* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Effect of Different Growth Promoters on the Cecal Microflora and
Survey
Document related concepts
Transcript
461 Bulgarian Journal of Agricultural Science, 12 (2006), 461-474 National Centre for Agrarian Sciences Effect of Different Growth Promoters on the Cecal Microflora and Performance of Broiler Chickens S. A. DENEV TrakiaUniversity, Agricultural Faculty, BG-6000 Stara Zagora, Bulgaria Abstract DENEV, S. A., 2006. Effect of different growth promoters on the cecal microflora and performance of broiler chickens. Bulg. J. Agric. Sci., 12: 461-474 The objective of this study was an evaluation of the effects of different growth promoters: probiotic Lacto-Sacc® (Alltech, Inc. USA), nutritive premix Pharmastim and nutritive antibiotic Avilamycin on the cecal microflora and performance of broiler chickens. The experiment was conducted using four feeding groups of one-d-old broiler chicken: Group I: Basal diet without supplement (Untreated control); Groups II, III and IV: Basal diet supplemented with probiotic Lacto-Sacc®, (1.0 kg/t); nutritive premix Pharmastim, (0.3 kg/t); and nutritive antibiotic Avilamycin (5,0 mg/kg), respectively. The addition of Lacto-Sacc® to the basal diet significantly increased the count of lactobacilli and enterococci in the cecum of the broiler chickens and depressed the count of coliforms by the low pH of cecal content. There was no significant difference in the count of lactobacilli, enterococci, and coliforms present in the cecum of the untreated control, Pharmastim and Avilamycin-fed groups. Lacto-Sacc® enhanced growth of beneficial microorganisms, regulated the microbial environment in the cecum. Lacto-Sacc® significantly increased the body weight of broiler chickens (P<0.001), compared to the untreated control and the groups fed Pharmastim and Avilamycin. The Lacto-Sacc®, Pharmastim and Avilamycin supplements improved feed conversion ratio by 8.8, 1.1 and 1.9% (P<0.001), compared to the untreated control, respectively. These results suggested that feed additives based on microorganisms provide an alternative to antimicrobial substances in broiler nutrition. Probiotics, and especially Lacto-Sacc® can reduce or replace antibiotics used for growth promotion in broiler chickens. Key words: broilers, nutrition, cecal microflora, growth promoters, probiotics, antibiotics Introduction The microbial populations that inhabit the gastrointestinal tract (GIT) of poultry have been extensively studied in recent years (Barrow, 1992; Chiang and Hsien, 1995; Jin et al., 1996; Farran et al., 2004). Many workers have studied the cecal microbial This paper was presented as Poster: 1) At the Annual Meeting of the European Association for Animal Production 2004 (EAAP-2004), 5-9 Sep., Bled, Slovenia, Book of Abstracts No 10 (Eds. A. Hofer and etc.), Wageningen Academic Publishers, The Netherlands, pp 362. 2) At the 20th Alltech’s Annual Symposium “Nutritional Biotechnology in the Feed and Food Industries” May 24-26, 2004, Lexington, Kentucky, USA. Proceedings of the 20th Alltech’s Annual Symposium “Nutritional Biotechnology in the Feed and Food Industries”, Suppl. 1., (Eds. T.P.Lyons and K. Jacques), pp.46.v [email protected] 462 flora of the poultry. Several investigations on indigenous microflora of chickens, laying hens, turkeys, pheasants, and ducks have shown that the cecum contains the largest number of resident obligate and facultative anaerobic bacteria (Smith, 1965; Timms, 1968; Barnes and Impey, 1970, 1972; Barnes et al., 1972; Salanitro et al., 1974a, 1974b) that perform a number of beneficial functions. The mechanisms whereby these bacteria are maintained in the cecum are poorly understood (Barrow, 1992). Although many studies have been carried out in the intestinal flora of the bird and in particular the chicken, one is still far from understanding the factors that govern the presence of particular groups of microorganisms in various parts of the intestine and the role they may play in the nutrition and physiology of the bird. Intensive rearing conditions for livestock are believed to contribute to a delay or disturbance in the development of intestinal microflora. Chickens reared in commercial hatcheries are never exposed to bacteria from the mother hen and therefore the development of their intestinal microflora is delayed. At hatching, the intestinal tract is essentially sterile, but it becomes rapidly colonized by microorganisms from the environment (Stavric, 1994). Several weeks are required to establish an adult-type microflora in the cecum (Smith, 1965). Nurmi and Rantala (1973) recognized that the susceptibility of young chickens to colonization with pathogens was due to the delayed establishment of their microflora. The cecal microflora of the alimentary tract has a significant effect on the health and performance of poultry. There is considerable evidence that the cecal microflora exert protection against the establish- S. A. Denev ment of microbial pathogens such as Salmonella (Nurmi and Rantala, 1973) and Campylobacter (Barrow, 1992), which preferentially colonize the cecum. It is well established that the manipulation of the gastrointestinal microflora (GIM) of poultry has a significant effect on the growth rate and efficiency of feed utilization. However, the influences of antimicrobial agents on microbial population of the cecum are less understood. Antibiotics disturb the microbial balance in the cecum. The sensitivity of chickens to Salmonella