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Abstract

This study investigated the effect of probiotic (Bacillus licheniformis) and flavomycin combination, using different doses, on growth performance and intestinal histology of broilers. Two hundred and ten one-day-old unsexed broiler chickens were allocated into five groups (based on the dose of each additive per ton of feed): A received no additives; B (probiotic 1.6x1012 CFU + flavomycin 4 g); C (probiotic 1.6x1012 CFU + flavomycin 20 g); D (probiotic 3.2x1012 CFU/ton + flavomycin 4 g/ton); E (probiotic 3.2x1012 CFU + flavomycin 20 g). On day 21 of age, group E showed the lowest (p<0.05) body weight gain (BWG). Low feed intake (FI) was observed in group B, C, and D. Low values (p<0.05) of feed conversion ratio (FCR) were recorded in group B, C, and D. Group E showed the highest value (p<0.05) of FCR. On day 42 of age, FCR was noticeably lower (p<0.05) in group B, C and D compared to group A and E. All groups received feed additives revealed a significant rise (p<0.05) in villus height (VH). The crypt depth (CD) was higher in group B and C. Group D demonstrated higher (p<0.05) villus to crypt ratio (V:C). In conclusion, the combination of high dose of the probiotic, Bacillus licheniformis, and low dose of falvomycin had beneficial effects on the performance as well as the intestinal histology of broiler chickens. Not only did the coadministration of high doses of the two additives had no benefits on the performance at six weeks of age, but also might be detrimental during starter period.

Keywords: Bacillus licheniformis; Broilers; Flavomycin; Intestine; Performance

Introduction

The use of antibiotics as growth promotors has been restricted or even strictly prohibited in many countries worldwide. This is mainly due to the great concerns regarding the antibiotic residues [1-3]. It is well known that the antibiotic residues in poultry meat can pose a potential hazard to human health such as imbalance of intestinal microbiota, and bacterial resistance to antibiotics [4]. As a result, this situation has led to adopt alternative strategies [5]. One of these strategies is the use of natural feed additives such as probiotics [6]. Probiotics are defined as "live microorganisms which, when administered in adequate amounts, confer a health benefit on the host" [7]. Several microorganisms are utilized as probiotics, including Bacillus licheniformis bacteria [8]. Flavomycin, also known as flavophospholipol, is an antibiotic which considered as another possible alternative to several antibiotics used as growth promoters in poultry diet. It is noteworthy that flavomycin can be administered without concerns about tissue residues, due to the fact that its large molecules cannot be absorbed by the gastrointestinal tract of birds [9]. Consequently, the residues of flavomycin were not detected even when added to diet in high doses. For instance, an oral dose of 20 ppm of falvomycin did not cause tissue residues in chicken [10]. Moreover, flavomycin is characterized by its low impact on beneficial bacteria existed in broiler gut [10]. It has been also reported that falvomycin reduced the shedding of potentially pathogenic bacteria in chicken feces [11]. The use of probiotics and flavomycin in broilers has been reported to improve intestinal health along with intestinal morphology, both of which resulted in the enhancement of nutrient absorption and subsequently growth performance [12-15]. On the other hand, results obtained by using either probiotics or flavomycin were sometimes controversial [16]. However, the benefits emerged from utilizing probiotics and flavomycin as feed additives may well justify the continuity of research on their positive effects [8,17]. In this respect, little is known about the use of the two additives when given in combination despite the availability of a commercial product containing both flavomycin and Bacillus licheniformis. Study conducted by wang et al. [8] on such product revealed a synergistic effects of the probiotic Bacillus licheniformis (1.35×1012 CFU/ton) and falvomycin (5 g/ton) on broiler chickens as compared to the administration of falvomycin alone. However, it is envisioned that more benefits could be obtained by determining the optimum dose of each additive when fed in combination. Therefore, the present study aimed at examining the effect of different combinations of probiotic (Bacillus licheniformis) and falvomycin on the performance and the duodenal histology of broiler chickens.

Materials and Methods

Experimental design: The experiment was carried out in closed house at Animal Production Research Center, Poultry Research Section, Sudan. Two hundred and ten one-day-old unsexed broiler chicks (Arbor acres) were utilized in this study. The birds were weighted individually to obtain the average initial body weight. Afterwards, the chicks were allocated randomly into five treatment groups, using randomized complete block design. Each group comprised 42 birds which were divided into six replicates of 7 birds each. The replicate was represented by a pen with a dimensions of 1 × 1 m2 and wood shavings on the floor The five dietary treatment groups were: group A received no additives; group B received Bacillus licheniformis 1.6x1012 CFU/ton of feed + flavomycin 4 g/ton of feed; group C received Bacillus licheniformis 1.6x1012 CFU/ton of feed + flavomycin 20 g/ton of feed; group D received Bacillus licheniformis 3.2x1012 CFU/ton of feed + flavomycin 4 g/ton of feed; group E received 3.2x1012 CFU/ton of feed + flavomycin 20 g/ton of feed (Table 1). The high and low doses of the probiotic were determined according to the guidelines given by the manufacturer. The low dose of falvomycin in this study was approximately similar to the dose utilized by Wang et al. [8]. The high dose of flavomycin was calculated based on its safe use, in particular the lack of tissue residues [10].

The basal diet given to the chicks was calculated to meet the nutrients requirement of broilers in the starter (1 to 23 days of age) and finisher (24 to 42 days of age) periods (Table 2) as recommended by the National Research Council [18]. Mash feed was then formulated using a horizontal mixture. Birds were allowed to access water and feed ad libitum. Continuous lighting program was applied throughout the experiment. The house temperature had been set at 33 °C from day 1 to day 3 before it was reduced gradually to 24 °C until the end of the experiment.

