Effect of a synbiotic on the intestinal microflora of chickens1

©2014 Poultry Science Association, Inc.

Effect of a synbiotic on the intestinal microflora of chickens1 Seyed Morteza Dibaji,* Alireza Seidavi,*2 Leila Asadpour,† and Fernando Moreira da Silva‡2

Primary Audience: Agricultural Faculty, Poultry Farmers, Poultry Microbiologists SUMMARY The current experiment was conducted to evaluate the effect of a synbiotic (Biomin Imbo) on intestinal microflora of Ross broiler chickens. A total of 200 male chickens were randomly divided into 20 groups of 10 birds each, kept in 20 pens (1.5 × 1 m each), and fed for 42 d at different synbiotic levels. Treatments included (1) a basal diet without synbiotics (control), (2) a basal diet with synbiotic levels proposed by the manufacturer, (3) a basal diet with synbiotic levels 25% lower than those proposed by the manufacturer, (4) a basal diet with synbiotic levels 50% higher than those proposed by the manufacturer, and (5) a basal diet with synbiotic levels 25% higher than those proposed by the manufacturer. At the end of the 42-d feeding period, 1 bird was randomly selected from each experimental unit, humanely euthanized, and the cecum was removed to measure the microbial population. The cecal contents were collected in discharge containers for microbial cultures, and counts were conducted after microbial culture. The addition of the synbiotic reduced Escherichia coli and total coliform populations in the intestines of broiler chickens. Conversely, different levels of probiotic increased the numbers of lactobacilli in the intestine of broiler chickens. Concentrations of the synbiotic higher than the recommended levels in the diet increased the lactic acid bacteria population in the gut of broiler chickens. Key words: synbiotic, intestinal microflora, broiler chicken 2014 J. Appl. Poult. Res. 23:1–6 http://dx.doi.org/10.3382/japr.2012-00709

DESCRIPTION OF PROBLEM The population of the world has increased from 3 billion in 1959 to more than 7 billion in March 2012. As the world’s population grows, 1

hunger persists in many locations and almost 1 billion people are reported to be malnourished [1]. By 2050, farmers will need to double crop production to meet the demand. In this context, the world needs food products with annual

This is an Open Access article distributed under the terms of the Creative Commons Attribution-Noncommercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 2 Corresponding authors: [email protected] and [email protected]

Downloaded from http://japr.oxfordjournals.org/ at National Chung Hsing University Library on April 10, 2014

*Department of Animal Science, and †Department of Veterinary Science, Rasht Branch, Islamic Azad University, Rasht 41335-3516, Iran; and ‡University of the Azores, Faculty of Agrarian Sciences, Department of Animal Reproduction, Research Centre for Agricultural and Environmental Science and Technology of the Azores, Angra do Heroísmo 9700, Portugal

2

thereby increasing the resistance to disease [9]. This study was designed to evaluate the effect of synbiotic Biomin Imbo on intestinal microflora of broiler chickens.

MATERIALS AND METHODS At the Faculty of Agriculture, Islamic Azad University, 200 one-day-old male Ross 308 chicks were randomly divided into 20 groups of 10 birds each and kept in 20 pens (1.5 × 1 m each) in the same room separated from each other by a metal mesh wall. Birds received 22L:2D throughout the study period. Room temperature was maintained at 30 to 33°C for the first week and then reduced gradually 2.8°C every week to 20°C; this temperature was then maintained for the duration of the study. Each treatment included 4 repetitions (10 birds per repetition), where birds had ad libitum access to feed and water provided in chute feeders for 42 d, with different synbiotic levels (Biomin Imbo [10]). • Treatment 1: Basal diet without synbiotic (control). • Treatment 2: Standard Ross diet based on catalog (base) with the levels of Biomin Imbo recommended by the manufacturer (0.1% for the starter period, 0.05% for the growth period, and 0.025% for the finisher period). • Treatment 3: Standard Ross diet based on catalog (base) with a 25% reduction in the level of the synbiotic recommended by the manufacturer (0.075% for the starter period, 0.0375% for the growth period, and 0.01875% for the finisher period). • Treatment 4: Standard Ross diet based on catalog (base) with a 50% increase in the level of the synbiotic recommended by the manufacturer (0.15% for the starter period, 0.075% for the growth period, and 0.0375% for the finisher period). • Treatment 5: Standard Ross diet based on catalog (base) with a 25% increase in the level of the synbiotic recommended by the manufacturer (0.125% for the starter period, 0.0625% for the growth period, and 0.03125% for the finisher period). Composition of the basal diet and its nutritive characteristics are represented in Table 1 and Table 2, respectively.