infection increases after antibiotic treatment (Stavric, 1994). Although antibiotics have been used as therapeutic agents and growth promoters since the 1950s, there has been concern that their usage as growth promoters could result in the development of resistant populations of bacteria, making subsequent therapy with antibiotics difficult (Fuller, 1989; Skinner, 2003). Furthermore, consumers' concern regarding antibiotic residues in meat has also been growing. Because of the growing concern over the transmission and the proliferation of resistant zoonotic bacteria via the food chain, the European Union (EU) decided in 1999 to ban 4 commonly used antimicrobial growth promoters (AGP) (virginiamycin, spiramycin, tylosin, and zinc bacitracin). During 2003, the EU also signalised its intention to remove the other AGP by January 2006 (flavomycin and avilamycin/monensin and salinomycin). Currently, there are a number of nonantibiotic alternatives. Some of these alternatives may include significant changes in husbandry practices or the strategic use of probiotics, prebiotics, acidifiers, enzymes, herbal products, microflora enhancers, and immuno-modulators (Ferket, 2004). Effect of Different Growth Promoters on the Cecal Microflora and Performance... Modulation of the intestinal bacteria towards a "healthy" community by feeding live bacteria (so called "probiotics") or speciality carbohydrates supporting beneficial bacteria (so called "prebiotics") is currently under active research (Apajalahti et al., 2004.). The target of such nutraceutical products is to improve gastrointestinal health by selecting for beneficial microflora and suppressing known intestinal and food-borne pathogens. The interest in the use of probiotics as substitute is not surprising, especially in view of some claims that they offer a similar growth promoter benefit (Simon, 2003). The main benefits attributed to probiotics as natural feed additives are: restoration and maintenance of the proper balance of the microflora in the intestinal tract during times of stress, diseases and following antibiotic therapy. Probiotics act as natural growth promoters, and improve the general health and performance of poultry (Dawson, 2001). Many reports and reviews have shown that the oral administration of probiotics improve growth rate, feed conversion (Jernigan et al., 1985; Hussein and Ashry, 1991; Cho et al., 1992; Baidya et al., 1993; Va Holourek, 1993; Alvares et al., 1994; Manickan et al., 1994; Chiang and Hsieh, 1995; Denev, 1996, 1997; Georgieva et al., 2000; Simon, 2003; Skiner, 2003; Edens, 2003) and enhance immune response and health (Perdigon et al., 1995; Maasen et al., 2000; Sotirov et al., 2000, 2001; Vitini et al., 2001 Denev, 2000, 2003). For the above reasons, there is a wide interest in replacing AGP with more natural feed additives - probiotics. Although the cecum is an area of high microbial activity in the intestine of chickens, the effects of such factors as a diet on the cecal microflora is still not properly understood, because a number of predomi- 463 nant anaerobic bacteria present have not yet been isolated and characterized. There is also little information available describing the predominant species of bacteria occurring in the GIT of broiler chickens fed different growth promoters, especially nutritive antibiotics as well as probiotics. The objective of the current study was to examine the effect of probiotic LactoSacc®, a nutritive premix Pharmastim аnd Avilamycin supplementation on the cecal microflora and growth performance of broiler chickens. Materials and Methods Chickens, Diet and Husbandry Two hundred and forty-one-d-old female broiler chickens (Arbor Acres) supplied by a commercial broiler hatchery were used in the current experiment. An environmentally controlled research building with floor pens was used to house 60 birds per pen (0.16 m2 per bird). Litter was new pine wood shavings. Birds were randomly assigned to treatment pens with empty barrier pens between treatment pens. An initial brooding temperature of 35oC was maintained for the newly hatched chickens and reduced in weekly increments to a constant temperature of 25oC at 3 weeks of age. After that, environmental temperature and ventilation were regulated according to standard management practices for bird age and external weather. Lighting was continuous and feed and water were provided ad libitum. The chickens were divided into four groups of 60 chickens each. During the starter (0 to 28 d) and finisher (29 to 49 d) periods, all groups were fed the basal starter or finisher diets. The diet composition is presented in Table 1. We compared four feeding groups: Group I: Basal diet 464 without supplement (Untreated control); Group II: Basal diet supplemented with Lacto-Sacc® (Аlltech Inc., USA) (1.0 kg/t), contained per kg): Lactobacillus acidophilus (NLC 84), minimum 110 billion (CFU); Streptococcus faecium (NCS 97), minimum 77 billion CFU; Yeast culture Saccharomyces cerevisiaе, minimum. 