Growth Performance: Growth performance was evaluated according to the calculations of body weight gain (BWG), feed intake (FI), and feed conversion ratio (FCR) at the end of weeks 3 and 6 of age (days 21 and 42).

Samples Collection: On day 42, 6 broiler chickens from each group were selected (one bird from each replicate). Birds were then slaughtered manually by making a fast-single ventral cut in the neck using a sharp knife with straight surface. The small intestine was detached from the other visceral organs and samples from the mid of the duodenum were taken for histological investigations. Samples were fixed in 10% neutral buffered formalin for 5 days, and processed for routine histological technique. Thirty tissue sections (6 for each treatment) were stained with hematoxylin and eosin. Sections were then examined under light microscope connected to a digital camera (Optica microscopes, Italy). Histological measurements were then determined using a software program provided with the camera. Ten well-oriented villi with prominent lamina propria were selected from each section. The villus height (VH) was measured from the base to the tip of each villus. In addition, ten well-defined intestinal crypts were also selected for measurement in each section. Therefore, a total of 600 readings (300 for villi and 300 for intestinal crypts) were carried out; each group was representing by 120 readings (60 for villi and 60 for intestinal crypts).

Statistical Analysis: Statistical analysis was conducted using one-way ANOVA in statistical software (SPSS version 21.0, IBM Corporation, New York, USA). Significant differences were determined at p<0.05 and Duncan’s multiple range test was applied to separate the treatment means.

Results

Growth performance: Table 3 shows the growth performance of the broiler chickens utilized in this study. On day 21 of age, group D showed the highest (p<0.05) BWG (591.7 g) whereas the lowest (p<0.05) BWG was observed in group E (499.9 g). Remarkable reduction (p<0.05) in FI was observed in group B (714.2 g), C (656.0 g), and D (695.4 g) when compared to group A (837.9 g) and E (822.5 g). Significantly lower values (p<0.05) of FCR were recorded in group B (1.25), C (1.17), and D (1.17) compared to group A (1.47). The highest value (p<0.05) of FCR was noted in group E (1.64). On day 42 of age, the highest (p<0.05) BWG was observed in group B (2244.8 g). Significantly lower (p<0.05) FI was noted in group B (3298.1 g), C (3255.5 g), and D (3246.4 g) when compared to FI in group A (3434.6 g). Broilers in groups B, C and D displayed noticeably lower (p<0.05) FCR (1.47,1.49, 1.48 respectively) as compared to FCR in group A (1.68) and E (1.62).

Intestinal histology: Table 4 shows the histological parameters of the duodenum of the broiler chickens in this study. All treatment groups received feed additives revealed a significant rise (p<0.05) in VH when compared to control group. In addition, the VH in group C (1735.7 µm) and D (1655.7 µm) was significantly higher as compared to group B (1506.2 µm) and E (1439.1 µm). The CD was remarkably (p<0.05) higher in group B (208.4 µm) and C (214.0 µm) when compared to CD in group A (189.9 µm). The CD in group D (199.5 µm) and E (197.5 µm) did not show a significant difference (p>0.05) as compared to control group (189.9 µm). Birds in group D had substantially higher (p<0.05) V:C (8.59) compared to birds in group A (7.28), B (7.33), and E (7.71).

Discussion

Similarly, most of the combinations of Bacillus licheniformis and falvomycin utilized in this study displayed marked improvement in the growth performance of broilers during the first three weeks of age. In contrast, it has been reported that the combination of flavomycin and Bacillus licheniformis had no discernable improvement in the performance of broilers during starter period [8]. In this study, the combination of higher doses of the two additives during the same period might have been even detrimental to the broiler chicks as they exhibited poor FCR compared to broilers given neither of the additives. However, the aforementioned negative impact of the combination of these additives during the relatively fast-growing stage of chickens warrant further investigations. In this study, the combinations containing different doses of Bacillus licheniformis and falvomycin had substantial beneficial results on growth performance at six weeks of age as compared to birds received no additives. This is in accord with the finding reported by Wang et al. [18] who stated that the dietary addition of Bacillus licheniformis and flavomycin in combination was more effective compared to flavomycin when given alone. Additionally, the combined effect of probiotics and flavomycin on body weight gain has been also reported in broiler chickens at day 38 of age [19]. On the other hand, despite the positive effects of the two additives when given in combination, it is essential to indicate that the coadministration of high doses of Bacillus licheniformis and falvomycin in this study had no beneficial effects on growth performance of broiler chickens at six weeks of age. It is well documented that the histological features of the small intestine of broiler chicks are improved by dietary addition of either probiotics, Bacillus spp. [8,17] or flavomycin [8,20]. In this study, all treatment groups received feed additives have shown remarkable increase in VH as compared to the group received no additives. Besides, the V:C was essentially more pronounced in chickens received higher dose of the probiotic and lower dose of falvomycin. Taken into account the results of the growth performance in this study, it is most likely that the combination of higher dose of probiotic and lower dose of falvomycin had more beneficial effects not only on performance but also on intestinal histology of broiler chickens. In conclusion, the present study revealed that the combination of higher dose of the probiotic, Bacillus licheniformis, and lower dose of falvomycin had beneficial effects on the performance as well as the intestinal histology of broiler chickens. In contrast, the coadministration of high doses of the probiotic and falvomycin had no benefits on the growth performance at six weeks of age. The high doses of the two additives might have been even detrimental during starter period.

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