Downloaded from http://japr.oxfordjournals.org/ at National Chung Hsing University Library on April 10, 2014

growth of 2.5% for the next 10 yr [2]. Considering these statistics, many researchers and nutrition experts believe that poultry production can play a role in increasing food production. Poultry can make positive contributions to the diet of those with low incomes, as it is consistently high quality, low in saturated fats, as well as more healthy and affordable when compared with other meats. Thus, it is a source of essential nutrients and sought after worldwide. In addition, as chicken has an FCR of less than 2.0, which is much lower than other livestock, poultry can play an important role providing an inexpensive protein source and alleviating world hunger. To protect health and enhance the performance of chickens, such as immune response, intestinal health status, reducing morbidity and mortality, improving digestion, and feed conversion, additives (e.g., antibiotics) are sometimes used, possibly causing gastrointestinal disturbances and adding to concern over antibiotic resistance. Probiotic bacteria are a sustainable approach to modulate the gut microflora toward a more favorable composition. Probiotics should contribute to efficient production in a sustainable way, promoting healthy and robust animals [3]. Probiotics are considered live microorganisms that are thought to be beneficial to the host organism. The concept behind probiotics was introduced in the early 20th century by Elie Metchnikoff, known as the father of probiotics. Probiotics were thought to benefit the host by improving its intestinal microbial balance, thus inhibiting pathogens and toxin-producing bacteria [4]. Today, specific health effects are being investigated and documented, including alleviation of chronic intestinal inflammatory diseases [5], and prevention and treatment of pathogeninduced diarrhea [6], urogenital infections [7], and atopic diseases [8]. The probiotic part of Biomin Imbo is Enterococcus faecium (5 × 1110 cfu/kg), which helps to prevent the growth of pathogenic microorganisms such as Salmonella [9]. This probiotic also contains fructo-oligosaccharides that stimulate the growth of bifidobacteria, which, along with Enterococcus faecum, cause intestinal microflora stability and act as a barrier against pathogens. Other components of this probiotic (extracted from seaweed) stimulate the activity of macrophages and lymphocytes of the immune system,

JAPR: Research Report

Dibaji et al.: SYNBIOTIC IN CHICKEN

3

Table 1. Composition of the basal diet used in the experiment Starter

Grower

Finisher

Corn Soybean meal Fish meal Meat meal Oil dl-Met l-Lys l-Thr Ca 22%, P 18% CaCO3 KHCO3 NaCl Vitamin and mineral mixture1 Total

46.09 40.00 3.00 3.00 4.56 0.29 0.04 0.03 0.99 0.98 0.05 0.37 0.60 100

50.91 35.00 3.00 3.00 5.45 0.23 — — 0.75 0.76 0.03 0.37 0.50 100

48.88 39.97 — — 7.38 0.17 — — 1.64 1.00 — 0.45 0.50 100

1

Vitamin A: 5,000 IU/g; vitamin D3: 500 IU/g; vitamin E: 3 mg/g; vitamin K3: 1.5 mg/g; vitamin B2: 1 mg/g; calcium pantothenate: 4 mg/g; niacin: 15 mg/g; vitamin B6: 13 mg/g; Cu: 3 mg/g; Zn: 15 mg/g; Mn: 20 mg/g; Fe: 10 mg/g; K: 0.3 mg/g.