1 980 billion cells, and enzymes; Group III: Basal diet supplemented with Pharmastim (Biovet, Bg), (0.3 kg/t), contained antibiotic Flavophospholipolum (Flavomycine) 2,0 and Excipiens ad 100.0), Group IV: Basal diet supplemented with Avilamycin (Elanco, USA) (5.0 mg/kg active substance). Sampling procedures At the end of the experimental period, (49 d of age) five birds from each group were randomly selected and killed by CO2 asphyxiation for collection of cecal contents. The intestinal tract was removed and placed in a container under a flow of oxygen-free CO2. By using aseptic techniques, both cecum with contents were ligated, removed from the dissected body cavity, and the contents were squeezed into a beaker being flushed with CO2. The cecal contents were collected aseptically, mixed, while being flushed with CO2, before the dilutions were made. A pH-meter with a microelectrode measured the pH of cecal contents from each group. The test method was that used by Barnes et al. (1979). Microbiological analyses Media. The count of viable microbes in the cecal contents were determined using various media: 1) The Anaerobic dilution solution (ADS) used in this work was a modification of the dilution blank of Holdeman and Moore (1977) containing S. A. Denev (g/l): K2HPO4 x 3H2O, 4.105; KH2PO4, 1.636; cysteine hydrochloride, 0.5; gelatin, 2.0; and resazurin, 1.0. The solution was pre-reduced before use; 2) VLMH Medium (Barnes and Impey, 1970), containing (g/l): tryptone, 10.0; NaCI, 5.0; beef extract, 3.0; yeast extract, 5.0; cysteine hydrochloride, 0.4; glucose, 2.5 (pH 7.2±0.2)) enriched with menadione (0.5 µg/ml) and hemin (5.0 µg/ml) and solidified with 1.5% agar (Schneitz et al., 1981) was used to enumerate the total anaerobic and aerobic counts. The Anaerobic dilution solution and VLMH Medium were prepared from individual ingredients (Merck, Germany); 3) Rogosa Agar (Difco Laboratories, USA) was used for enumeration of lactobacilli; 4) Slanetz and Bartley Medium (Oxoid UK, CM377) was used for enumeration of enterococci; 5) MacConkey Agar (Difco Laboratories, USA) for coliforms bacteria; 6) Bacto Brilliant Green agar (Difco Laboratories, USA) for Salmonella spp. Enumeration Methods. Serial 10-fold dilutions were prepared with sterile prereduced ADS. All dilution steps and inoculation of media were carried out under anaerobic conditions according to procedures described by Moore and Holdeman (1974). The serially diluted cecal contents were used for the enumeration of the anaerobic and facultative anaerobic organisms. All the plates used for the enumeration of the cecal microflora were freshly prepared and poured a day before use, pre-dried at 60oC for 15 minutes and stored in the anaerobic conditions. The diluted cecal contents were incorporated into plates of VLMH Medium, Rogosa Agar, Slanetz and Bartley Medium and MacConkey's Agar, and Bacto Brilliant Green agar to enumerate the total anaerobic and facultative anaerobic organisms, Effect of Different Growth Promoters on the Cecal Microflora and Performance... lactobacilli, enterococci, coliforms, and Salmonella counts respectively. The plates were incubated anaerobically in Gas Pak® anaerobic system (BBL Microbiology Systems, Cockeysville, MD. USA.) at 37oC for 5 days (Total anaerobic count); at 37 oC for 48 h (lactobacilli and enterococci), and aerobically at 37oC for 24 h (facultative aerobic count, coliforms and Salmonella). The total colony counts per g of undiluted cecal content from each medium were obtained as the weighed mean from two or three highest duplicate dilutions that showed growth. Performance data During the 7 weeks experimental period, the following performance criteria were studied: a) Live body weight (LBW) was recorded individually before feeding to the nearest 0.1 g at the beginning of the trial (after hatching) and then to the nearest 0.01 kg at 1, 2, 3, 4, 5, 6, and 7 weeks of age; b) Daily weight gain (DWG), kg; c) Feed conversion ratio (FCR) was calculated on the basis of kg of feed/kg weight gain. Statistical analysis All bacterial populations estimates were calculated and reported on a per g (wet weight) basis of cecal content. The mean and standard deviation of the bacterial counts (CFU/g) were calculated using logarithmic values. Student's t-test, conducted using Microsoft Excel was used to determine the significance of the differences between the experimental groups. Results and Discussion The composition of the cecal microflora of broiler chickens is shown in Table 1. The total viable count of obligate and 465 facultative anaerobes in the cecal contents was high in all experimental groups. These bacteria formed the major part of the cecal flora. There was no significant difference in the total count of bacteria present in the cecum of the control and experimental groups. Many other investigations have shown that the cecum contains the largest number of bacteria, most of which are obligate and facultative anaerobes (Barnes and Impey, 1970; Barnes et al., 1972; Salanitro et al., 1978; Sofos et al., 1985). The results of the present study on the bacterial counts of obligate and facultative anaerobes in cecal contents of broiler chickens indicate also that Pharmastim and Avilamycin have no inhibitory effect on cecal microflora. According to Bare and Wiseman (1964) the mechanisms that have been proposed to explain the growth promoting effect of antibiotics usually involve inhibition or stimulation of microorganisms in the intestinal tract. Ilieva et al. (1997) reported that the nutritive effect of Avilamycin is a result of its favorable effect on the intestinal microflora. Lactic acid bacteria, particularly lactobacilli, constitute an important group of microorganisms colonizing the GIT of chickens (Smith, 1965; Barnes, 1979). Mead and Adams (1975) have shown that the number of lactobacilli in the cecal tract of young chickens increases during the first 14d from 510 to 910/g feces. Lactobacillus acidophilus, Lactobacillus salivarius and Lactobacillus reuteri