feed conversion and slightly increased body weight of chickens.Crina Poultry preparation, a blend of essential oil compounds, improved feed efficiency and reduced digesta viscosity (Francesch et al., 1999).Moreover the preparation prevented the colonization of the intestine of broilers by C. perfringens (Williams and Losa, 2001a).Acid Lac Dry, a mixture of organic acids, was found to numerically reduce C. perfringens counts but not to the same extend as the antibiotic (Van Campenhout et al., 2001).In our study chickens fed diets supplemented with fungal mycelium and probiotic had reduced number of C. perfringens, but still the numbers were higher than in birds receiving 10 ppm virginiamycin.In the studies of Stutz and Lawton (1984a) virginiamycin supplemented at levels of 55 and 16.5 ppm significantly improved weight gain and decreased C. perfringens counts.Body weight gain of birds fed SP diet supplemented with antibiotic as compared to a SP diet without antibiotic was significantly increased.In other dietary treatments no such a significant effect of the antibiotic was found.The inverse correlation between body weight of broilers and ileal C. perfringens counts observed in our study is consistent with observations of Stutz and Lawton (1984a).They found highly significant inverse correlations (r=-0.879;P<0.01) in broilers between weight gain and C. perfringens counts.
Results from the studies on the effects of probiotic cultures on animal production are often contradictory.The beneficial effect of probiotic cultures on growth performance and intestinal microbiota have been documented (Tortureo, 1973;Pascual et al., 1999;Jin et al., 2000).However, there have also been reports of no significant differences in weight gains of chickens fed diets with or without Lactobacillus cultures (Watkins and Kratzer, 1984;Maiolino et al., 1992).We observed no improvements neither in performance, toe ash nor in P and Ca retention in broilers fed probiotic supplemented diet as compared to the standard phosphorus diet.Only digesta viscosity of birds receiving probiotic were lower than in birds fed the standard phosphorus diet.
The study presented here confirmed a high efficacy of a novel, not registered feed additive -A.niger mycelium, fed at a level of 1.5% in enhancing growth performance of chickens.Additionally, it effectively inhibited growth of C. perfringens thus preventing growth depression.Since A. niger mycelium caused reduction in intestinal viscosity to a much higher extent than phytase, probiotic or antibiotic and was found to be high in xylanase, β-glucanase, cellulase activities (data not presented) it could probably replace NSP degrading enzymes and increase nutrient digestibility.It needs to be emphasized, however, that A. niger mycelium was added to the low phosphorus control diet, whereas other experimental diets subjected to microbiological analysis contained the standard phosphorus level.Concluding, the A. niger mycelium seems to be a highly effective biocatalyst with a possible probiotic role.

TABLE 2
Performance and toe ash of broilers fed maize-wheat-soyabean meal diets enriched with phytase, antibiotic, probiotic and fungal mycelium from hatch to 21 d of age 1 two-way Anova (factors: treatment, antibiotic) was performed on data excluding the Myc and Pr diets TABLE 3 Digesta viscosities, P and Ca retention and Clostridium perfringens counts of broilers fed maizewheat-soyabean meal diets enriched with phytase, antibiotic, probiotic and fungal mycelium from hatch to 21 d of age 1 + probiotic added to the drinking water 1.5 ml/l; 9. high phosphorus diet (HP); 10.HP + 10 mg virginiamycin/kg diet 2