Birds were in pens for the entire 42 d. Seed containers and water bottles were cleaned using a disinfectant solution of Despadak (ratio of 1:100 [11]). In addition, facilities (walls, ceilings, and shelves) were sprayed with the same disinfectant. Manure produced during the experiment was collected after the study ended. At the end of the trial, a bird from each repetition was euthanized and the cecum was removed for further culture. Use and care of birds

in this study was approved by the Islamic Azad University Ethics Committee. The experimental procedures described herein were also approved by this committee, and care was taken to minimize the number of birds used. Agar plates were streaked with cecal contents and sent to the laboratory of nutrition and dairy industry from the Islamic Azad University, about 100 m from bird housing. To determine bacterial growth and colony counts, the agar

Table 2. Nutritional composition (calculated) of basal diets used in the experiment Ingredient (%, unless otherwise noted) ME (kcal/kg) CP Lys Met Met + Cys Thr Trp Arg Ile Val Leu Ca (%) Available P Na K Cl Dietary cation-anion balance (mEq/kg) Choline (g/kg) Linoleic acid CF EE

Starter

Grower

Finisher

3,025 24.9 1.41 0.67 1.05 1.98 0.30 1.68 1.04 1.60 1.99 1.05 0.50 0.23 1.00 0.30 272.12 1.48 1.21 3.78 6.84

3,150 23 1.26 0.59 0.94 0.87 0.27 1.54 0.95 1.07 1.87 0.90 0.45 0.23 0.90 0.30 244.55 1.37 1.27 3.52 7.87

3,200 22 1.22 0.50 0.85 0.85 0.28 1.51 0.94 1.03 1.82 0.85 0.42 0.20 0.93 0.30 242.77 1.37 1.24 3.73 9.22

Downloaded from http://japr.oxfordjournals.org/ at National Chung Hsing University Library on April 10, 2014

Ingredient (%)

JAPR: Research Report

4

RESULTS AND DISCUSSION Microflora identified in each treatment (total bacteria, E. coli, coliform bacteria, lactobacilli, and lactic acid bacteria) are summarized in Table 3. Additions of Biomin Imbo increased the

amount of the total bacteria in the gut of broilers (P < 0.05). Comparison of averages obtained from the experiments showed that treatment 3, the recommended dosage, had the most influence on the total bacteria (Table 3). Concerning the effect of different synbiotic levels on E. coli and coliform populations, results showed that additions of this probiotic reduced E. coli bacteria and total coliform populations in the intestines of broiler chickens (P < 0.05). Different levels of probiotics increased the numbers of lactobacilli in the intestine of broiler chickens (P < 0.05). Adding a higher concentration than the recommended levels increased the lactic acid bacteria population in gut of broiler chickens (P < 0.01). In contrast, adding recommended or levels below recommendation did not change the populations of lactic acid bacteria. Based on our experiments, we found that the supplement used had statistically significant effects. Total bacteria and lactobacilli increased in all treatments. Escherichia coli and coliform populations decreased in all treatments. Lactic acid bacteria increased when the probiotic was added in excess of recommended dosages (P < 0.05). Razavi et al. [13] studied the effect of probiotics and virginiamycin on the lactobacilli population in the ileum of broiler chickens and reported that the highest population was observed in chickens that consume probiotics and antibiotics. Probiotics and can improve performance in the ileum; however, organic acids and virginiamycin did not improve performance compared with control treatment. Abrishami et al. [14] studied the effect of different prebiotics (Fermecto) and probiotics (cultured intestinal contents) on the performance and microbial population of the digestive system. Supplementing with Fermecto, the number of lactobacilli increased aerobic bacteria up to 42 d in the digestive system. Adding cultured intestinal contents increased the number of lactobacilli and aerobic bacteria up to 42 d in the digestive system. Those authors reported a significant effect in increasing the number of lactobacilli and aerobic bacteria. Adding Fermecto as a prebiotic (at the suggested level based on the source) can cause performance improvement and partial change in bacterial population. Other related studies included projects by Sabouni et al. [15], who demonstrated that pro-