been shown to be the most common species in the chicken gut by Sara et al. (1985). The colonization of the GIT by lactobacilli has long been believed to promote health and productivity. The experimental data obtained by the microbiological analysis indicated that the 466 S. A. Denev Table 1 Composition of basal starter and finisher diets Ingredient, % Maize Soybean meal (41% CP* ) Sunflower meal (38% CP*) Fish meal Animal Fat Vitamin mineral premix* Sodium chloride CaCO3 Dicalcium phosphate DL-Methionine Total: Calculated nutrient content: ME, MJ/kg Crude protein, % Lysine, % Methionine + cystine, % Calcium, % Phosphorus (available), % Starter (To 28/d) 52.99 31.15 5.00 5.00 3.00 0.50 0.30 0.60 1.40 0.06 100.00 Finisher (29 to 49/d) 61.89 24.35 5.00 3.00 3.00 0.50 0.30 0.60 1.30 0.06 100.00 3003.00 22.20 1.28 0.90 0.99 0.45 3085.00 18.82 1.01 0.75 0.90 0.40 Vitamin-mineral premix supplied the following vitamins and minerals per kg of diet: vitamin A, 200 000 IU; vitamin D3, 500 000 IU; vitamin E, 4 200 mg; vitamin K3, 400 mg; vitamin B1, 200 mg; vitamin B2, 1000 mg, vitamin B6, 400 mg; vitamin B12, 3 mg; vitamin PP, 5 000 mg; Calcium D-pantotenic acid, 2 000 mg; Folic acid, 100 mg; Cholin chloride, 80 000 mg; Fe, 5 462 mg; Mn, 10 342 mg; Cu, 926 mg; Zn, 12 727 mg; Co, 54,5 mg; I, 137 mg, and Se, 18,3 mg. * Lactobacillus population increased predominantly when broiler chickens consumed a diet supplemented with probiotic. The results presented in Table 2 show clearly that the lactobacilli were the most numerous microorganisms present in the cecum of broiler chicks receiving a basal diet with the microbial supplement LactoSacc ® . Lacto-Sacc ® significantly increased the total number of lactobacilli in the cecal content of broiler chicks (P<0.001). Bonomi et al. (1995), and Maruta et al. (1996) observed similar effects of Lacto-Sacc® (Paik et al., 1990) and other directly fed microbials (Edens, 2003). The counts of lactobacilli were significantly lower (P<0.001) in the cecal contents of the untreated control, Pharmastim and Avilamycin-fed chickens. However, no significant difference was observed in the count of lactobacilli present in the cecum 467 Effect of Different Growth Promoters on the Cecal Microflora and Performance... Table 2 ® Effect of Lacto-Sacc , Pharmastim and Avilamycin on the cecal microflora broiler chickens* Bacterial counts** Total anaerobes Facultative Anaerobes Lactobacilli Enterococci Coliforms Salmonella Untreated Control a Lacto-Sacc® Pharmastim a Avilamycin a 11.663±0.018 9.595±0.005a 11.533±0.250 9.485±0.015a 11.468±0.180 11.762±0.150a 9.433±0.016a 9.689±0.006a 6.878±0.003a 6.422±0.024a 5.826±0.006a 0 8.467±0.015b 7.039±0.035b 3.969±0.020b 0 6.764±0.069a 6.307±0.066a 5.656±0.012a 0 6.608±0.013a 6.460±0.176a 5.666±0.008a 0 * Mean of five chickens ± SE of the mean. ** Log10/g (wet weight) of cecal contents a – d: Different letters indicate significant differences (P < 0.001) of the control, Pharmastim and Avilamycin-fed groups. These results indicated that the above nutritive antibiotics do not inhibit the population of Lactobacilli in the cecal content of broiler chickens. Duta and Devriese (1981) also reported that lactobacilli isolated from cecum of poultry are resistant to some growth promoting agents, particularly bacitracin, nitrovin, carbadox, flavomycin, lincomycin, oleandomycin, spiramicin, and tylosin. Enterococci form a significant part of the intestinal flora of the bird throughout life (Barnes et al., 1978). The results of the present study (Table 2) indicate that Lacto-Sacc® increases the number of enterococci population in the cecum of broiler chickens. In the control, Pharmas-tim and Avilamycin-treated groups, the number of cecal Enterococci was significantly lower in comparison with the Lacto-Sacc® - fed chickens P <0.001). However, no observable significant difference in the counts of Enterococci between the control, and Pharmastim and Avilamycin-fed broiler chickens were found. One of the most important functions of probiotics is the control or elimination of enteropathogens (Fuller, 1989; Gibson et al., 1997; Denev, 1996, 1977, 2000, 2003; Edens, 2003). The mechanism by which this occurs is still not well elucidated. Among the suggestions that have been considered are competition for limited carbon sources, and the production of antibacterial compounds (organic acids, hydrogen peroxide, bacteriocins). The present study has shown that the probiotic Lacto-Sacc® significantly decreased the number of coliforms in the cecal content of broiler chicks compared to the control, Pharmastim and Avilamycin-fed chicks (P<0.001). Chapman (1989), Paik et al. (1990) and Bonomi et al. (1995) have also reported that the addition of probiotics to the diet increased the development and activity of intestinal microflora, by increasing the number of LAB, including lactobacilli and by decreasing the number of coliforms, particularly E. coli. Many other investigators have studied the potentials of probiotics, which 468 DWG was obtained by Gous (1990); Wiseman (1990); McGinnis (1992), Gippert et al. (1992), Gippert and Bodrogil (1992); Samanta and Biswas (1995), Wambeke et al. (1995); Vladimirova and Sourdjiyska (1996); Edens (2003). The LBW and DWG of Pharmastim, and Avilamycin fed broiler chickens were low and unaffected by treatment. The difference between groups with and without nutritive antibiotics (untreated control) was not significant. The results concerning the effect of the nutritive antibiotic Avilamycin that we obtained is different from the ones reported by Jambos et al. (1995). Chapman (1989) obtained similar results as the results achieved by us with other nutritive antibiotics in comparison with probiotics. Ayed et al. (2004) reported that Avilamycin decreases chicken growth performance and has undesirable side effect on health. The supplementation of probiotic Lac6.4 6.3 6.2 6.1 6 5.9 5.8 5.7 Avilamycin Pharmastim Lacto-Sacc 5.6 Control Cecal pH exert in vitro inhibitory effects toward enteric microorganisms (Gilliland and Speck, 1979; Edens, 2003) and in vivo growth competition with E. coli in gnotobiotic chickens (Watkins et al., 1982; Watkins and Miller, 1983). No cecal Salmonella was found from birds fed the different supplements at 49 d of age (Table 2). The inclusion of Lacto-Sacc® in the diet of broiler chickens decreased significantly (P<0.001) the pH of the cecal content in comparison with the control, Pharmastim, and Avilamycin-fed chickens (Figure 1). The low pH produced in the cecum by the probiotic microorganisms was partly responsible for the suppression of coliforms in the cecum. The feeding of Lacto-Sacc® may have greater significance in birds with enteric disease caused by pathogenic E. coli (Kovacz-Zomborszky et al., 1994). Kumprecht et al. (1994) and Rada et al. (1995) also reported that including probiotics in the poultry diet decreases pH in the cecum. Similar results were observed with the nutritive premix Pharmastim compared to the control (P 0.05). There was no significant effect of Avilamycin on the pH of the cecal contents in broiler chickens. The effects of Lacto-Sacc®, Pharmastim and Avilamycin on broiler performance are presented in Table 3. These data indicate that at the end of the experimental period the probiotic Lacto-Sacc® had a beneficial effect on growth performance. It significantly increased the LBW and DWG of broiler chickens (P<0.001). The LBW of Lacto-Sacc® fed chicks were 7.5, 3.4, and 3.6 % higher in comparison with the untreated control, Pharmastim and Avilamycin fed chicks respectively. A similar beneficial effect of Lacto-Sacc® and other probiotics on poultry LBW and S. A. Denev Fig. 1. Effect of different supplements on the cecal pH of broiler chickens 469 Effect of Different Growth Promoters on the Cecal Microflora and Performance... to-Sacc® to the basal diet improved the efficiency of feed conversion in comparison with the untreated control, Pharmastim and Avilamycin fed broiler chickens (Table 3). During the experimental period, the best feed conversion was appeared chickens fed Lacto-Sacc® (2.077 kg) which needed 8.8% less feed for 1.0 kg weight gain, in comparison with the untreated control (2.277 kg). The differences among the untreated control and other experimental groups were less - 1.1% (2.251 kg Pharmastim supplemented) and 1.9% (2.233 kg Avilamycin supplemented). The better results in body growth and feed efficiency obtained in the LactoSacc® fed chickens are most probably due to the beneficial effect of this probiotic on cecal microflora particularly on the Lactobacilli and the inhibition of enteropathogenic, and harmful bacteria. On the other hand, Lacto-Sacc® contains some enzymes - amylase, protease, and cellulase that improve digestibility and feed conversion of the diet. Mortality was not affected by any of the dietary treatment during the trial, which is in agreement with Farran et al. (2004) who used prebiotics, Avilamycin, Flavomycin, and Parks et al. (2001), who used mannanoligosaccharides, Bambermy-cin and Virginiamycin. Zulkifli et al. (2000) when feeding broiler diets supplemented with Lactobacillus spp. cultures or oxytetracycline reported similar lack of effect on mortality. According to Wenk (1990) and Edens (2003) feed additives based on microorganisms provide an alternative to antimicrobial growth promoters in animal nutrition. The probiotics can reduce or replace antibiotics used for growth promotion in broiler chickens. The present study demonstrated that Lacto-Sacc® enhanced growth of beneficial microorganisms and regulated the microbial environment in the cecum of broilers. This probiotic significantly increased the number of lactobacilli and enterococci and depressed the number of coliforms and Salmonella spp. by the low pH of ce- Table 3 Effect of Lacto-Sacc®, Pharmastim and Avilamycin on broiler performance* Day Control Lacto-Sacc ® Pharmastim Avilamycin Live body weight, g 1 7 28 38.08 37.73 103.00±2.00a 110.00±1.90a 818.00±8.60a 37.88 38.08 100.00±2.05a 99.00±1.72a 897.00±11.78b 836.00±16.30a 811.00±8.20a 42 1494.00±13.88a 1560.00±15.47b 1479.00±22.49a 1435.00±12.29c 49 1875.00±20.56a 1810.00±28.76a 1807.00±15.19a 2016.00±21.48b Feed conversion ratio, g feed/g gain ** 1-28 28-49 1.970 (100) 2.501 (100) 1.769 (89.8) 2.315 (92.6) 1.894 (96.1) 2.543 (101.7) 1.949 (98.9) 2.454 (98.1) 1-49 2.277 (100) 2.077 (91.2) 2.251 (98.9) 2.233 (98.1) * - Georgieva et al. (2000); ** - (% ; a – d: Different letters indicate significant differences (P < 0.001) 470 cal content. There was no significant difference in the number of lactobacilli, enterococci, and coliforms present in the cecum of the untreated control, Pharmastim, and Avilamycin-fed groups. LactoSacc® significantly increased the LBW of broiler chicks by 7.5; 3.4 and 3.6 % respectively - compared to the untreated control and the groups fed Pharmastim and Avilamycin. The Lacto-Sacc ® , Pharmastim and Avilamycin supplements improved feed conversion by 8.8; 1.1 and 1.9% (compared to the untreated