Downloaded from http://japr.oxfordjournals.org/ at National Chung Hsing University Library on April 10, 2014

plates streaked on the site were used. Collecting tubes were weighed, wrapped in an aluminum sheet, and autoclaved for 10 min. The culture media were prepared 24 h before collected samples were poured into petri dishes. de Man, Rogosa, Sharpe agar was used to culture lactobacilli, eosin methylene blue was used to culture Escherichia coli, MacConkey agar was used to culture coliforms, and nutrient agar was used to culture total aerobic bacteria. Samples were transferred to the laboratory in the listed tubes and weighed again. The amount of sample in each tube was calculated from the difference between these 2 values. Tubes were shaken for approximately 30 min; the action was performed for bacteria isolated from gastrointestinal contents and preparation of suspension. One milliliter was removed from the prepared suspension and added to 9 mL of PBS in the other tube. The suspension was prepared from 10−1 dilutions and serial dilutions were done (10−2, 10−3, 10−4, 10−5, and 10−6). Next, 100 μL was removed from dilutions (10−4, 10−5, and 10−6) and poured into the petri dish previously prepared containing the medium and completely distributed to all parts of the medium. Lactobacilli were incubated at 37°C in anaerobic conditions for 72 h. An anaerobic jar was used to create anaerobic conditions. Total aerobic bacteria were incubated at 37°C under aerobic conditions for 48 h. Counting of bacteria in the petri dishes was done by a colony counter. Bacterial counts were reported as logarithm number of bacteria per 1-g sample. Data were analyzed by SPSS [12] statistical software, and averages were compared by the Tukey test. The statistical design was Xij = μ + Tj + eij, where Xij is the number of each control in the experiment; μ represents the overall mean; Tj is the effect of each group of the experimental diet; and eij is the effect of the error. The total value of any observed treatment effects and the average test error was the result of the whole population. Before performing statistical analysis of data, all data were tested by a normality test.

8.448b ± 7.331 8.441b ± 7.065 8.462b ± 7.390 8.542ab ± 7.293 8.591a ± 3.983 7.715b ± 7.106 7.855ab ± 7.250 7.822ab ± 7.217 8.112a ± 7.271 8.051ab ± 6.757 a,b

Treatment means that are not followed by the same letter differ significantly from one another (P < 0.05).

8.035a ± 6.662 7.544ab ± 2.893 6.753b ± 7.000 7.829ab ± 7.039 6.761b ± 7.021 8.004b ± 7.212 8.725a ± 0.222 8.726a ± 0.179 8.689a ± 7.937 8.621a ± 6.991 Control Recommended amount by company 25% lower than the recommended amount by company 50% higher than the recommended amount by company 25% higher than the recommended amount by company 1 2 3 4 5

Treatment

Description

8.037a ± 6.815 7.647b ± 5.509 7.554b ± 6.487 7.420b ± 6.706 7.459b ± 6.149

Coliforms bacteria (log cfu/g) Escherichia coli (log cfu/g) Total bacteria (log cfu/g) Table 3. Mean comparison (± SEM) of cecum microflora among treatments