control), respectively. These results suggested that feed additives based on microorganisms provide an alternative to antimicrobial substances in poultry nutrition. The probiotic Lacto-Sacc® can reduce or replace antibiotics used for growth promotion in broiler chickens. Conclusions The addition of probiotic Lacto-Sacc® to the basal diet significantly increased the count of lactobacilli and enterococci in the cecum of the broiler chickens and depressed the count of coliforms by the low pH of cecal content. There was no significant difference in the count of lactobacilli, enterococci, and coliforms present in the cecum of the untreated control, Pharmastim and Avilamycin-fed groups. Lacto-Sacc® enhanced growth of beneficial microorganisms, regulated the microbial environment in the cecum. Lacto-Sacc® significantly increased the body weight of broiler chickens (P<0.001), compared to the untreated control and the groups fed Pharmastim and Avilamycin. The Lacto-Sacc®, Pharmastim and Avilamycin supplements improved feed conversion ratio by 8.8, 1.1 and 1.9% (P<0.001), S. A. Denev compared to the untreated control, respectively. These results suggested that feed additives based on microorganisms provide an alternative to antimicrobial substances in broiler nutrition. Probiotics, and especially Lacto-Sacc® can reduce or replace antibiotics used for growth promotion in broiler chickens. Acknowledgements The author express gratitude to L. Gati (The Eastern European Director of Alltech, Kft., Budapest, Hungary) for supplying the Alltech's product LactoSacc®, to Lina Popova (MSc.), Department of Microbiology, Agricultural Faculty, Trakia University, Stara Zagora 6000, Bulgaria) for her excellent technical assistance, and to Prof. F. W. Edens (Department of Poultry Science, North Carolina State University, USA) for his constructive comments on the manuscript. References Alvares, L. C., E. M. Barrera and E. A. Gonzales, 1994. Evaluation of growth promoters for broiler chickens. Veterinary (Mexico), 25: 141-144. Apajalahti, J., A. Kettunen, and H. Graham, 2004. Characteristics of the gastrointestinal microbial communities, with special reference to the chicken. World's Poultry Science Journal, 60: 223-232. Ayed, M.H., Z. Laamari, and B. Rekik, 2004. Effects of incorporating an antibiotic "Avilamycin" and a probiotic "Activis" in broiler diets. Proceedings, Western Section, American Society of Animal Science, 55: 237-240. Baidya, N., L. Mandal, and G. C. Banerjee, 1993. Efficiency of feeding antibiotics and probiotics in broilers. Journal of Veterinary and Animal Science, 24: 120-124. Effect of Different Growth Promoters on the Cecal Microflora and Performance... Bare, L. N. and R. F. Wiseman, 1964. Delayed appearance of Lactobacilli in the intestines of chicks reared in a "new" environment. Applied Microbiology, 12: 457-459 Barnes, E. M., 1979. The intestinal microflora of poultry and game birds during life and after storage. Journal of Applied Bacteriology, 46: 407-419. Barnes, E. M. and C. S. Impey, 1970. The isolation and properties of the predominant anaerobic bacteria in the cecum of chickens and turkey. British Poultry Science, 11: 467-481. Barnes, E. M. and C. S. Impey, 1972. Some properties of the nonsporing anaerobes from poultry caeca. Journal of Applied Bacteriology, 35: 241-251. Barnes, E. M., G. C. Mead, D. A. Bartum, and G. C. Harry, 1972. The intestinal flora of the chicken in the period of 2 to 6 weeks of age with particular reference to the anaerobic bacteria. British Poultry Science, 13: 311-326. Barnes, E. M., G. C. Mead, C. S. Impey and B. W. Adams, 1978. The effect of dietary bacitracin on the incidence of Streptococcus faecalis subspecies liquefaciens and related streptococci in the intestines of young chicks. British Poultry Science, 19: 713-723. Barnes, E. M., C. S. Impey and B. J. H. Stevens, 1979. Factors affecting the incidence and antisalmonella activity of the anaerobic cecal flora of the young chick. Journal of Hygiene, 82: 263-283. Barrow, P. A., 1992. Probiotics for chickens. In: R. Fuller (Editor). Probiotics. The Scientific Basis, Chapman & Hall, London, UK, pp. 223-257. Bonomi, A., A. Quarantelli, and C. Cerati, 1995. Use of Bacillus subtilis. Rivista di Scienza dell' Allimentazione, 24: 583-594. Chapman, J. D., 1989: Probiotics, acidifiers and yeast culture: A place for natural additives in pig and poultry production. Proceedings No 119, Animal Feeds. Biological Addi- 471 tives. 13 June. Post Graduate Committee in Veterinary Science, University of Sydney, Australia, pp. 63-77. Chiang, S. H. and W. M. Hsien, 1995. Effect of direct-fed microorganisms on broiler growth performance and litter ammonia level. Asian-Australasian Journal of Animal Sciences, 8: 159-162. Cho, K. H., U. T. Lee, C. K. Yang, D. Y. Ryu, Y. S. Kim, and Y. D. Yoon, 1992. The effect of Lactobacillus casei (TSC) for growth promotion in broiler chicken. Korean Journal of Veterinary Public Health, 16: 55-59. Dawson, K. A., 2001. Use of probiotics in poultry feed. Proceedings of Multu-State Poultry Meeting, May 22-24, KY, USA, pp. 1-12. Denev, S. A., 1996. Probiotics - Past, Present and Future. Bulgarian Journal of Agricultural Science, 2: 445-474. Denev, S. A., 1997. Probiotics - Present and Future. In: S.A. Denev (Editor). Proceedings of the First Alltech's Research Seminar "Biotechnology in the Feed Industry", 4th March, Sofia, Bulgaria, pp. 67-78. Denev, S. A., 2000: Role of Lactobacilli in Human and Animal Health. Animal Science Journal, 71 (6): 549-562. (Jp). Denev, S. A., 2003. Role of Lactobacilli in Gastrointestinal Ecosystem. In: A. Galabov (Editor). Proceedings of Tenth Congress of the Bulgarian Microbiologists, Plovdiv, October 9-12, Bulgaria, pp. 75-76. Dutta, G. N. and L. A. Devriese, 1981. Sensitivity and resistance to growth promoting agents in animal lactobacilli. Journal of Applied Bacteriology, 51: 283-288. Edens, F. W. 2003. An alternative for antibiotic use in poultry: probiotics. Revista Brasiliera de Ciencia Avicola, 5 (2): 56-80. Farran, M. T., R. Y. Usayran, R.Y. Eid, E. K. Barbour, A. H. Darwish, and V. M. Ashkarian, 2004. Effect of prebiotics, avilamycin, and flavomycin on broiler performance and cecal flora. Proceeding of XXII World's Poultry Congress, June 8-13, 472 Istanbul, Turkey, pp. 234-237. Ferket, P. R., 2004. Alternatives to antibiotics in poultry production: responses, practical experience and recommendations. In: T. P. Lyons and K. A. Jacques (Editors). Nutritional Biotechnology in the Feed and Food Industries. Proceedings of Alltech's 20th Annual Symposium. Nottingham University Press, Nottingham, UK, pp. 69-80. Fuller, R., 1989. Probiotics in man and animals. Journal of Applied Bacteriology, 66: 365-378. Georgieva, V., S. A. Denev, and B. Marinov, 2000: Effect of some probiotic and nutritive means on chicken broiler productivity. Animal Science, 3: 19-23 (Bg). Gibson, G. R., J. M. Saavedra, S. Macfarlane, and G.T. Macfarlane, 1997. Probiotics and intestinal infections. In: R. Fuller (Editor), Probiotics, Vol. 2, Application and practical aspects, Chapman & Hall, London, UK, pp. 11-39. Gilliland, S. E., and M. L. Speck, 1977. Antagonistic action of Lactobacillus acidophilus toward intestinal and foodborne pathogens in associative cultures. Journal of Food Protection, 40: 820-823. Gippert, T., G. Bodrogie, and H. Farkas, 1992. Effect of Lacto-Sacc on Performance of Peking Ducks Reared in Partially Intensive Conditions. In: T. P. Lyons (Editor). Biotechnology in the Feed Industry. Proceedings of Alltech's Eight Annual Symposium, Nicholasville, Kentucky, USA, pp. 50-51. Gippert, T., and G. Bodrogie, 1992. Lacto-Sacc in the feeding of broiler duck. Proceedings 9th International Symposium of Waterfowl, Pisa, Italy, pp. 16-18. Gous, R., 1990. Efficiency of Acid-Pack 4-Way and Lacto-Sacc in Broiler Production. In: T. P. Lyons (Editor). Biotechnology in the Feed Industry. Proceedings of Alltech's Eight Annual Symposium, Nicholasville, Kentucky, USA, pp. 493-494. Holdeman, L. V., and W. E. C. Moore, 1977. Anaerobe Laboratory Manual, fourth ed. S. A. Denev Virginia Polytechnic Institute and State Univ. Anaerobe Laboratory, Blacksburg, USA, 425 pp. Hussein, H.H., and M. A. El-Ashry, 1991. Some studies on the beneficial effect of Lactobacillus concentrate supplementation on broiler performance. Egyptian Journal of Animal Production, 28: 85-91. Ilieva, Y., M. Veleva, T. Dakova, K. Kirov, P. Dilov, and G. Monov, 1997. Testing of nutritive effect of Avilamycin in pigs. Animal Science, 1-2: 24-28 (Bg). Jambros, D., M. Kirchebner, O. J. Wiliczkiewcz, and J. Skorupinska, 1995. Effect of application of Avilamycin (Maxus) and different levels of crude protein in concentrate mixtures on the nitrogen excretion and retention and on the apparent digestibility of amino acids in broiler chickens. Proceedings 10th European Symposium on Poultry Nutrition., 15-19th October, AntalyaTurkey, pp. 375-376. Jernigan, M. A., R. D. Miles, and A. S. Arafa, 1985. Probiotics in poultry nutrition- review. World's Poulrty Science Journal, 41: 99-107. Jin, L.Z., Y. W. Ho, N. Abdullah, and S. Jalaludin, 1996. Influence of dried Bacillus subtilis and Lactobacilli cultures on intestinal microflora and performance in broilers. Asian-Australasian Journal of Animal Sciences, 8: 159-162. Kovacz-Zomborszky, M., F. Kreizinger, S.Gombos, and Z. Zomborszky, 1994. Data on the effects of the probiotic Lacto-Sacc. Acta Veterinary Hungary, 42: 3-14. Kumprecht, I., P. Zobas, Z. Gasnarek, and E. Robosova, 1994. The effect of continuous application of probiotics preparations based on Saccharomyces cerevisiae var. elipsoideus and Streptococcus faecium C68 (SF 68) on chicken broilers yield. Zivocisna Vyroba, 39: 491-503. Maassen, C. B., C. Van Holten-Neelen, F. Balk, M.J. Glashouwer, L.J. Leer, J. D. Laman, W. J. Boersma, and E. Claassen, 2000. Effect of Different Growth Promoters on the Cecal Microflora and Performance... Strain-dependent induction of cytokine profiles in the gut by orally administered Lactobacillus strains. Vaccine, 18: 2613-2623. Manickam, R., K. Viswanatham, and M. Mohan, 1994. Effect of probiotics in broiler performance. Indian Veterinary Journal, 71: 737-739. Maruta, K., H. Miyazaki, S. Masuda, M. Takahashi, T. Marubashi, Y. Tanado, H. Mrkvanova, J. Vosirek, J. Marvanova, and J. Bis, 1991. The effect of Lactiferm and Probioticum preparations on digestive tract microflora in broiler chickens. Zivocisna Vyroba, 53: 59:66. McGinnis, K., 1992. Lacto-Sacc Versus Bacitracin in Broiler Starter Diets. In: T. P. Lyons (Editor). Biotechnology in The Feed Industry, Proceedings of Alltech's Eight Annual Symposium, Nicholasville, Kentucky, U.S.A., pp. 49. Mead, G.C., and B. W. Adams, 1975. Some observations on the cecal microflora of the chick during the first two weeks of life. British Poultry Science, 16: 169-176. Moore, W. E. C., and L.V. Holdeman, 1974: Human fecal flora: the normal flora of 20 Japanese-Hawaiians. Applied Microbiology, 27: 961-979. Nurmi, E. and M. Rantala, 1973. New aspects of Salmonella infection in broiler production. Nature, 241: 210-211. Paik, I. K., Y. J. Choi, and C. J. Kim, 1990. Effect of Supplementation of Probiotics (Lacto-Sacc) and Organic Acids (Acid-Pak 4-Way) in Broiler Chickens. In: T. P. Lyons (Editor). Biotechnology in The Feed Industry. Proceedings of Alltech's Sixth Annual Symposium, Nicholasville, Kentucky, U.S.A., pp. 491-492. Parks, A.W., J. L. Grimes, P. R. Ferket, and A. S. Fairchild, 2001. The effect of mannanoligosaccharides, bambermycins and virginiamycin on performance of large male market turkeys. Poultry Science, 80: 718-723. 