5 biotics and prebiotics improve the number of lactobacilli and total bacteria in the ileum. Kabir et al. [16] demonstrated that probiotics are able to eliminate harmful pathogens through competition for joining to small gut. Broiler chickens fed probiotics had histological changes in the intestine, including increased villi length and cellular levels. In addition, chickens fed with lactobacilli have fewer coliforms in their feces. Intestinal microflora can also be improved by changing intestinal acidity by increasing lactic acid concentration in the intestine, reducing the activities of harmful bacteria (Salmonella and E. coli), and increasing lactobacilli [17]. Chichlowski et al. [18] reported that use of probiotic and prebiotic products can improve intestine bacteria balance. Overall, the results of the above experiments [13–22] agree with our results. The use of the present probiotic resulted in an increase in total bacteria and gram-positive bacteria, such as lactobacilli and lactic acid bacteria, but reduced coliform and E. coli. Our results do not agree with Khalaji et al. [21] and Rakhshan et al. [23], possibly because those researchers used different additives. Khalaji et al. [21] studied the effect of the prebiotic Technomoss on the digestive health of broiler chickens. According to the poultry industry, gastrointestinal disease is one of the most important problems, and concerns of resistance to antibiotics also exist. Therefore, a new trend has emerged toward the use of probiotics. The effect of a moss prebiotic on gut morphology and microbial intestine population in broiler chickens was studied by Rakhshan et al. [23]. No significant difference was observed in the microbial number of lactobacilli and coliforms of intestinal contents among the different groups [23]. According to available reports, increasing congenital malformations, the occurrence of chronic diseases, and some other complications in humans could be avoided or reduced if the use of antibiotics was reduced and if probiotics and synbiotics, as alternatives, were more accepted [24].

CONCLUSIONS AND APPLICATIONS

1. In the present study, positive results were obtained when male Ross 308 chicks were fed a standard diet with a probiotic

Downloaded from http://japr.oxfordjournals.org/ at National Chung Hsing University Library on April 10, 2014

Lactobacillus bacteria (log cfu/g)

Lactic acid bacteria (log cfu/g)

Dibaji et al.: SYNBIOTIC IN CHICKEN

JAPR: Research Report

6



REFERENCES AND NOTES 1. Alexander, R. 2013. Does a child die of hunger every 10 seconds? BBC NEWS Magazine. Accessed Oct. 15, 2013. http://www.bbc.co.uk/news/magazine-22935692. 2. Mack, M. 2009. Role of technology is crucial in improving food security. Syngenta CEO in USDA Outlook Forum, Washington, DC. Syngenta International AG, Basel, Switzerland. 3. Brittain, A., C. S. Lee, and P. J. O’Brien. 2002. Microbial contributions to aquatic nutrition. Page 220 in Microbial Approaches to Aquatic Nutrition in Environmentally Sound Aquaculture Production Systems. C. S. Lee and P. O’Bryen, ed. World Aquaculture Society, Baton Rouge, LA. 4. Metchnikoff, E. 1907. Essais optimistes. Paris. The prolongation of life. Optimistic studies. Chalmers. P. Mitchel, ed. Springer Publishing Co. Inc., New York, NY. 5. Mach, T. 2006. Clinical usefulness of probiotics in inflammatory bowel diseases. J. Physiol. Pharmacol. 57(Suppl. 9):23–33. 6. Yan, F., and D. B. Polk. 2006. Probiotics as functional food in the treatment of diarrhea. Curr. Opin. Clin. Nutr. Metab. Care 9:717–721. 7. Reid, G. 2008. Probiotic lactobacilli for urogenital health in women. J. Clin. Gastroenterol. 42(Suppl 3 Pt 2):S234–S236. 8. Vanderhoof, J. A. 2008. Probiotics in allergy management. J. Pediatr. Gastroenterol. Nutr. 47(Suppl 2):S38–S40. 9. Guarnera, F., and G. J. Schaafsmab. 1988. Probiotics. Int. J. Food Microbiol. 39:237–238. 10. Biomin Holding GmbH, Herzogenburg, Austria. 11. Lab. Calier S.A., Barcelona, Spain. 12. SPSS Inc., Chicago, IL. 13. Razavi, S. H., H. A. Shahriar, Y. Ebrahimnejad, A. Ahmadzadeh, M. Ghaderi, S. Goli, and V. N. Rezaee. 2010. The effect of probiotic, organic acid, prebiotic and antibiotic on performance and Lactobacillus population in ileum of broiler chicken. Pages 994–997 in 4th Iranian Animal Science Congress, Tehran University, Tehran-Karaj, Iran. 14. Abrishami, M. H., V. K. Zareaha, H. Kermanshahi, and V. M. Pilehvar. 2010. The effect of different prebiotic