473 Perdigon, G., S. Alvarez, M. Rachid, G. Aguero, and N.Gobrato, 1995. Immune System Stimulation by Probiotics. Journal of Dairy Science, 78: 1597-1606. Rada, V., M. Marounek, I. Rychly, D. Santruckova, and K. Vorisek, 1995. Effect of Lactobacillus salivarius administration on microflora in the crop and caeca of broiler chickens. Journal of Animal Feed Science, 4: 161-170. Salanitro, J.P., I. G. Fairchilds, and Y. D. Zgorninski, 1974a. Isolation, culture characteristics, and identification of anaerobic bacteria from the chicken cecum. Applied Microbiology, 27: 678-687. Salanitro, J.P., I. G. Blake, and P. A. Muirhead, 1974b. Studies on the cecal microflora of commercial broiler chickens. Applied Microbiology, 28: 439-447. Salanitro, J.P., I. G. Blake, P.A. Muirhead, M. Maglio, and J.R. Goodman, 1978. Bacteria isolated from the duodenum, ileum, and cecum of young chicks. Applied and Environmental Microbiology, 4: 782-790. Samanta, M., and P. Biswas, 1995. Effect of feeding probiotic and lactic acid on the performance of broilers. Indian Journal of Poultry Science, 30: 145-147. Sarra, P.G., F., Dellagio, and V. Bottazzi, 1985. Taxonomy of Lactobacilli isolated from the alimentary tract of chickens. Systematic and Applied Microbiology, 6: 86-89. Schneitz, C., E. Seuna, and A. Rizzo, 1981. The anaerobically cultured cecal flora of adult fowls that protects chickens from Salmonella infections. Acta Pathologica and Microbiologica Scandinavica. Sect. B., 8: 109-116. Simon, O., 2003. Probiotics in poultry production. Proceedings of International Seminar "Role of Probiotics in Animal Nutrition and their Link to the Demands of European Consumers, February 11, Lelystad, The Netherlands, pp. 1-6. Skinner, J., 2003. Raising poultry without antibiotics. Proceedings of Midwest Poul- 474 try Federation Convention, St. Paul, Minnesota, USA, March 18-20, pp. 1-4. Smith, H. W., 1965. Observations on the flora of the alimentary tract of animals and factors affecting the composition. Journal of Pathology and Bacteriology, 89: 95-122. Sofos, J. N., D. J. Fagerberg, and C. L. Quarles, 1985. Effects of sorbic acid feed fungistat on the intestinal microflora of floor-reared broiler chickens. Poultry Science, 64: 832-840. Sotirov, L., S. A. Denev, V. Georgieva, 2000. Effect of different growth promoters on lysozyme and compliment activity of broiler Chicks. Bulgarian Journal of Agriculture Science, 6: 75-82. Sotirov, L., S. A. Denev, I. Tsachev, M. Lalev, M. Oblakova, 2001. Effect of different growth promoters on lysozyme and compliment activity. II. Studing in turkeys. Revue de Medicine Veterinary, 152, (1): 67-70. Stavric, S., 1994. Probiotics in Salmonella control: Do they work? Proceedings 9th European Poultry Conference, Vol. 2. WPSA, Glasgow, 7-12th August, Walker&Connell Ltd. Darvel, UK, pp. 98-101. Timms, L., 1968. Observation on the bacterial flora of the alimentary tract in three ages groups of normal chickens. British Veterinary Journal, 124: 470-477. Va Holourek, J., 1993. Influence of different biostimulators on growth and feed consumption on broiler fowls. Zivocisna Viroba, 55: 211-220. Vitini, E., S. Alvares, M. Medina, M. Medici, M. V. Budeguer, and G. Perdigon, 2000. Gut mucosal immunostimulation by lactic acid bacteria. Biocell, 24:223-232. Vladimirova, L., and S. Sourdjiyska, 1996: Tests on the effect from adding probiotics S. A. Denev to the combined feeds for chicks. Animal Science, (1-2): 36-39 (Bg). Wambeke, F. V., J. Peeters, and F. VanWambeke, 1995. The effect of Paciflor R. on the performances, carcass composition, and caecal bacterial numbers of broilers. Archiv fur Geflugelkunde, 59: 125-129. Watkins, B. A., B. F. Miller, and D. H. Neil, 1982: In vivo effects of Lactobacillus acidophilus against pathogenic Escherichia coli in gnotobiotic chicks. Poultry Science, 61: 1298-1308. Watkins, B. A., and B. F. Miller, 1983: Competitive gut exclusion of avian pathogens by Lactobacillus acidophilus in gnotobiotic chicks. Poultry Science, 62: 1772-1779. Wenk, C., 1990. Yeast Cultures, Lactobacilli and a Mixture of Enzymes in Diets for Growing Pigs and Chickens under Swiss Conditions: Influence on the Utilization of the Nutrients and Energy. In: T. P. Lyons (Editor), Biotechnology in The Feed Industry, Proceedings of Alltech's Sixth Annual Symposium, Nicholasville, Kentucky, U.S.A., pp. 315-329. Wiseman, J., 1990. Acid-Pack 4-Way and Lacto-Sacc for broilers: effects on liveweight gain, intake and efficiency. In: T. P. Lyons (Editor). Biotechnology in The Feed Industry. Proceedings of Alltech's Sixth Annual Symposium, Nicholasville, Kentucky, U.S.A., pp. 525-526. Zulkifli, I. N. Abdullah, A. N. Mohd, and Y. W. Ho, 2000. Growth performance and immune response of two commercial broiler strains fed diet containing Lactobacillus cultures and oxytetracycline under heat stress conditions. British Poultry Science, 41: 593-597. Received February, 2, 2006; accepted April, 23, 2006.