Feremco and perobiotic levels of cultured intestinal contents on performance and bacterial population of digestive system. Pages 699–702 in 4th Iranian Animal Science Congress, Tehran University, Tehran-Karaj, Iran. 15. Sabouni, S., N. Eila, M. Aalehi, and V. B. Gholamhossein. 2010. The effect of probiotics and prebiotic diet on performance and bacterial population and morphology of gut ileum of broiler chicken. Pages 1070–1077 in 4th Iranian Animal Science Congress, Tehran University, Tehran-Karaj, Iran. 16. Kabir, S., M. M. Rahman, M. B. Rahman, and S. U. Ahmad. 2004. The dynamics of probiotics on growth performance and immune in broiler. Jpn. Poult. Sci. 3:61–64. 17. Vegas, J. L. 2004. Prebiotics, probiotic, acid fires and antibiotic growth promoters. Pages 339–346 in Poultry Diseases: A Guide for Farmers and Poultry Professionals. International Book Distributing Co., Charbagh, Lucknow, India. 18. Chichlowski, M., W. J. Croom, F. W. Edens, B. W. McBride, R. Qiu, C. C. Chiang, L. R. Daniel, G. B. Havenstein, and M. D. Koci. 2007. Microarchitecture and spatial relationship between bacteria and ileal, cecal, and colonic epithelium in chicks fed a direct-fed microbial, PrimaLac, and salinomycin. Poult. Sci. 86:1121–1132. 19. Willis, W. L., and L. Reid. 2008. Investigating the effects of dietary probiotic feeding regimens on broiler chicken production and Campylobacter jejuni presence. Poult. Sci. 87:606–611. 20. Lee, K. W., S. H. Lee, H. S. Lillehoj, G. X. Li, S. I. Jang, U. S. Babu, M. S. Park, D. K. Kim, E. P. Lillehoj, A. P. Neumann, T. G. Rehberger, and G. R. Siragusa. 2010. Effects of direct-fed microbials on growth performance, gut morphometry, and immune characteristics in broiler chickens. Poult. Sci. 89:203–216. 21. Kannan, M., R. Karunakaran, V. Balakirishnan, and T. G. Parabhaken. 2005. Influence of probiotics supplementation on lipid profile of broilers. Int. J. Poult. Sci. 4:994– 997. 22. Khalaji, S., M. Zaghari, and V. S. Nezafati. 2010. The effect of probiotics Tecnomoss on digestive health, safety and performance of broiler chicken. Pages 207–209 in 4th Iranian Animal Science Congress, Tehran University, Tehran-Karaj, Iran. 23. Rakhshan, M., S. Shivazad, M. Mousavi, and M. Zaghari. 2010. The effect of probiotics teknomoss on gut morphology and intestinal bacterial population in broiler chicken. Pages 703–706 in 4th Iranian Animal Science Congress, Tehran University, Tehran-Karaj, Iran. 24. Azimi, J. 2008. Study of the effect of probiotics on poultry feed. MSc Diss. Tehran University, Tehran, Iran.

Acknowledgments

This manuscript is obtained from the MS thesis of Seyed Morteza Dibaji at Islamic Azad University. We are grateful to Islamic Azad University for support. The Research Centre for Agricultural and Environmental Science and Technology of the Azores is also fully acknowledged.

Downloaded from http://japr.oxfordjournals.org/ at National Chung Hsing University Library on April 10, 2014

added at 50% of the level recommended by the manufacturer, increasing beneficial bacteria such as lactobacilli and lactic acid. 2. Because good microbes help develop a strong immune system, reduce morbidity and mortality, and improve digestion and feed conversion, the use of this probiotic ameliorates the bird’s performance, thereby reducing production costs.